Molecular Networks Controlling Dynamic Chromosome Behaviors during
Development
Our research focuses on the interplay between the structure of chromosomes and their
function. Chromosomes undergo dynamic behaviors during development to ensure
genome stability and accurate cell fate decisions. We study inter-related molecular networks
that control diverse chromosome behaviors: chromosome counting to determine sexual
fate; X-chromosome remodeling to achieve X-chromosome repression during dosage
compensation, an epigenetic process; chromosome cohesion to tether and release
replicated chromosomes for reducing genome copy number during germ cell formation;
and chromosome compaction to control gene expression, chromosome segregation, and
recombination between maternal and paternal chromosomes. We have found that the
developmental control of gene expression is achieved through chromatin modifications that
affect chromosome structure with epigenetic consequences. We have also established
robust procedures for targeted genome editing across nematode species diverged by 300
MYR to study the evolution of sex determination and dosage compensation. We combine
genetic, genomic, proteomic, biochemical, and cell biological approaches to study these
questions in the model organism Caenorhabditis elegans, a round worm, and its related
nematode species.



Counting Chromosomes to Determine Sex: Molecular Antagonism between
X-Chromosome, Autosome Signals Specifies Nematode Sex
Many organisms determine sexual fate by a chromosome-counting mechanism that
distinguishes one X chromosome from two. Embryos with one X become males, while those
with two become females. We dissected the molecular mechanism by which the nematode
C. elegans counts its sex chromosomes to discern how small changes in the concentrations
of molecular signals are translated into dramatically different developmental fates. C.
elegans tallies X-chromosome number relative to the ploidy, the sets of autosomes (X:A
signal). It discriminates with high fidelity between tiny differences in the signal: 2X:3A
embryos (ratio 0.67) become males, while 3X:4A embryos (ratio 0.75) become
hermaphrodites (Figure 1).



































We showed that a set of X-linked genes called X-signal elements (XSEs) communicates
X-chromosome dose by repressing the master sex-determination switch gene xol-1 in a
cumulative, dose-dependent manner. XOL-1, a GHMP kinase, is activated in 1X:2A embryos
(1 dose of XSEs) to set the male fate but repressed in 2X:2A embryos (2 doses of XSEs) to
promote the hermaphrodite fate, including the activation of X-chromosome dosage
compensation. We also showed that the dose of autosomes is communicated by a set of
autosomal signal elements (ASEs) that also act in a cumulative, dose-dependent manner to
counter XSEs by stimulating xol-1 transcription. We have explored the biochemical basis by
which XSEs counter ASEs to determine sex. Analysis in vitro showed that XSEs (nuclear
receptors and homeodomain proteins) and ASEs (T-box and zinc-finger proteins) bind
directly to at least 5 distinct sites in xol-1 regulatory DNA to counteract each other's activities
and thereby regulate xol-1 transcription (Figure 2). Analysis in vivo showed that disrupting
ASE and XSE binding sites recapitulated the mis-regulation of xol-1 transcription caused by
disrupting the cognate signal element genes. XSE and ASE binding sites are distinct and
non-overlapping, suggesting that direct competition for xol-1 binding is not the mechanism
by which XSEs counter ASEs. Instead, XSEs likely antagonize ASEs by recruiting cofactors with
reciprocal activities that induce opposite transcriptional states. The X:A balance is thus
communicated in part through multiple antagonistic molecular interactions carried out on a
single promoter, revealing how small differences in X:A values can elicit different sexual
fates. We are currently identifying potential coactivators and corepressors and directing
efforts toward understanding the evolution of the X:A signal across nematode species.

Although most XSEs repress xol-1 by regulating transcription, one XSE, an RNA binding
protein, represses xol-1 by binding to an alternatively spliced intron and blocking its proper
splicing, thereby generating a non-functional transcript with an in-frame stop codon (Figure
2). This second tier of repression enhances the fidelity of the counting process.

The concept of a sex signal comprising competing XSEs and ASEs arose as a theory for fruit
flies one century ago, and it subsequently became entrenched in textbooks. Ironically, the
recent work of others showed the fly sex signal does not fit this simple paradigm, but our
work shows the worm signal does.

X-Chromosome Dosage Compensation: Repressing X Chromosomes via Molecular
Machines.
Organisms that use sex chromosomes to determine sexual fate evolved the essential,
chromosome-wide regulatory process called dosage compensation to balance
X-chromosome gene expression between the sexes. Strategies for dosage compensation
differ from worms to mammals, but invariably a regulatory complex is targeted to X
chromosomes of one sex to modulate transcription along the entire chromosome. The
heritable, regulation of X-chromosome expression during dosage compensation is
exemplary for dissecting the coordinate regulation of gene expression over large
chromosomal territories and the role of chromosome structure in regulating gene
expression.

We defined the C. elegans dosage compensation complex (DCC) and showed it is
homologous to condensin, a conserved protein complex that mediates the compaction,
resolution, and segregation of mitotic and meiotic chromosomes from yeast to humans
(Figure 3). The DCC binds to both X chromosomes of hermaphrodites to reduce
transcription by half (Figure 3). Failure to reduce expression kills hermaphrodites. Most DCC
condensin subunits also control the structure and function of mitotic and meiotic
chromosomes by participating in two other distinct condensin complexes (Figure 3). Not
only has the DCC co-opt subunits of condensin to control gene expression, it co-opted a
subunit from the MLL/COMPASS complex, a histone modifying complex, to help recruit
condensin subunits to rex sites.

We found that the DCC condensin subunits are recruited specifically to hermaphrodite X
chromosomes by sex-specific DCC subunits that trigger binding to cis-acting regulatory
elements on X, called rex and dox sites. rex (recruitment elements on X) sites recruit the DCC
in an autonomous, sequence-dependent manner using DNA motifs highly enriched on X
chromosomes. The DCC spreads to dox (dependent on X) sites, which reside in promoters of
active genes and bind the DCC robustly only when linked to rex sites.
































Dynamic Control of X-Chromosome Conformation and Repression by a Histone
H4K20me Demethylase.
We recently found that DCC subunit DPY-21 has a histone demethylase activity that is
responsible for the selective enrichment of H4K20me1 on X chromosomes of XX embryos
upon DCC binding.  X-ray crystallography and biochemical assays of DPY-21 revealed a novel
subfamily of Jumonji C histone demethylases that converts H4K20me2 to H4K20me1. 
Selective inactivation of demethylase activity in vivo by genome editing eliminated
H4K20me1 enrichment on X, elevated X-linked gene expression, reduced X-chromosome
compaction, and disrupted X-chromosome topology by weakening TAD boundaries.  These
findings, among others, demonstrate the direct impact of chromatin modification on
higher-order chromosome structure in long-range regulation of gene expression.

H4K20me1 is also enriched on the mammalian inactive X chromosome, but the role of this
enrichment in mammalian transcriptional silencing is not known, nor is a selective reagent
available to test its role.  We showed that the mouse homolog of the DCC subunit also has
H4K20me2 demethylase activity.  Hence the worm system holds great promise for
understanding the effects of histone modification in mammals.

Unexpectedly, DPY-21 associates with autosomes but not X chromosomes of germ cells in a
DCC-independent manner to enrich H4K20me1 and facilitate chromosome compaction. 
Thus, DPY-21 is an adaptable chromatin regulator that is harnessed during development for
distinct biological functions.  In both somatic cells and germ cells, H4K20me1 enrichment
modulates 3D chromosome architecture to carry out these functions.































Step of transcription controlled by the DCC.
We have dissected a key aspect of the dosage compensation mechanism by determining the
step of transcription controlled by the DCC to repress X-chromosome gene expression. This
work was performed in collaboration with John Lis' lab at Cornell University. In principle, the
DCC could control any step of transcription: recruitment of RNA polymerase II (Pol II) to the
promoter, initiation of transcription, escape of Pol II from the promoter or pause sites,
elongation of RNA transcripts, or termination of transcription. The mechanism had been
elusive in C. elegans due to improper annotation of transcription start sites (TSSs). Nascent
RNA transcripts from most nematode genes undergo rapid co-transcriptional processing in
which the 5' end is replaced by a common 22-nucleotide leader RNA through a
trans-splicing mechanism, thereby destroying all knowledge of TSSs and promoters.

To understand the step of transcription controlled by the DCC, we first devised a general
strategy for mapping transcription start sites and created an invaluable nematode TSS data
set. The TSS mapping strategy, called GRO-cap, recovered nascent RNAs with 5'-caps prior to
processing. We then determined the genome-wide distribution, orientation, and quantity of
transcriptionally-engaged RNA Polymerase II (Pol II) relative to TSSs in wild-type and
DC-defective animals using GRO-seq (global run-on sequencing).

We found that promoters are unexpectedly far upstream from the 5' ends of mature
mRNAs, and promoter-proximal Pol II pausing occurs only in starved larvae and is rare in C.
elegans embryos, unlike in most metazoans. These results indicated that enhancement of
promoter pausing in XX embryos cannot be the mechanism of reducing transcription during
dosage compensation. In contrast, control of pausing is a common mechanism for
controlling transcription of developmental regulatory genes in most metazoans and is
thought to be the mechanism of dosage compensation in fruit flies.

Then, by comparing the location and density of transcriptionally engaged Pol II in wild-type
and dosage-compensation-defective embryos, we found that the step of transcription
controlled by the dosage compensation process is the recruitment of Pol II. That is, C.
elegans equalizes X-chromosome-wide gene expression between the sexes by reducing
Pol II recruitment to the promoters of X-linked genes in XX embryos by about half. One of
our research directions is to dissect the mechanisms by which the DCC limits Pol II
recruitment.

Our data set also enabled us to analyze starvation-controlled gene regulation in
collaboration with Ryan Baugh's lab at Duke University. We found a new phenomenon of Pol
II docking, the stable association of Pol II upstream of the transcription start sites, and
hence sites of pausing. We found that docked Pol II accumulates, without initiating,
upstream of inactive growth genes that are turned off during starvation are activated upon
feeding. We found that Pol II pausing occurs at active stress-response genes that are
downregulated upon feeding. Hence, growth and stress genes are controlled by distinct
mechanisms to coordinate gene expression with nutrient availability.

DCC recruitment and binding to X chromosomes.
We showed that many of the DCC recruitment (rex) sites have a DNA motif (called MEX) that
is highly enriched on X compared to autosomes and is essential for DCC binding to a subset
of rex sites. However, not all rex sites have this motif. We recently defined new principles by
which the DCC is recruited to X chromosomes, including the identification of a new,
essential DCC binding motif (MEX II) that is enriched on X. We found that MEX II acts in
combination with MEX to foster high-affinity binding at some rex sites but also acts alone at
other rex sites to foster stable binding. We demonstrated these DCC binding principles by
using DCC binding assays in vivo and in vitro.

We also showed that SUMOylation of specific DCC subunits is essential for sex-specific
assembly and function of the DCC on X. Depletion of SUMO in vivo severely disrupts DCC
binding and causes changes in X-linked gene expression similar to those caused by deleting
the genes that encode DCC subunits. Three DCC subunits undergo SUMOylation, one
subunit essential for DCC loading and two subunits that are integral to the condensin
portion of the DCC.

DCC SUMOylation is triggered by the signal that initiates DCC assembly onto X. The initial
step of assembly--binding of X-targeting factors to rex sites--is independent of SUMOylation,
but robust binding of the complete complex requires SUMOylation. One of SUMOylated
DCC subunits also participates in condensin complexes essential for chromosome
segregation, but its SUMOylation occurs only in the context of the DCC. Our results reinforce
a newly emerging theme in which multiple proteins of a complex are collectively
SUMOylated in response to a specific stimulus, leading to accelerated complex formation
and enhanced function.

Condensin-driven remodeling of X-chromosome topology during dosage
compensation.
The three-dimensional organization of a genome plays a critical role in regulating gene
expression, yet little is known about the machinery and mechanisms that determine
higher-order chromosome structure. The involvement of bona fide condensin subunits in
dosage compensation together with our observation that the DCC acts at a distance to
regulate gene expression suggested that the DCC might alter the topology of X
chromosomes to reduce gene expression chromosome wide.

Using genome-wide chromosome conformation capture techniques (in collaboration with
Job Dekker's lab at U. Mass. Worcester) with single-cell fluorescence in situ hybridization
and RNA-seq to compare chromosome structure and gene expression in wild-type and
dosage-compensation-defective embryos, we showed that the DCC remodels X
chromosomes of hermaphrodites into a unique, sex-specific spatial conformation, distinct
from autosomes, using its highest-affinity rex sites to facilitate long-range interactions
across X. Dosage-compensated X chromosomes consist of self-interacting domains (~ 1 Mb)
resembling mammalian Topologically Associating Domains (TADs). TADs on X have stronger
boundaries and more regular spacing than those on autosomes. Many TAD boundaries on X
coincide with the highest-affinity rex sites, and these boundaries become diminished or lost
in mutants lacking DCC binding, causing the structure of X to resemble that of autosomes.
These results predicted that deletion of an endogenous rex site at a DCC-dependent
boundary should disrupt the boundary. As predicted, Cas9-mediated deletion of a rex site
greatly diminished the boundary, further demonstrating the condensin-driven remodeling
of X-chromosome topology during dosage compensation. Thus, condensin acts as a key
structural element to reorganize interphase chromosomes and thereby regulate gene
expression. Prior to our work, no molecular trigger or set of DNA binding sites was known to
cause a comparably strong effect on TAD structure in higher eukaryotes. Our understanding
of the topology of dosage-compensated X chromosomes provides fertile ground to decipher
the detailed mechanistic relationship between higher-order chromosome structure and
chromosome-wide regulation of gene expression.












































Targeted Genome-editing Across Highly Diverged Nematode Species.
Thwarted by the lack of reverse genetic approaches to enable cross-species comparisons of
gene function, we established robust strategies for targeted genome editing across
nematode species diverged by 300 MYR. In our initial work, a collaboration with Sangamo
BioSciences, we used engineered nucleases containing fusions between the DNA cleavage
domain of the enzyme FokI and a custom-designed DNA binding domain: either zinc-finger
motifs for zinc-finger nucleases or transcription activator-like effector domains for TALE
nucleases (TALENs). In those experiments, we allowed the DNA double-strand breaks to be
repaired imprecisely by non-homologous end joining (NHEJ) to create mutations in precise
locations.

We then extended the use of TALENs to achieve precise insertion and deletion of desired
sequences by introducing single-stranded or double-stranded templates to generate precise
insertions or deletions through homology directed repair (HDR), the first demonstration of
HDR using ZFNs or TALENs in the nematode community. We then adopted the use of the
CRISPR-associated nuclease Cas9 because of the ease in making RNA guides to program
target specificity.

Despite successful application of Cas9 technology, predicting DNA targets and guide RNAs
that support efficient genome editing was problematic. We then devised a strategy for
high-frequency genome editing (both NHEJ and HDR) at all targets tested. The key
innovation was designing guide RNAs with a GG motif at the 3' end of their target-specific
sequences. This design increased the frequency of mutagenesis 10-fold. The ease of mutant
recovery was further enhanced by combining this efficient guide design with a
co-conversion strategy, in which targets of interest are analyzed in animals exhibiting a
dominant phenotype caused by Cas9-dependent editing of an unrelated target.

Evolution cis-acting Regulatory Sites that Control Dosage Compensation.
Mechanisms that specify sexual fate and compensate for X-chromosome dose have
diverged rapidly across species compared to other developmental processes, making it
particularly informative to study these rapidly changing processes over short evolutionary
time scales. Application of our genome editing strategies to C. briggsae revealed that the
core dosage compensation machinery and key components of the genetic hierarchy that
controls dosage compensation and sex determination were conserved across the 30 MYR
separation between C. elegans and C. briggsae. In contrast, the set of cis-acting elements on
X that recruit the DCC (rex sites) has diverged, retaining no functional overlap. ChIP-seq
analysis defined the C. briggsae DCC binding sites, and in vivo binding assays confirmed the
ability of these sites to recruit the DCC when detached from X in C. briggsae but not in C.
elegans, and vice versa. The evolution of these sites differs dramatically from the highly
conserved DCC binding sites used by equivalently diverged fruit fly species and from the
unchanged target sites of conserved transcription factors that control multiple
developmental processes from flies to humans. Hence, the divergence in DCC binding
specificity across nematode species provides a powerful opportunity to understand the
path and timing for the concerted change in hundreds of DNA target sites and the evolution
of X chromosomes. We have extended our analysis of DCC binding specificity to other
nematode species and have shown that rex sites have diverged functionally at least three
times in 30 MYR of evolutionary history.

Tethering Replicated Chromosomes via Cohesin to Ensure Genome Stability during
Meiosis.
Faithful segregation of chromosomes during cell division is essential for genome stability.
Accurate chromosome segregation is required both for the proliferative cell divisions that
produce daughter cells during mitosis and the two sequential divisions that produce
haploid sperm and eggs from diploid germline stem cells during meiosis. Approximately
30% of human zygotes have abnormal chromosome content at conception due to defects in
meiosis. Such aneuploidy is a leading cause of miscarriages and birth defects and arises, in
part, from defects in sister chromatid cohesion (SCC). SCC tethers replicated sister
chromatids prior to cell divisions to ensure proper chromosome segregation. In humans,
SCC is established in the developing germ cells of a fetus and must be maintained until
ovulation in adults. This long-lived SCC is established and maintained by cohesin complexes,
evolutionarily conserved protein complexes structurally related to condensin (Figure 6).





































Studies in budding yeast showed that mitotic and meiotic cohesins are distinct but differ
only in a single subunit called the kleisin. During yeast meiosis, a single cohesin complex
carries out all aspects of SCC. In contrast, our work in nematodes shows that regulation of
meiotic SCC in higher eukaryotes is more complex. We found that multiple functionally
specialized cohesin complexes mediate the establishment and two-step release of SCC that
underlies the production of haploid gametes (Figure 6). The meiotic complexes differ by a
single kleisin subunit, and the kleisin influences nearly all aspects of meiotic cohesin
function: the mechanisms for loading cohesins onto chromosomes, for triggering
DNA-bound cohesins to become cohesive, and for releasing cohesins in a temporal- and
location-specific manner (Figure 7). One kleisin triggers cohesion just after the
chromosomes replicate, as in yeast. Unexpectedly, the other triggers cohesion in a
replication-independent manner, only after programmed DSBs are made during meiosis to
initiate recombination between homologous maternal and paternal chromosomes. Thus,
break-induced cohesion is essential for tethering replicated meiotic chromosomes. Later,
recombination stimulates separase-independent removal of the two different cohesin
complexes from reciprocal chromosomal territories flanking the crossover site. This
region-specific removal likely underlies the two-step separation of homologs and sisters.
Unexpectedly, one cohesin complex also performs cohesion-independent functions in
synaptonemal complex assembly. Our findings establish a new model for cohesin function
in meiosis: the choreographed actions of multiple cohesins, endowed with unexpectedly
specialized functions by their kleisins, underlie the stepwise separation of homologous
chromosomes and then sister chromatids required for reduction of genome copy number.
This model diverges significantly from that in yeast but likely applies to plants and
mammals, which utilize similar meiotic kleisins.

Meiotic Chromosome Structures Constrain and Respond to Designation of Crossover
Sites.
Crossover recombination events between homologous chromosomes are required to form
chiasmata, temporary connections between homologues that ensure their proper
segregation at meiosis I. Despite this requirement for crossovers and an excess of the
double-strand DNA breaks that are the initiating events for meiotic recombination, most
organisms make very few crossovers per chromosome pair. Moreover, crossovers tend to
inhibit the formation of other crossovers nearby on the same chromosome pair, a poorly
understood phenomenon known as crossover interference. We showed (in collaboration
with the Villenueve lab at Stanford) that the synaptonemal complex, a meiosis-specific
structure that assembles between aligned homologous chromosomes, both constrains and
is altered by crossover recombination events. Partial depletion of the synaptonemal
complex central region proteins attenuates crossover interference, increasing crossovers
and reducing the effective distance over which interference operates, indicating that
synaptonemal complex proteins limit crossovers. Moreover, we showed that crossovers are
associated with a local 0.4-0.5-micrometre increase in chromosome axis length. We
proposed that meiotic crossover regulation operates as a self-limiting system in which
meiotic chromosome structures establish an environment that promotes crossover
formation, which in turn alters chromosome structure to inhibit other crossovers at
additional sites.

Ironically, the effect of depleting condensin I or condensin II on increasing crossovers
appears to occur by a different mechanism, because the sites of extra crossovers are not
marked by the same molecular markers as the crossovers created by reducing the
synaptonemal complex. We are investigating the crossover pathway employed to achieve
these extra, non-interfering crossovers in condensin mutants.

Figure 6. Multiple cohesin complexes tether
meiotic chromosomes. (Click to enlarge.)
Figure 2. Model depicting the molecular antagonism between
XSEs and ASEs that determines sex.  (Click to enlarge.)
Research
Figure 5. DCC modulates spatial organization of X chromosomes. (Click to enlarge.)
Figure 3. Biochemically distinct condensin complexes with
interchangeable subunits control chromosome structure
throughout C. elegans development. (Click to enlarge.)
Molecular Networks Controlling Dynamic Chromosome Behaviors during Development
Our research focuses on the interplay between the structure of chromosomes and their function. Chromosomes undergo dynamic
behaviors during development to ensure genome stability and accurate cell fate decisions. We study inter-related molecular
networks that control diverse chromosome behaviors: chromosome counting to determine sexual fate; X-chromosome-wide
repression during dosage compensation to balance gene expression between the sexes; chromosome cohesion to tether and
release replicated chromosomes for reducing genome copy number during germ cell formation; and chromosome compaction to
control gene expression, chromosome segregation, and recombination between maternal and paternal chromosomes. We have also
established robust procedures for targeted genome editing across nematode species diverged by 300 MYR.



Counting Chromosomes to Determine Sex: Molecular Antagonism between X-Chromosome, Autosome Signals Specifies
Nematode Sex
Many organisms determine sexual fate by a chromosome-counting mechanism that distinguishes one X chromosome from two.
Embryos with one X become males, while those with two become females. We dissected the molecular mechanism by which the
nematode C. elegans counts its sex chromosomes to discern how small changes in the concentrations of molecular signals are
translated into dramatically different developmental fates. C. elegans tallies X-chromosome number relative to the ploidy, the sets of
autosomes (X:A signal). It discriminates with high fidelity between tiny differences in the signal: 2X:3A embryos (ratio 0.67) become
males, while 3X:4A embryos (ratio 0.75) become hermaphrodites (Figure 1).




















We showed that a set of X-linked genes called X-signal elements (XSEs) communicates X-chromosome dose by repressing the master
sex-determination switch gene xol-1 in a cumulative, dose-dependent manner. XOL-1, a GHMP kinase, is activated in 1X:2A embryos
(1 dose of XSEs) to set the male fate but repressed in 2X:2A embryos (2 doses of XSEs) to promote the hermaphrodite fate, including
the activation of X-chromosome dosage compensation. We also showed that the dose of autosomes is communicated by a set of
autosomal signal elements (ASEs) that also act in a cumulative, dose-dependent manner to counter XSEs by stimulating xol-1
transcription. We have explored the biochemical basis by which XSEs counter ASEs to determine sex. Analysis in vitro showed that
XSEs (nuclear receptors and homeodomain proteins) and ASEs (T-box and zinc-finger proteins) bind directly to at least 5 distinct sites
in xol-1 regulatory DNA to counteract each other's activities and thereby regulate xol-1 transcription (Figure 2). Analysis in vivo
showed that disrupting ASE and XSE binding sites recapitulated the mis-regulation of xol-1 transcription caused by disrupting the
cognate signal element genes. XSE and ASE binding sites are distinct and non-overlapping, suggesting that direct competition for
xol-1 binding is not the mechanism by which XSEs counter ASEs. Instead, XSEs likely antagonize ASEs by recruiting cofactors with
reciprocal activities that induce opposite transcriptional states. The X:A balance is thus communicated in part through multiple
antagonistic molecular interactions carried out on a single promoter, revealing how small differences in X:A values can elicit different
sexual fates. We are currently identifying potential coactivators and corepressors and directing efforts toward understanding the
evolution of the X:A signal across nematode species.

Although most XSEs repress xol-1 by regulating transcription, one XSE, an RNA binding protein, represses xol-1 by binding to an
alternatively spliced intron and blocking its proper splicing, thereby generating a non-functional transcript with an in-frame stop
codon (Figure 2). This second tier of repression enhances the fidelity of the counting process.

The concept of a sex signal comprising competing XSEs and ASEs arose as a theory for fruit flies one century ago, and it
subsequently became entrenched in textbooks. Ironically, the recent work of others showed the fly sex signal does not fit this simple
paradigm, but our work shows the worm signal does.

X-Chromosome Dosage Compensation: Repressing X Chromosomes via Molecular Machines.
Organisms that use sex chromosomes to determine sexual fate evolved the essential, chromosome-wide regulatory process called
dosage compensation to balance X-chromosome gene expression between the sexes. Strategies for dosage compensation differ
from worms to mammals, but invariably a regulatory complex is targeted to X chromosomes of one sex to modulate transcription
along the entire chromosome. The heritable, regulation of X-chromosome expression during dosage compensation is exemplary for
dissecting the coordinate regulation of gene expression over large chromosomal territories and the role of chromosome structure in
regulating gene expression.

We defined the C. elegans dosage compensation complex (DCC) and showed it is homologous to condensin, a conserved protein
complex that mediates the compaction, resolution, and segregation of mitotic and meiotic chromosomes from yeast to humans
(Figure 3). The DCC binds to both X chromosomes of hermaphrodites to reduce transcription by half (Figure 3). Failure to reduce
expression kills hermaphrodites. Most DCC condensin subunits also control the structure and function of mitotic and meiotic
chromosomes by participating in two other distinct condensin complexes (Figure 3). Not only has the DCC co-opt subunits of
condensin to control gene expression, it co-opted a subunit from the MLL/COMPASS complex, a histone modifying complex, to help
recruit condensin subunits to rex sites.

We found that the DCC condensin subunits are recruited specifically to hermaphrodite X chromosomes by sex-specific DCC subunits
that trigger binding to cis-acting regulatory elements on X, called rex and dox sites. rex (recruitment elements on X) sites recruit the
DCC in an autonomous, sequence-dependent manner using DNA motifs highly enriched on X chromosomes. The DCC spreads to
dox (dependent on X) sites, which reside in promoters of active genes and bind the DCC robustly only when linked to rex sites.




















Step of transcription controlled by the DCC. We have dissected a key aspect of the dosage compensation mechanism by determining
the step of transcription controlled by the DCC to repress X-chromosome gene expression. This work was performed in
collaboration with John Lis' lab at Cornell University. In principle, the DCC could control any step of transcription: recruitment of RNA
polymerase II (Pol II) to the promoter, initiation of transcription, escape of Pol II from the promoter or pause sites, elongation of RNA
transcripts, or termination of transcription. The mechanism had been elusive in C. elegans due to improper annotation of
transcription start sites (TSSs). Nascent RNA transcripts from most nematode genes
undergo rapid co-transcriptional processing in which the 5' end is replaced by a common 22-nucleotide leader RNA through a
trans-splicing mechanism, thereby destroying all knowledge of TSSs and promoters.

To understand the step of transcription controlled by the DCC, we first devised a general strategy for mapping transcription start
sites and created an invaluable nematode TSS data set. The TSS mapping strategy, called GRO-cap, recovered nascent RNAs with
5'-caps prior to processing. We then determined the genome-wide distribution, orientation, and quantity of
transcriptionally-engaged RNA Polymerase II (Pol II) relative to TSSs in wild-type and DC-defective animals using GRO-seq (global
run-on sequencing).

We found that promoters are unexpectedly far upstream from the 5' ends of mature mRNAs, and promoter-proximal Pol II pausing
occurs only in starved larvae and is rare in C. elegans embryos, unlike in most metazoans. These results indicated that enhancement
of promoter pausing in XX embryos cannot be the mechanism of reducing transcription during dosage compensation. In contrast,
control of pausing is a common mechanism for controlling transcription of developmental regulatory genes in most metazoans and
is thought to be the mechanism of dosage compensation in fruit flies.

Then, by comparing the location and density of transcriptionally engaged Pol II in wild-type and dosage-compensation-defective
embryos, we found that the step of transcription controlled by the dosage compensation process is the recruitment of Pol II. That is,
C. elegans equalizes X-chromosome-wide gene expression between the sexes by reducing Pol II recruitment to the promoters of
X-linked genes in XX embryos by about half. One of our research directions is to dissect the mechanisms by which the DCC limits Pol
II recruitment.

Our data set also enabled us to analyze starvation-controlled gene regulation in collaboration with Ryan Baugh's lab at Duke
University. We found a new phenomenon of Pol II docking, the stable association of Pol II upstream of the transcription start sites,
and hence sites of pausing. We found that docked Pol II accumulates, without initiating, upstream of inactive growth genes that are
turned off during starvation are activated upon feeding. We found that Pol II pausing occurs at active stress-response genes that are
downregulated upon feeding. Hence, growth and stress genes are controlled by distinct mechanisms to coordinate gene expression
with nutrient availability.

DCC recruitment and binding to X chromosomes. We showed that many of the DCC recruitment (rex) sites have a DNA motif (called
MEX) that is highly enriched on X compared to autosomes and is essential for DCC binding to a subset of rex sites. However, not all
rex sites have this motif. We recently defined new principles by which the DCC is recruited to X chromosomes, including the
identification of a new, essential DCC binding motif (MEX II) that is enriched on X. We found that MEX II acts in combination with MEX
to foster high-affinity binding at some rex sites but also acts alone at other rex sites to foster stable binding. We demonstrated these
DCC binding principles by using DCC binding assays in vivo and in vitro.

We also showed that SUMOylation of specific DCC subunits is essential for sex-specific assembly and function of the DCC on X.
Depletion of SUMO in vivo severely disrupts DCC binding and causes changes in X-linked gene expression similar to those caused by
deleting the genes that encode DCC subunits. Three DCC subunits undergo SUMOylation, one subunit essential for DCC loading and
two subunits that are integral to the condensin portion of the DCC.

DCC SUMOylation is triggered by the signal that initiates DCC assembly onto X. The initial step of assembly--binding of X-targeting
factors to rex sites--is independent of SUMOylation, but robust binding of the complete complex requires SUMOylation. One of
SUMOylated DCC subunits also participates in condensin complexes essential for chromosome segregation, but its SUMOylation
occurs only in the context of the DCC. Our results reinforce a newly emerging theme in which multiple proteins of a complex are
collectively SUMOylated in response to a specific stimulus, leading to accelerated complex formation and enhanced function.

Condensin-driven remodeling of X-chromosome topology during dosage compensation. The three-dimensional organization of a
genome plays a critical role in regulating gene expression, yet little is known about the machinery and mechanisms that determine
higher-order chromosome structure. The involvement of bona fide condensin subunits in dosage compensation together with our
observation that the DCC acts at a distance to regulate gene expression suggested that the DCC might alter the topology of X
chromosomes to reduce gene expression chromosome wide.

Using genome-wide chromosome conformation capture techniques (in collaboration with Job Dekker's lab at U. Mass. Worcester)
with single-cell fluorescence in situ hybridization and RNA-seq to compare chromosome structure and gene expression in wild-type
and dosage-compensation-defective embryos, we showed that the DCC remodels X chromosomes of hermaphrodites into a unique,
sex-specific spatial conformation, distinct from autosomes, using its highest-affinity rex sites to facilitate long-range interactions
across X. Dosage-compensated X chromosomes consist of self-interacting domains (~ 1 Mb) resembling mammalian Topologically
Associating Domains (TADs). TADs on X have stronger boundaries and more regular spacing than those on autosomes. Many TAD
boundaries on X coincide with the highest-affinity rex sites, and these boundaries become diminished or lost in mutants lacking DCC
binding, causing the structure of X to resemble that of autosomes. These results predicted that deletion of an endogenous rex site at
a DCC-dependent boundary should disrupt the boundary. As predicted, Cas9-mediated deletion of a rex site greatly diminished the
boundary, further demonstrating the condensin-driven remodeling of X-chromosome topology during dosage compensation. Thus,
condensin acts as a key structural element to reorganize interphase chromosomes and thereby regulate gene expression. Prior to
our work, no molecular trigger or set of DNA binding sites was known to cause a comparably strong effect on TAD structure in
higher eukaryotes. Our understanding of the topology of dosage-compensated X chromosomes provides fertile ground to decipher
the detailed mechanistic relationship between higher-order chromosome structure and chromosome-wide regulation of gene
expression.























Targeted Genome-editing Across Highly Diverged Nematode Species. Thwarted by the lack of reverse genetic approaches to enable
cross-species comparisons of gene function, we established robust strategies for targeted genome editing across nematode species
diverged by 300 MYR. In our initial work, a collaboration with Sangamo BioSciences, we used engineered nucleases containing
fusions between the DNA cleavage domain of the enzyme FokI and a custom-designed DNA binding domain: either zinc-finger
motifs for zinc-finger nucleases or transcription activator-like effector domains for TALE nucleases (TALENs). In those experiments,
we allowed the DNA double-strand breaks to be repaired imprecisely by non-homologous end joining (NHEJ) to create mutations in
precise locations.

We then extended the use of TALENs to achieve precise insertion and deletion of desired sequences by introducing single-stranded
or double-stranded templates to generate precise insertions or deletions through homology directed repair (HDR), the first
demonstration of HDR using ZFNs or TALENs in the nematode community. We then adopted the use of the CRISPR-associated
nuclease Cas9 because of the ease in making RNA guides to program target specificity.

Despite successful application of Cas9 technology, predicting DNA targets and guide RNAs that support efficient genome editing was
problematic. We then devised a strategy for high-frequency genome editing (both NHEJ and HDR) at all targets tested. The key
innovation was designing guide RNAs with a GG motif at the 3' end of their target-specific sequences. This design increased the
frequency of mutagenesis 10-fold. The ease of mutant recovery was further enhanced by combining this efficient guide design with
a co-conversion strategy, in which targets of interest are analyzed in animals exhibiting a dominant phenotype caused by
Cas9-dependent editing of an unrelated target.

Evolution cis-acting Regulatory Sites that Control Dosage Compensation. Mechanisms that specify sexual fate and compensate for
X-chromosome dose have diverged rapidly across species compared to other developmental processes, making it particularly
informative to study these rapidly changing processes over short evolutionary time scales. Application of our genome editing
strategies to C. briggsae revealed that the core dosage compensation machinery and key components of the genetic hierarchy that
controls dosage compensation and sex determination were conserved across the 30 MYR separation between C. elegans and C.
briggsae. In contrast, the set of cis-acting elements on X that recruit the DCC (rex sites) has diverged, retaining no functional overlap.
ChIP-seq analysis defined the C. briggsae DCC binding sites, and in vivo binding assays confirmed the ability of these sites to recruit
the DCC when detached from X in C. briggsae but not in C. elegans, and vice versa. The evolution of these sites differs dramatically
from the highly conserved DCC binding sites used by equivalently diverged fruit fly species and from the unchanged target sites of
conserved transcription factors that control multiple developmental processes from flies to humans. Hence, the divergence in DCC
binding specificity across nematode species provides a powerful opportunity to understand the path and timing for the concerted
change in hundreds of DNA target sites and the evolution of X chromosomes. We have extended our analysis of DCC binding
specificity to other nematode species and have shown that rex sites have diverged functionally at least three times in 30 MYR of
evolutionary history.

Tethering Replicated Chromosomes via Cohesin to Ensure Genome Stability during Meiosis.  Faithful segregation of chromosomes
during cell division is essential for genome stability. Accurate chromosome segregation is required both for the proliferative cell
divisions that produce daughter cells during mitosis and the two sequential divisions that produce haploid sperm and eggs from
diploid germline stem cells during meiosis. Approximately 30% of human zygotes have abnormal chromosome content at
conception due to defects in meiosis. Such aneuploidy is a leading cause of miscarriages and birth defects and arises, in part, from
defects in sister chromatid cohesion (SCC). SCC tethers replicated sister chromatids prior to cell divisions to ensure proper
chromosome segregation. In humans, SCC is established in the developing germ cells of a fetus and must be maintained until
ovulation in adults. This long-lived SCC is established and maintained by cohesin complexes, evolutionarily conserved protein
complexes structurally related to condensin (Figure 5).




















Studies in budding yeast showed that mitotic and meiotic cohesins are distinct but differ only in a single subunit called the kleisin.
During yeast meiosis, a single cohesin complex carries out all aspects of SCC. In contrast, our work in nematodes shows that
regulation of meiotic SCC in higher eukaryotes is more complex. We found that multiple functionally specialized cohesin complexes
mediate the establishment and two-step release of SCC that underlies the production of haploid gametes (Figure 5). The meiotic
complexes differ by a single kleisin subunit, and the kleisin influences nearly all aspects of meiotic cohesin function: the
mechanisms for loading cohesins onto chromosomes, for triggering DNA-bound cohesins to become cohesive, and for releasing
cohesins in a temporal- and location-specific manner (Figure 6). One kleisin triggers cohesion just after the chromosomes replicate,
as in yeast. Unexpectedly, the other triggers cohesion in a replication-independent manner, only after programmed DSBs are made
during meiosis to initiate recombination between homologous maternal and paternal chromosomes. Thus, break-induced cohesion
is essential for tethering replicated meiotic chromosomes. Later, recombination stimulates separase-independent removal of the
two different cohesin complexes from reciprocal chromosomal territories flanking the crossover site. This region-specific removal
likely underlies the two-step separation of homologs and sisters. Unexpectedly, one cohesin complex also performs
cohesion-independent functions in synaptonemal complex assembly. Our findings establish a new model for cohesin function in
meiosis: the choreographed actions of multiple cohesins, endowed with unexpectedly specialized functions by their kleisins,
underlie the stepwise separation of homologous chromosomes and then sister chromatids required for reduction of genome copy
number. This model diverges significantly from that in yeast but likely applies to plants and mammals, which utilize similar meiotic
kleisins.

Meiotic Chromosome Structures Constrain and Respond to Designation of Crossover Sites. Crossover recombination events between
homologous chromosomes are required to form chiasmata, temporary connections between homologues that ensure their proper
segregation at meiosis I. Despite this requirement for crossovers and an excess of the double-strand DNA breaks that are the
initiating events for meiotic recombination, most organisms make very few crossovers per chromosome pair. Moreover, crossovers
tend to inhibit the formation of other crossovers nearby on the same chromosome pair, a poorly understood phenomenon known
as crossover interference. We showed (in collaboration with the Villenueve lab at Stanford) that the synaptonemal complex, a
meiosis-specific structure that assembles between aligned homologous chromosomes, both constrains and is altered by crossover
recombination events. Partial depletion of the synaptonemal complex central region proteins attenuates crossover interference,
increasing crossovers and reducing the effective distance over which interference operates, indicating that synaptonemal complex
proteins limit crossovers. Moreover, we showed that crossovers are associated with a local 0.4-0.5-micrometre increase in
chromosome axis length. We proposed that meiotic crossover regulation operates as a self-limiting system in which meiotic
chromosome structures establish an environment that promotes crossover formation, which in turn alters chromosome structure to
inhibit other crossovers at additional sites.

Ironically, the effect of depleting condensin I or condensin II on increasing crossovers appears to occur by a different mechanism,
because the sites of extra crossovers are not marked by the same molecular markers as the crossovers created by reducing the
synaptonemal complex. We are investigating the crossover pathway employed to achieve these extra, non-interfering crossovers in
condensin mutants.
Research
Featured Science
Examples of Current Projects
Figure 2. Model depicting the molecular antagonism between
XSEs and ASEs that determines sex.  (Click to enlarge.)
Examples of Current Projects
Figure 1. The X:A sex determining signal. (Click to enlarge.)
Figure 7. Divergent kleisin subunits of cohesin specify distinct
mechanisms that tether and release meiotic chromosomes. 
Molecular Networks Controlling Dynamic
Chromosome Behaviors during Development
Our research focuses on the interplay
between the structure of chromosomes and
their function. Chromosomes undergo
dynamic behaviors during development to
ensure genome stability and accurate cell fate
decisions. We study inter-related molecular
networks that control diverse chromosome
behaviors: chromosome counting to
determine sexual fate; X-chromosome
remodeling to achieve X-chromosome
repression during dosage compensation, an
epigenetic process; chromosome cohesion to
tether and release replicated chromosomes
for reducing genome copy number during
germ cell formation; and chromosome
compaction to control gene expression,
chromosome segregation, and recombination
between maternal and paternal
chromosomes. We have found that the
developmental control of gene expression is
achieved through chromatin modifications
that affect chromosome structure with
epigenetic consequences. We have also
established robust procedures for targeted
genome editing across nematode species
diverged by 300 MYR to study the evolution of
sex determination and dosage compensation.
We combine genetic, genomic, proteomic,
biochemical, and cell biological approaches to
study these questions in the model organism
Caenorhabditis elegans, a round worm, and
its related nematode species.


Counting Chromosomes to Determine Sex:
Molecular Antagonism between
X-Chromosome, Autosome Signals Specifies
Nematode Sex
Many organisms determine sexual fate by a
chromosome-counting mechanism that
distinguishes one X chromosome from two.
Embryos with one X become males, while
those with two become females. We dissected
the molecular mechanism by which the
nematode C. elegans counts its sex
chromosomes to discern how small changes
in the concentrations of molecular signals are
translated into dramatically different
developmental fates. C. elegans tallies
X-chromosome number relative to the ploidy,
the sets of autosomes (X:A signal). It
discriminates with high fidelity between
tiny differences in the signal: 2X:3A embryos
(ratio 0.67) become males, while 3X:4A
embryos (ratio 0.75) become hermaphrodites
(Figure 1).























We showed that a set of X-linked genes called
X-signal elements (XSEs) communicates
X-chromosome dose by repressing the master
sex-determination switch gene xol-1 in a
cumulative, dose-dependent manner. XOL-1, a
GHMP kinase, is activated in 1X:2A embryos (1
dose of XSEs) to set the male fate but
repressed in 2X:2A embryos (2 doses of XSEs)
to promote the hermaphrodite fate, including
the activation of X-chromosome dosage
compensation. We also showed that the dose
of autosomes is communicated by a set of
autosomal signal elements (ASEs) that also act
in a cumulative, dose-dependent manner to
counter XSEs by stimulating xol-1
transcription. We have explored the
biochemical basis by which XSEs counter ASEs
to determine sex. Analysis in vitro showed
that XSEs (nuclear receptors and
homeodomain proteins) and ASEs (T-box and
zinc-finger proteins) bind directly to at least 5
distinct sites in xol-1 regulatory DNA to
counteract each other's activities and thereby
regulate xol-1 transcription (Figure 2). Analysis
in vivo showed that disrupting ASE and XSE
binding sites recapitulated the mis-regulation
of xol-1 transcription caused by disrupting the
cognate signal element genes. XSE and ASE
binding sites are distinct and non-overlapping,
suggesting that direct competition for xol-1
binding is not the mechanism by which XSEs
counter ASEs. Instead, XSEs likely antagonize
ASEs by recruiting cofactors with reciprocal
activities that induce opposite transcriptional
states. The X:A balance is thus communicated
in part through multiple antagonistic
molecular interactions carried out on a single
promoter, revealing how small differences in
X:A values can elicit different sexual fates. We
are currently identifying potential coactivators
and corepressors and directing efforts toward
understanding the evolution of the X:A signal
across nematode species.

Although most XSEs repress xol-1 by
regulating transcription, one XSE, an RNA
binding protein, represses xol-1 by binding to
an alternatively spliced intron and blocking its
proper splicing, thereby generating a
non-functional transcript with an in-frame
stop codon (Figure 2). This second tier of
repression enhances the fidelity of the
counting process.

The concept of a sex signal comprising
competing XSEs and ASEs arose as a theory
for fruit flies one century ago, and it
subsequently became entrenched in
textbooks. Ironically, the recent work of others
showed the fly sex signal does not fit this
simple paradigm, but our work shows the
worm signal does.

X-Chromosome Dosage Compensation:
Repressing X Chromosomes via Molecular
Machines.
Organisms that use sex chromosomes to
determine sexual fate evolved the essential,
chromosome-wide regulatory process called
dosage compensation to balance
X-chromosome gene expression between the
sexes. Strategies for dosage compensation
differ from worms to mammals, but invariably
a regulatory complex is targeted to X
chromosomes of one sex to modulate
transcription along the entire chromosome.
The heritable, regulation of X-chromosome
expression during dosage compensation is
exemplary for dissecting the coordinate
regulation of gene expression over large
chromosomal territories and the role of
chromosome structure in regulating gene
expression.

We defined the C. elegans dosage
compensation complex (DCC) and showed it is
homologous to condensin, a conserved
protein complex that mediates the
compaction, resolution, and segregation of
mitotic and meiotic chromosomes from yeast
to humans (Figure 3). The DCC binds to both X
chromosomes of hermaphrodites to reduce
transcription by half (Figure 3). Failure to
reduce expression kills hermaphrodites. Most
DCC condensin subunits also control the
structure and function of mitotic and meiotic
chromosomes by participating in two other
distinct condensin complexes (Figure 3). Not
only has the DCC co-opt subunits of
condensin to control gene expression, it
co-opted a subunit from the MLL/COMPASS
complex, a histone modifying complex, to
help recruit condensin subunits to rex sites.

We found that the DCC condensin subunits
are recruited specifically to hermaphrodite X
chromosomes by sex-specific DCC subunits
that trigger binding to cis-acting regulatory
elements on X, called rex and dox sites. rex
(recruitment elements on X) sites recruit the
DCC in an autonomous, sequence-dependent
manner using DNA motifs highly enriched on
X chromosomes. The DCC spreads to dox
(dependent on X) sites, which reside in
promoters of active genes and bind the DCC
robustly only when linked to rex sites.






















Dynamic Control of X-Chromosome
Conformation and Repression by a Histone
H4K20me Demethylase.
We recently found that DCC subunit DPY-21
has a histone demethylase activity that is
responsible for the selective enrichment of
H4K20me1 on X chromosomes of XX embryos
upon DCC binding.  X-ray crystallography and
biochemical assays of DPY-21 revealed a novel
subfamily of Jumonji C histone demethylases
that converts H4K20me2 to H4K20me1. 
Selective inactivation of demethylase activity
in vivo by genome editing eliminated
H4K20me1 enrichment on X, elevated X-linked
gene expression, reduced X-chromosome
compaction, and disrupted X-chromosome
topology by weakening TAD boundaries. 
These findings, among others, demonstrate
the direct impact of chromatin modification
on higher-order chromosome structure in
long-range regulation of gene expression.

H4K20me1 is also enriched on the
mammalian inactive X chromosome, but the
role of this enrichment in mammalian
transcriptional silencing is not known, nor is a
selective reagent available to test its role.  We
showed that the mouse homolog of the DCC
subunit also has H4K20me2 demethylase
activity.  Hence the worm system holds great
promise for understanding the effects of
histone modification in mammals.

Unexpectedly, DPY-21 associates with
autosomes but not X chromosomes of germ
cells in a DCC-independent manner to enrich
H4K20me1 and facilitate chromosome
compaction.  Thus, DPY-21 is an adaptable
chromatin regulator that is harnessed during
development for distinct biological functions. 
In both somatic cells and germ cells,
H4K20me1 enrichment modulates 3D
chromosome architecture to carry out these
functions.


















Step of transcription controlled by the DCC.
We have dissected a key aspect of the dosage
compensation mechanism by determining the
step of transcription controlled by the DCC to
repress X-chromosome gene expression. This
work was performed in collaboration with
John Lis' lab at Cornell University. In principle,
the DCC could control any step of
transcription: recruitment of RNA polymerase
II (Pol II) to the promoter, initiation of
transcription, escape of Pol II from the
promoter or pause sites, elongation of RNA
transcripts, or termination of transcription.
The mechanism had been elusive in C. elegans
due to improper annotation of transcription
start sites (TSSs). Nascent RNA transcripts
from most nematode genes undergo rapid
co-transcriptional processing in which the 5'
end is replaced by a common 22-nucleotide
leader RNA through a trans-splicing
mechanism, thereby destroying all knowledge
of TSSs and promoters.

To understand the step of transcription
controlled by the DCC, we first devised a
general strategy for mapping transcription
start sites and created an invaluable
nematode TSS data set. The TSS mapping
strategy, called GRO-cap, recovered nascent
RNAs with 5'-caps prior to processing. We
then determined the genome-wide
distribution, orientation, and quantity of
transcriptionally-engaged RNA Polymerase II
(Pol II) relative to TSSs in wild-type and
DC-defective animals using GRO-seq (global
run-on sequencing).

We found that promoters are unexpectedly
far upstream from the 5' ends of mature
mRNAs, and promoter-proximal Pol II pausing
occurs only in starved larvae and is rare in C.
elegans embryos, unlike in most metazoans.
These results indicated that enhancement of
promoter pausing in XX embryos cannot be
the mechanism of reducing transcription
during dosage compensation. In contrast,
control of pausing is a common mechanism
for controlling transcription of developmental
regulatory genes in most metazoans and is
thought to be the mechanism of dosage
compensation in fruit flies.

Then, by comparing the location and density
of transcriptionally engaged Pol II in wild-type
and dosage-compensation-defective embryos,
we found that the step of transcription
controlled by the dosage compensation
process is the recruitment of Pol II. That is, C.
elegans equalizes X-chromosome-wide gene
expression between the sexes by reducing Pol
II recruitment to the promoters of X-linked
genes in XX embryos by about half. One of
our research directions is to dissect the
mechanisms by which the DCC limits Pol II
recruitment.

Our data set also enabled us to analyze
starvation-controlled gene regulation in
collaboration with Ryan Baugh's lab at Duke
University. We found a new phenomenon of
Pol II docking, the stable association of Pol II
upstream of the transcription start sites, and
hence sites of pausing. We found that docked
Pol II accumulates, without initiating,
upstream of inactive growth genes that are
turned off during starvation are activated
upon feeding. We found that Pol II pausing
occurs at active stress-response genes that
are downregulated upon feeding. Hence,
growth and stress genes are controlled by
distinct mechanisms to coordinate gene
expression with nutrient availability.

DCC recruitment and binding to X
chromosomes.
We showed that many of the DCC recruitment
(rex) sites have a DNA motif (called MEX) that
is highly enriched on X compared to
autosomes and is essential for DCC binding to
a subset of rex sites. However, not all rex sites
have this motif. We recently defined new
principles by which the DCC is recruited to X
chromosomes, including the identification of a
new, essential DCC binding motif (MEX II) that
is enriched on X. We found that MEX II acts in
combination with MEX to foster high-affinity
binding at some rex sites but also acts alone at
other rex sites to foster stable binding. We
demonstrated these DCC binding principles by
using DCC binding assays in vivo and in vitro.

We also showed that SUMOylation of specific
DCC subunits is essential for sex-specific
assembly and function of the DCC on X.
Depletion of SUMO in vivo severely disrupts
DCC binding and causes changes in X-linked
gene expression similar to those caused by
deleting the genes that encode DCC subunits.
Three DCC subunits undergo SUMOylation,
one subunit essential for DCC loading and two
subunits that are integral to the condensin
portion of the DCC.

DCC SUMOylation is triggered by the signal
that initiates DCC assembly onto X. The initial
step of assembly--binding of X-targeting
factors to rex sites--is independent of
SUMOylation, but robust binding of the
complete complex requires SUMOylation. One
of SUMOylated DCC subunits also participates
in condensin complexes essential for
chromosome segregation, but its
SUMOylation occurs only in the context of the
DCC. Our results reinforce a newly emerging
theme in which multiple proteins of a complex
are collectively SUMOylated in response to a
specific stimulus, leading to accelerated
complex formation and enhanced function.

Condensin-driven remodeling of
X-chromosome topology during dosage
compensation.
The three-dimensional organization of a
genome plays a critical role in regulating gene
expression, yet little is known about the
machinery and mechanisms that determine
higher-order chromosome structure. The
involvement of bona fide condensin subunits
in dosage compensation together with our
observation that the DCC acts at a distance to
regulate gene expression suggested that the
DCC might alter the topology of X
chromosomes to reduce gene expression
chromosome wide.

Using genome-wide chromosome
conformation capture techniques (in
collaboration with Job Dekker's lab at U. Mass.
Worcester) with single-cell fluorescence in situ
hybridization and RNA-seq to compare
chromosome structure and gene expression
in wild-type and dosage-compensation-
defective embryos, we showed that the DCC
remodels X chromosomes of hermaphrodites
into a unique, sex-specific spatial
conformation, distinct from autosomes, using
its highest-affinity rex sites to facilitate
long-range interactions across X.
Dosage-compensated X chromosomes consist
of self-interacting domains (~ 1 Mb)
resembling mammalian Topologically
Associating Domains (TADs). TADs on X have
stronger boundaries and more regular
spacing than those on autosomes. Many TAD
boundaries on X coincide with the
highest-affinity rex sites, and these boundaries
become diminished or lost in mutants lacking
DCC binding, causing the structure of X to
resemble that of autosomes. These results
predicted that deletion of an endogenous rex
site at a DCC-dependent boundary should
disrupt the boundary. As predicted,
Cas9-mediated deletion of a rex site greatly
diminished the boundary, further
demonstrating the condensin-driven
remodeling of X-chromosome topology during
dosage compensation. Thus, condensin acts
as a key structural element to reorganize
interphase chromosomes and thereby
regulate gene expression. Prior to our work,
no molecular trigger or set of DNA binding
sites was known to cause a comparably strong
effect on TAD structure in higher eukaryotes.
Our understanding of the topology of
dosage-compensated X chromosomes
provides fertile ground to decipher the
detailed mechanistic relationship between
higher-order chromosome structure and
chromosome-wide regulation of gene
expression.



























Targeted Genome-editing Across Highly
Diverged Nematode Species.
Thwarted by the lack of reverse genetic
approaches to enable cross-species
comparisons of gene function, we established
robust strategies for targeted genome editing
across nematode species diverged by 300
MYR. In our initial work, a collaboration with
Sangamo BioSciences, we used engineered
nucleases containing fusions between the
DNA cleavage domain of the enzyme FokI and
a custom-designed DNA binding domain:
either zinc-finger motifs for zinc-finger
nucleases or transcription activator-like
effector domains for TALE nucleases (TALENs).
In those experiments, we allowed the DNA
double-strand breaks to be repaired
imprecisely by non-homologous end joining
(NHEJ) to create mutations in precise locations.

We then extended the use of TALENs to
achieve precise insertion and deletion of
desired sequences by introducing
single-stranded or double-stranded templates
to generate precise insertions or deletions
through homology directed repair (HDR), the
first demonstration of HDR using ZFNs or
TALENs in the nematode community. We then
adopted the use of the CRISPR-associated
nuclease Cas9 because of the ease in making
RNA guides to program target specificity.

Despite successful application of Cas9
technology, predicting DNA targets and guide
RNAs that support efficient genome editing
was problematic. We then devised a strategy
for high-frequency genome editing (both NHEJ
and HDR) at all targets tested. The key
innovation was designing guide RNAs with a
GG motif at the 3' end of their target-specific
sequences. This design increased the
frequency of mutagenesis 10-fold. The ease of
mutant recovery was further enhanced by
combining this efficient guide design with a
co-conversion strategy, in which targets of
interest are analyzed in animals exhibiting a
dominant phenotype caused by Cas9-
dependent editing of an unrelated target.

Evolution cis-acting Regulatory Sites that
Control Dosage Compensation.
Mechanisms that specify sexual fate and
compensate for X-chromosome dose have
diverged rapidly across species compared to
other developmental processes, making it
particularly informative to study these rapidly
changing processes over short evolutionary
time scales. Application of our genome editing
strategies to C. briggsae revealed that the core
dosage compensation machinery and key
components of the genetic hierarchy that
controls dosage compensation and sex
determination were conserved across the 30
MYR separation between C. elegans and C.
briggsae. In contrast, the set of cis-acting
elements on X that recruit the DCC (rex sites)
has diverged, retaining no functional overlap.
ChIP-seq analysis defined the C. briggsae DCC
binding sites, and in vivo binding assays
confirmed the ability of these sites to recruit
the DCC when detached from X in C. briggsae
but not in C. elegans, and vice versa. The
evolution of these sites differs dramatically
from the highly conserved DCC binding sites
used by equivalently diverged fruit fly species
and from the unchanged target sites of
conserved transcription factors that control
multiple developmental processes from flies
to humans. Hence, the divergence in DCC
binding specificity across nematode species
provides a powerful opportunity to
understand the path and timing for the
concerted change in hundreds of DNA target
sites and the evolution of X chromosomes. We
have extended our analysis of DCC binding
specificity to other nematode species and
have shown that rex sites have diverged
functionally at least three times in 30 MYR of
evolutionary history.

Tethering  Replicated Chromosomes via
Cohesin to Ensure Genome Stability during
Meiosis.
Faithful segregation of chromosomes during
cell division is essential for genome stability.
Accurate chromosome segregation is required
both for the proliferative cell divisions that
produce daughter cells during mitosis and the
two sequential divisions that produce haploid
sperm and eggs from diploid germline stem
cells during meiosis. Approximately 30% of
human zygotes have abnormal chromosome
content at conception due to defects in
meiosis. Such aneuploidy is a leading cause of
miscarriages and birth defects and arises, in
part, from defects in sister chromatid
cohesion (SCC). SCC tethers replicated sister
chromatids prior to cell divisions to ensure
proper chromosome segregation. In humans,
SCC is established in the developing germ cells
of a fetus and must be maintained until
ovulation in adults. This long-lived SCC is
established and maintained by cohesin
complexes, evolutionarily conserved protein
complexes structurally related to condensin
(Figure 6).
























Studies in budding yeast showed that mitotic
and meiotic cohesins are distinct but differ
only in a single subunit called the kleisin.
During yeast meiosis, a single cohesin
complex carries out all aspects of SCC. In
contrast, our work in nematodes shows that
regulation of meiotic SCC in higher eukaryotes
is more complex. We found that multiple
functionally specialized cohesin complexes
mediate the establishment and two-step
release of SCC that underlies the production
of haploid gametes (Figure 6). The meiotic
complexes differ by a single kleisin subunit,
and the kleisin influences nearly all aspects of
meiotic cohesin function: the mechanisms for
loading cohesins onto chromosomes, for
triggering DNA-bound cohesins to become
cohesive, and for releasing cohesins in a
temporal- and location-specific manner
(Figure 7). One kleisin triggers cohesion just
after the chromosomes replicate, as in yeast.
Unexpectedly, the other triggers cohesion in a
replication-independent manner, only after
programmed DSBs are made during meiosis
to initiate recombination between
homologous maternal and paternal
chromosomes. Thus, break-induced cohesion
is essential for tethering replicated meiotic
chromosomes. Later, recombination
stimulates separase-independent removal of
the two different cohesin complexes from
reciprocal chromosomal territories flanking
the crossover site. This region-specific
removal likely underlies the two-step
separation of homologs and sisters.
Unexpectedly, one cohesin complex also
performs cohesion-independent functions in
synaptonemal complex assembly. Our
findings establish a new model for cohesin
function in meiosis: the choreographed
actions of multiple cohesins, endowed with
unexpectedly specialized functions by their
kleisins, underlie the stepwise separation of
homologous chromosomes and then sister
chromatids required for reduction of genome
copy number. This model diverges
significantly from that in yeast but likely
applies to plants and mammals, which utilize
similar meiotic kleisins.

Meiotic Chromosome Structure Constrain
and Respond to Designation of Crossover
Sites.
Crossover recombination events between
homologous chromosomes are required to
form chiasmata, temporary connections
between homologues that ensure their proper
segregation at meiosis I. Despite this
requirement for crossovers and an excess of
the double-strand DNA breaks that are the
initiating events for meiotic recombination,
most organisms make very few crossovers per
chromosome pair. Moreover, crossovers tend
to inhibit the formation of other crossovers
nearby on the same chromosome pair, a
poorly understood phenomenon known as
crossover interference. We showed (in
collaboration with the Villenueve lab at
Stanford) that the synaptonemal complex, a
meiosis-specific structure that assembles
between aligned homologous chromosomes,
both constrains and is altered by crossover
recombination events. Partial depletion of the
synaptonemal complex central region
proteins attenuates crossover interference,
increasing crossovers and reducing the
effective distance over which interference
operates, indicating that synaptonemal
complex proteins limit crossovers. Moreover,
we showed that crossovers are associated
with a local 0.4-0.5-micrometre increase in
chromosome axis length. We proposed that
meiotic crossover regulation operates as a
self-limiting system in which meiotic
chromosome structures establish an
environment that promotes crossover
formation, which in turn alters chromosome
structure to inhibit other crossovers at
additional sites.

Ironically, the effect of depleting condensin I
or condensin II on increasing crossovers
appears to occur by a different mechanism,
because the sites of extra crossovers are not
marked by the same molecular markers as
the crossovers created by reducing the
synaptonemal complex. We are investigating
the crossover pathway employed to achieve
these extra, non-interfering crossovers in
condensin mutants.
Figure 1. The X:A sex determining signal. (Click for legend.)
Figure 2. Model depicting the molecular
antagonism between XSEs and ASEs
that determines sex. (Click for legend.)
Figure 3. Biochemically distinct
condensin complexes with
interchangeable subunits control
chromosome structure
throughout C. elegans development.
Figure 5. DCC modulates spatial organization of X chromosomes.
(Click for legend.)

Figure 6. Multiple cohesin complexes
tether meiotic chromosomes.
Figure 7. Divergent kleisin subunits of cohesin specify distinct mechanisms that tether and release meiotic chromosomes.  (Click for legend.)
Figure 4. DPY-21 is a chromatin regulator that is harnessed during development for different biological functions. (Click for legend.)
Molecular Networks Controlling Dynamic Chromosome Behaviors during Development
Our research focuses on the interplay between the structure of chromosomes and their function. Chromosomes undergo dynamic behaviors during
development to ensure genome stability and accurate cell fate decisions. We study inter-related molecular networks that control diverse
chromosome behaviors: chromosome counting to determine sexual fate; X-chromosome remodeling to achieve X-chromosome repression during
dosage compensation, an epigenetic process; chromosome cohesion to tether and release replicated chromosomes for reducing genome copy
number during germ cell formation; and chromosome compaction to control gene expression, chromosome segregation, and recombination
between maternal and paternal chromosomes. We have found that the developmental control of gene expression is achieved through chromatin
modifications that affect chromosome structure with epigenetic consequences. We have also established robust procedures for targeted genome
editing across nematode species diverged by 300 MYR to study the evolution of sex determination and dosage compensation. We combine genetic,
genomic, proteomic, biochemical, and cell biological approaches to study these questions in the model organism Caenorhabditis elegans, a round
worm, and its related nematode species.



Counting Chromosomes to Determine Sex: Molecular Antagonism between X-Chromosome, Autosome Signals Specifies Nematode Sex
Many organisms determine sexual fate by a chromosome-counting mechanism that distinguishes one X chromosome from two. Embryos with one X
become males, while those with two become females. We dissected the molecular mechanism by which the nematode C. elegans counts its sex
chromosomes to discern how small changes in the concentrations of molecular signals are translated into dramatically different developmental
fates. C. elegans tallies X-chromosome number relative to the ploidy, the sets of autosomes (X:A signal). It discriminates with high fidelity between
tiny differences in the signal: 2X:3A embryos (ratio 0.67) become males, while 3X:4A embryos (ratio 0.75) become hermaphrodites (Figure 1).



















We showed that a set of X-linked genes called X-signal elements (XSEs) communicates X-chromosome dose by repressing the master
sex-determination switch gene xol-1 in a cumulative, dose-dependent manner. XOL-1, a GHMP kinase, is activated in 1X:2A embryos (1 dose of XSEs)
to set the male fate but repressed in 2X:2A embryos (2 doses of XSEs) to promote the hermaphrodite fate, including the activation of X-chromosome
dosage compensation. We also showed that the dose of autosomes is communicated by a set of autosomal signal elements (ASEs) that also act in a
cumulative, dose-dependent manner to counter XSEs by stimulating xol-1 transcription. We have explored the biochemical basis by which XSEs
counter ASEs to determine sex. Analysis in vitro showed that XSEs (nuclear receptors and homeodomain proteins) and ASEs (T-box and zinc-finger
proteins) bind directly to at least 5 distinct sites in xol-1 regulatory DNA to counteract each other's activities and thereby regulate xol-1 transcription
(Figure 2). Analysis in vivo showed that disrupting ASE and XSE binding sites recapitulated the mis-regulation of xol-1 transcription caused by
disrupting the cognate signal element genes. XSE and ASE binding sites are distinct and non-overlapping, suggesting that direct competition for xol-1
binding is not the mechanism by which XSEs counter ASEs. Instead, XSEs likely antagonize ASEs by recruiting cofactors with reciprocal activities that
induce opposite transcriptional states. The X:A balance is thus communicated in part through multiple antagonistic molecular interactions carried
out on a single promoter, revealing how small differences in X:A values can elicit different sexual fates. We are currently identifying potential
coactivators and corepressors and directing efforts toward understanding the evolution of the X:A signal across nematode species.

Although most XSEs repress xol-1 by regulating transcription, one XSE, an RNA binding protein, represses xol-1 by binding to an alternatively spliced
intron and blocking its proper splicing, thereby generating a non-functional transcript with an in-frame stop codon (Figure 2). This second tier of
repression enhances the fidelity of the counting process.

The concept of a sex signal comprising competing XSEs and ASEs arose as a theory for fruit flies one century ago, and it subsequently became
entrenched in textbooks. Ironically, the recent work of others showed the fly sex signal does not fit this simple paradigm, but our work shows the
worm signal does.

X-Chromosome Dosage Compensation: Repressing X Chromosomes via Molecular Machines.
Organisms that use sex chromosomes to determine sexual fate evolved the essential, chromosome-wide regulatory process called dosage
compensation to balance X-chromosome gene expression between the sexes. Strategies for dosage compensation differ from worms to mammals,
but invariably a regulatory complex is targeted to X chromosomes of one sex to modulate transcription along the entire chromosome. The heritable,
regulation of X-chromosome expression during dosage compensation is exemplary for dissecting the coordinate regulation of gene expression over
large chromosomal territories and the role of chromosome structure in regulating gene expression.

We defined the C. elegans dosage compensation complex (DCC) and showed it is homologous to condensin, a conserved protein complex that
mediates the compaction, resolution, and segregation of mitotic and meiotic chromosomes from yeast to humans (Figure 3). The DCC binds to both
X chromosomes of hermaphrodites to reduce transcription by half (Figure 3). Failure to reduce expression kills hermaphrodites. Most DCC condensin
subunits also control the structure and function of mitotic and meiotic chromosomes by participating in two other distinct condensin complexes
(Figure 3). Not only has the DCC co-opt subunits of condensin to control gene expression, it co-opted a subunit from the MLL/COMPASS complex, a
histone modifying complex, to help recruit condensin subunits to rex sites.

We found that the DCC condensin subunits are recruited specifically to hermaphrodite X chromosomes by sex-specific DCC subunits that trigger
binding to cis-acting regulatory elements on X, called rex and dox sites. rex (recruitment elements on X) sites recruit the DCC in an autonomous,
sequence-dependent manner using DNA motifs highly enriched on X chromosomes. The DCC spreads to dox (dependent on X) sites, which reside in
promoters of active genes and bind the DCC robustly only when linked to rex sites.





















Dynamic Control of X-Chromosome Conformation and Repression by a Histone H4K20me Demethylase.
We recently found that DCC subunit DPY-21 has a histone demethylase activity that is responsible for the selective enrichment of H4K20me1 on X
chromosomes of XX embryos upon DCC binding.  X-ray crystallography and biochemical assays of DPY-21 revealed a novel subfamily of Jumonji C
histone demethylases that converts H4K20me2 to H4K20me1.  Selective inactivation of demethylase activity in vivo by genome editing eliminated
H4K20me1 enrichment on X, elevated X-linked gene expression, reduced X-chromosome compaction, and disrupted X-chromosome topology by
weakening TAD boundaries.  These findings, among others, demonstrate the direct impact of chromatin modification on higher-order chromosome
structure in long-range regulation of gene expression.

H4K20me1 is also enriched on the mammalian inactive X chromosome, but the role of this enrichment in mammalian transcriptional silencing is not
known, nor is a selective reagent available to test its role.  We showed that the mouse homolog of the DCC subunit also has H4K20me2 demethylase
activity.  Hence the worm system holds great promise for understanding the effects of histone modification in mammals.

Unexpectedly, DPY-21 associates with autosomes but not X chromosomes of germ cells in a DCC-independent manner to enrich H4K20me1 and
facilitate chromosome compaction.  Thus, DPY-21 is an adaptable chromatin regulator that is harnessed during development for distinct biological
functions.  In both somatic cells and germ cells, H4K20me1 enrichment modulates 3D chromosome architecture to carry out these functions.


































Step of transcription controlled by the DCC.
We have dissected a key aspect of the dosage compensation mechanism by determining the step of transcription controlled by the DCC to repress
X-chromosome gene expression. This work was performed in collaboration with John Lis' lab at Cornell University. In principle, the DCC could control
any step of transcription: recruitment of RNA polymerase II (Pol II) to the promoter, initiation of transcription, escape of Pol II from the promoter or
pause sites, elongation of RNA transcripts, or termination of transcription. The mechanism had been elusive in C. elegans due to improper
annotation of transcription start sites (TSSs). Nascent RNA transcripts from most nematode genes undergo rapid co-transcriptional processing in
which the 5' end is replaced by a common 22-nucleotide leader RNA through a trans-splicing mechanism, thereby destroying all knowledge of TSSs
and promoters.

To understand the step of transcription controlled by the DCC, we first devised a general strategy for mapping transcription start sites and created an
invaluable nematode TSS data set. The TSS mapping strategy, called GRO-cap, recovered nascent RNAs with 5'-caps prior to processing. We then
determined the genome-wide distribution, orientation, and quantity of transcriptionally-engaged RNA Polymerase II (Pol II) relative to TSSs in
wild-type and DC-defective animals using GRO-seq (global run-on sequencing).

We found that promoters are unexpectedly far upstream from the 5' ends of mature mRNAs, and promoter-proximal Pol II pausing occurs only in
starved larvae and is rare in C. elegans embryos, unlike in most metazoans. These results indicated that enhancement of promoter pausing in XX
embryos cannot be the mechanism of reducing transcription during dosage compensation. In contrast, control of pausing is a common mechanism
for controlling transcription of developmental regulatory genes in most metazoans and is thought to be the mechanism of dosage compensation in
fruit flies.

Then, by comparing the location and density of transcriptionally engaged Pol II in wild-type and dosage-compensation-defective embryos, we found
that the step of transcription controlled by the dosage compensation process is the recruitment of Pol II. That is, C. elegans equalizes
X-chromosome-wide gene expression between the sexes by reducing Pol II recruitment to the promoters of X-linked genes in XX embryos by about
half. One of our research directions is to dissect the mechanisms by which the DCC limits Pol II recruitment.

Our data set also enabled us to analyze starvation-controlled gene regulation in collaboration with Ryan Baugh's lab at Duke University. We found a
new phenomenon of Pol II docking, the stable association of Pol II upstream of the transcription start sites, and hence sites of pausing. We found
that docked Pol II accumulates, without initiating, upstream of inactive growth genes that are turned off during starvation are activated upon feeding.
We found that Pol II pausing occurs at active stress-response genes that are downregulated upon feeding. Hence, growth and stress genes are
controlled by distinct mechanisms to coordinate gene expression with nutrient availability.

DCC recruitment and binding to X chromosomes.
We showed that many of the DCC recruitment (rex) sites have a DNA motif (called MEX) that is highly enriched on X compared to autosomes and is
essential for DCC binding to a subset of rex sites. However, not all rex sites have this motif. We recently defined new principles by which the DCC is
recruited to X chromosomes, including the identification of a new, essential DCC binding motif (MEX II) that is enriched on X. We found that MEX II
acts in combination with MEX to foster high-affinity binding at some rex sites but also acts alone at other rex sites to foster stable binding. We
demonstrated these DCC binding principles by using DCC binding assays in vivo and in vitro.

We also showed that SUMOylation of specific DCC subunits is essential for sex-specific assembly and function of the DCC on X. Depletion of SUMO in
vivo severely disrupts DCC binding and causes changes in X-linked gene expression similar to those caused by deleting the genes that encode DCC
subunits. Three DCC subunits undergo SUMOylation, one subunit essential for DCC loading and two subunits that are integral to the condensin
portion of the DCC.

DCC SUMOylation is triggered by the signal that initiates DCC assembly onto X. The initial step of assembly--binding of X-targeting factors to rex
sites--is independent of SUMOylation, but robust binding of the complete complex requires SUMOylation. One of SUMOylated DCC subunits also
participates in condensin complexes essential for chromosome segregation, but its SUMOylation occurs only in the context of the DCC. Our results
reinforce a newly emerging theme in which multiple proteins of a complex are collectively SUMOylated in response to a specific stimulus, leading to
accelerated complex formation and enhanced function.

Condensin-driven remodeling of X-chromosome topology during dosage compensation.
The three-dimensional organization of a genome plays a critical role in regulating gene expression, yet little is known about the machinery and
mechanisms that determine higher-order chromosome structure. The involvement of bona fide condensin subunits in dosage compensation
together with our observation that the DCC acts at a distance to regulate gene expression suggested that the DCC might alter the topology of X
chromosomes to reduce gene expression chromosome wide.

Using genome-wide chromosome conformation capture techniques (in collaboration with Job Dekker's lab at U. Mass. Worcester) with single-cell
fluorescence in situ hybridization and RNA-seq to compare chromosome structure and gene expression in wild-type and
dosage-compensation-defective embryos, we showed that the DCC remodels X chromosomes of hermaphrodites into a unique, sex-specific spatial
conformation, distinct from autosomes, using its highest-affinity rex sites to facilitate long-range interactions across X. Dosage-compensated X
chromosomes consist of self-interacting domains (~ 1 Mb) resembling mammalian Topologically Associating Domains (TADs). TADs on X have
stronger boundaries and more regular spacing than those on autosomes. Many TAD boundaries on X coincide with the highest-affinity rex sites, and
these boundaries become diminished or lost in mutants lacking DCC binding, causing the structure of X to resemble that of autosomes. These results
predicted that deletion of an endogenous rex site at a DCC-dependent boundary should disrupt the boundary. As predicted, Cas9-mediated deletion
of a rex site greatly diminished the boundary, further demonstrating the condensin-driven remodeling of X-chromosome topology during dosage
compensation. Thus, condensin acts as a key structural element to reorganize interphase chromosomes and thereby regulate gene expression. Prior
to our work, no molecular trigger or set of DNA binding sites was known to cause a comparably strong effect on TAD structure in higher eukaryotes.
Our understanding of the topology of dosage-compensated X chromosomes provides fertile ground to decipher the detailed mechanistic
relationship between higher-order chromosome structure and chromosome-wide regulation of gene expression.























Targeted Genome-editing Across Highly Diverged Nematode Species.
Thwarted by the lack of reverse genetic approaches to enable cross-species comparisons of gene function, we established robust strategies for
targeted genome editing across nematode species diverged by 300 MYR. In our initial work, a collaboration with Sangamo BioSciences, we used
engineered nucleases containing fusions between the DNA cleavage domain of the enzyme FokI and a custom-designed DNA binding domain: either
zinc-finger motifs for zinc-finger nucleases or transcription activator-like effector domains for TALE nucleases (TALENs). In those experiments, we
allowed the DNA double-strand breaks to be repaired imprecisely by non-homologous end joining (NHEJ) to create mutations in precise locations.

We then extended the use of TALENs to achieve precise insertion and deletion of desired sequences by introducing single-stranded or
double-stranded templates to generate precise insertions or deletions through homology directed repair (HDR), the first demonstration of HDR using
ZFNs or TALENs in the nematode community. We then adopted the use of the CRISPR-associated nuclease Cas9 because of the ease in making RNA
guides to program target specificity.

Despite successful application of Cas9 technology, predicting DNA targets and guide RNAs that support efficient genome editing was problematic. We
then devised a strategy for high-frequency genome editing (both NHEJ and HDR) at all targets tested. The key innovation was designing guide RNAs
with a GG motif at the 3' end of their target-specific sequences. This design increased the frequency of mutagenesis 10-fold. The ease of mutant
recovery was further enhanced by combining this efficient guide design with a co-conversion strategy, in which targets of interest are analyzed in
animals exhibiting a dominant phenotype caused by Cas9-dependent editing of an unrelated target.

Evolution cis-acting Regulatory Sites that Control Dosage Compensation.
Mechanisms that specify sexual fate and compensate for X-chromosome dose have diverged rapidly across species compared to other
developmental processes, making it particularly informative to study these rapidly changing processes over short evolutionary time scales.
Application of our genome editing strategies to C. briggsae revealed that the core dosage compensation machinery and key components of the
genetic hierarchy that controls dosage compensation and sex determination were conserved across the 30 MYR separation between C. elegans and
C. briggsae. In contrast, the set of cis-acting elements on X that recruit the DCC (rex sites) has diverged, retaining no functional overlap. ChIP-seq
analysis defined the C. briggsae DCC binding sites, and in vivo binding assays confirmed the ability of these sites to recruit the DCC when detached
from X in C. briggsae but not in C. elegans, and vice versa. The evolution of these sites differs dramatically from the highly conserved DCC binding
sites used by equivalently diverged fruit fly species and from the unchanged target sites of conserved transcription factors that control multiple
developmental processes from flies to humans. Hence, the divergence in DCC binding specificity across nematode species provides a powerful
opportunity to understand the path and timing for the concerted change in hundreds of DNA target sites and the evolution of X chromosomes. We
have extended our analysis of DCC binding specificity to other nematode species and have shown that rex sites have diverged functionally at least
three times in 30 MYR of evolutionary history.

Tethering Replicated Chromosomes via Cohesin to Ensure Genome Stability during Meiosis.
Faithful segregation of chromosomes during cell division is essential for genome stability. Accurate chromosome segregation is required both for the
proliferative cell divisions that produce daughter cells during mitosis and the two sequential divisions that produce haploid sperm and eggs from
diploid germline stem cells during meiosis. Approximately 30% of human zygotes have abnormal chromosome content at conception due to defects
in meiosis. Such aneuploidy is a leading cause of miscarriages and birth defects and arises, in part, from defects in sister chromatid cohesion (SCC).
SCC tethers replicated sister chromatids prior to cell divisions to ensure proper chromosome segregation. In humans, SCC is established in the
developing germ cells of a fetus and must be maintained until ovulation in adults. This long-lived SCC is established and maintained by cohesin
complexes, evolutionarily conserved protein complexes structurally related to condensin (Figure 6).




















Studies in budding yeast showed that mitotic and meiotic cohesins are distinct but differ only in a single subunit called the kleisin. During yeast
meiosis, a single cohesin complex carries out all aspects of SCC. In contrast, our work in nematodes shows that regulation of meiotic SCC in higher
eukaryotes is more complex. We found that multiple functionally specialized cohesin complexes mediate the establishment and two-step release of
SCC that underlies the production of haploid gametes (Figure 6). The meiotic complexes differ by a single kleisin subunit, and the kleisin influences
nearly all aspects of meiotic cohesin function: the mechanisms for loading cohesins onto chromosomes, for triggering DNA-bound cohesins to
become cohesive, and for releasing cohesins in a temporal- and location-specific manner (Figure 7). One kleisin triggers cohesion just after the
chromosomes replicate, as in yeast. Unexpectedly, the other triggers cohesion in a replication-independent manner, only after programmed DSBs
are made during meiosis to initiate recombination between homologous maternal and paternal chromosomes. Thus, break-induced cohesion is
essential for tethering replicated meiotic chromosomes. Later, recombination stimulates separase-independent removal of the two different cohesin
complexes from reciprocal chromosomal territories flanking the crossover site. This region-specific removal likely underlies the two-step separation
of homologs and sisters. Unexpectedly, one cohesin complex also performs cohesion-independent functions in synaptonemal complex assembly.
Our findings establish a new model for cohesin function in meiosis: the choreographed actions of multiple cohesins, endowed with unexpectedly
specialized functions by their kleisins, underlie the stepwise separation of homologous chromosomes and then sister chromatids required for
reduction of genome copy number. This model diverges significantly from that in yeast but likely applies to plants and mammals, which utilize similar
meiotic kleisins.

Meiotic Chromosome Structures Constrain and Respond to Designation of Crossover Sites.
Crossover recombination events between homologous chromosomes are required to form chiasmata, temporary connections between homologues
that ensure their proper segregation at meiosis I. Despite this requirement for crossovers and an excess of the double-strand DNA breaks that are
the initiating events for meiotic recombination, most organisms make very few crossovers per chromosome pair. Moreover, crossovers tend to
inhibit the formation of other crossovers nearby on the same chromosome pair, a poorly understood phenomenon known as crossover interference.
We showed (in collaboration with the Villenueve lab at Stanford) that the synaptonemal complex, a meiosis-specific structure that assembles
between aligned homologous chromosomes, both constrains and is altered by crossover recombination events. Partial depletion of the
synaptonemal complex central region proteins attenuates crossover interference, increasing crossovers and reducing the effective distance over
which interference operates, indicating that synaptonemal complex proteins limit crossovers. Moreover, we showed that crossovers are associated
with a local 0.4-0.5-micrometre increase in chromosome axis length. We proposed that meiotic crossover regulation operates as a self-limiting
system in which meiotic chromosome structures establish an environment that promotes crossover formation, which in turn alters chromosome
structure to inhibit other crossovers at additional sites.

Ironically, the effect of depleting condensin I or condensin II on increasing crossovers appears to occur by a different mechanism, because the sites
of extra crossovers are not marked by the same molecular markers as the crossovers created by reducing the synaptonemal complex. We are
investigating the crossover pathway employed to achieve these extra, non-interfering crossovers in condensin mutants.
Figure 4. DPY-21 is a chromatin regulator that is harnessed during development for different biological functions. DPY-21 crystal
structure and biochemical activity revealed a novel H4K20me2 Jumonji C demethylase.  In somatic cells, DPY-21 enriches H4K20me1 on X
chromosomes to repress gene expression.  H4K20me1 enrichment controls the higher-order structure of X chromosomes.  In germ cells,
DPY-21 enriches H4K20me1 on autosomes in a DCC-independent manner to compact autosomes. (Click to enlarge.)
Figure 5. DCC modulates spatial organization of X chromosomes. (Click to enlarge.)
Figure 6. Multiple cohesin complexes tether
meiotic chromosomes. (Click to enlarge.)
Figure 1. The X:A sex determining signal. (Click to enlarge.)
Figure 2. Model depicting the molecular antagonism between
XSEs and ASEs that determines sex.  (Click to enlarge.)
Figure 3. Biochemically distinct condensin complexes with interchangeable subunits control chromosome structure
throughout C. elegans development. (Click to enlarge.)
Figure 4. DPY-21 is a chromatin regulator that is harnessed during development for
different biological functions. DPY-21 crystal structure and biochemical activity
revealed a novel H4K20me2 Jumonji C demethylase.  In somatic cells, DPY-21 enriches
H4K20me1 on X chromosomes to repress gene expression. H4K20me1 enrichment
controls the higher-order structure of X chromosomes.  In germ cells, DPY-21 enriches
H4K20me1 on autosomes in a DCC-independent manner to compact autosomes.
Molecular Networks Controlling Dynamic Chromosome Behaviors during Development
Our research focuses on the interplay between the structure of chromosomes and their function. Chromosomes undergo
dynamic behaviors during development to ensure genome stability and accurate cell fate decisions. We study inter-related
molecular networks that control diverse chromosome behaviors: chromosome counting to determine sexual fate;
X-chromosome remodeling to achieve X-chromosome repression during dosage compensation, an epigenetic process;
chromosome cohesion to tether and release replicated chromosomes for reducing genome copy number during germ cell
formation; and chromosome compaction to control gene expression, chromosome segregation, and recombination between
maternal and paternal chromosomes. We have found that the developmental control of gene expression is achieved through
chromatin modifications that affect chromosome structure with epigenetic consequences. We have also established robust
procedures for targeted genome editing across nematode species diverged by 300 MYR to study the evolution of sex
determination and dosage compensation. We combine genetic, genomic, proteomic, biochemical, and cell biological approaches
to study these questions in the model organism Caenorhabditis elegans, a round worm, and its related nematode species.



Counting Chromosomes to Determine Sex: Molecular Antagonism between X-Chromosome, Autosome Signals
Specifies Nematode Sex
Many organisms determine sexual fate by a chromosome-counting mechanism that distinguishes one X chromosome from two.
Embryos with one X become males, while those with two become females. We dissected the molecular mechanism by which the
nematode C. elegans counts its sex chromosomes to discern how small changes in the concentrations of molecular signals are
translated into dramatically different developmental fates. C. elegans tallies X-chromosome number relative to the ploidy, the
sets of autosomes (X:A signal). It discriminates with high fidelity between tiny differences in the signal: 2X:3A embryos (ratio 0.67)
become males, while 3X:4A embryos (ratio 0.75) become hermaphrodites (Figure 1).




















We showed that a set of X-linked genes called X-signal elements (XSEs) communicates X-chromosome dose by repressing the
master sex-determination switch gene xol-1 in a cumulative, dose-dependent manner. XOL-1, a GHMP kinase, is activated in
1X:2A embryos (1 dose of XSEs) to set the male fate but repressed in 2X:2A embryos (2 doses of XSEs) to promote the
hermaphrodite fate, including the activation of X-chromosome dosage compensation. We also showed that the dose of
autosomes is communicated by a set of autosomal signal elements (ASEs) that also act in a cumulative, dose-dependent manner
to counter XSEs by stimulating xol-1 transcription. We have explored the biochemical basis by which XSEs counter ASEs to
determine sex. Analysis in vitro showed that XSEs (nuclear receptors and homeodomain proteins) and ASEs (T-box and
zinc-finger proteins) bind directly to at least 5 distinct sites in xol-1 regulatory DNA to counteract each other's activities and
thereby regulate xol-1 transcription (Figure 2). Analysis in vivo showed that disrupting ASE and XSE binding sites recapitulated
the mis-regulation of xol-1 transcription caused by disrupting the cognate signal element genes. XSE and ASE binding sites are
distinct and non-overlapping, suggesting that direct competition for xol-1 binding is not the mechanism by which XSEs counter
ASEs. Instead, XSEs likely antagonize ASEs by recruiting cofactors with reciprocal activities that induce opposite transcriptional
states. The X:A balance is thus communicated in part through multiple antagonistic molecular interactions carried out on a
single promoter, revealing how small differences in X:A values can elicit different sexual fates. We are currently identifying
potential coactivators and corepressors and directing efforts toward understanding the evolution of the X:A signal across
nematode species.

Although most XSEs repress xol-1 by regulating transcription, one XSE, an RNA binding protein, represses xol-1 by binding to an
alternatively spliced intron and blocking its proper splicing, thereby generating a non-functional transcript with an in-frame stop
codon (Figure 2). This second tier of repression enhances the fidelity of the counting process.

The concept of a sex signal comprising competing XSEs and ASEs arose as a theory for fruit flies one century ago, and it
subsequently became entrenched in textbooks. Ironically, the recent work of others showed the fly sex signal does not fit this
simple paradigm, but our work shows the worm signal does.

X-Chromosome Dosage Compensation: Repressing X Chromosomes via Molecular Machines.
Organisms that use sex chromosomes to determine sexual fate evolved the essential, chromosome-wide regulatory process
called dosage compensation to balance X-chromosome gene expression between the sexes. Strategies for dosage compensation
differ from worms to mammals, but invariably a regulatory complex is targeted to X chromosomes of one sex to modulate
transcription along the entire chromosome. The heritable, regulation of X-chromosome expression during dosage compensation
is exemplary for dissecting the coordinate regulation of gene expression over large chromosomal territories and the role of
chromosome structure in regulating gene expression.

We defined the C. elegans dosage compensation complex (DCC) and showed it is homologous to condensin, a conserved protein
complex that mediates the compaction, resolution, and segregation of mitotic and meiotic chromosomes from yeast to humans
(Figure 3). The DCC binds to both X chromosomes of hermaphrodites to reduce transcription by half (Figure 3). Failure to reduce
expression kills hermaphrodites. Most DCC condensin subunits also control the structure and function of mitotic and meiotic
chromosomes by participating in two other distinct condensin complexes (Figure 3). Not only has the DCC co-opt subunits of
condensin to control gene expression, it co-opted a subunit from the MLL/COMPASS complex, a histone modifying complex, to
help recruit condensin subunits to rex sites.

We found that the DCC condensin subunits are recruited specifically to hermaphrodite X chromosomes by sex-specific DCC
subunits that trigger binding to cis-acting regulatory elements on X, called rex and dox sites. rex (recruitment elements on X)
sites recruit the DCC in an autonomous, sequence-dependent manner using DNA motifs highly enriched on X chromosomes.
The DCC spreads to dox (dependent on X) sites, which reside in promoters of active genes and bind the DCC robustly only when
linked to rex sites.




















Dynamic Control of X-Chromosome Conformation and Repression by a Histone H4K20me Demethylase.
We recently found that DCC subunit DPY-21 has a histone demethylase activity that is responsible for the selective enrichment of
H4K20me1 on X chromosomes of XX embryos upon DCC binding.  X-ray crystallography and biochemical assays of DPY-21
revealed a novel subfamily of Jumonji C histone demethylases that converts H4K20me2 to H4K20me1.  Selective inactivation of
demethylase activity in vivo by genome editing eliminated H4K20me1 enrichment on X, elevated X-linked gene expression,
reduced X-chromosome compaction, and disrupted X-chromosome topology by weakening TAD boundaries.  These findings,
among others, demonstrate the direct impact of chromatin modification on higher-order chromosome structure in long-range
regulation of gene expression.

H4K20me1 is also enriched on the mammalian inactive X chromosome, but the role of this enrichment in mammalian
transcriptional silencing is not known, nor is a selective reagent available to test its role.  We showed that the mouse homolog of
the DCC subunit also has H4K20me2 demethylase activity.  Hence the worm system holds great promise for understanding the
effects of histone modification in mammals.

Unexpectedly, DPY-21 associates with autosomes but not X chromosomes of germ cells in a DCC-independent manner to enrich
H4K20me1 and facilitate chromosome compaction.  Thus, DPY-21 is an adaptable chromatin regulator that is harnessed during
development for distinct biological functions.  In both somatic cells and germ cells, H4K20me1 enrichment modulates 3D
chromosome architecture to carry out these functions.






























Step of transcription controlled by the DCC.
We have dissected a key aspect of the dosage compensation mechanism by determining the step of transcription controlled by
the DCC to repress X-chromosome gene expression. This work was performed in collaboration with John Lis' lab at Cornell
University. In principle, the DCC could control any step of transcription: recruitment of RNA polymerase II (Pol II) to the promoter,
initiation of transcription, escape of Pol II from the promoter or pause sites, elongation of RNA transcripts, or termination of
transcription. The mechanism had been elusive in C. elegans due to improper annotation of transcription start sites (TSSs).
Nascent RNA transcripts from most nematode genes undergo rapid co-transcriptional processing in which the 5' end is replaced
by a common 22-nucleotide leader RNA through a trans-splicing mechanism, thereby destroying all knowledge of TSSs and
promoters.

To understand the step of transcription controlled by the DCC, we first devised a general strategy for mapping transcription start
sites and created an invaluable nematode TSS data set. The TSS mapping strategy, called GRO-cap, recovered nascent RNAs with
5'-caps prior to processing. We then determined the genome-wide distribution, orientation, and quantity of
transcriptionally-engaged RNA Polymerase II (Pol II) relative to TSSs in wild-type and DC-defective animals using GRO-seq (global
run-on sequencing).

We found that promoters are unexpectedly far upstream from the 5' ends of mature mRNAs, and promoter-proximal Pol II
pausing occurs only in starved larvae and is rare in C. elegans embryos, unlike in most metazoans. These results indicated that
enhancement of promoter pausing in XX embryos cannot be the mechanism of reducing transcription during dosage
compensation. In contrast, control of pausing is a common mechanism for controlling transcription of developmental regulatory
genes in most metazoans and is thought to be the mechanism of dosage compensation in fruit flies.

Then, by comparing the location and density of transcriptionally engaged Pol II in wild-type and dosage-compensation-defective
embryos, we found that the step of transcription controlled by the dosage compensation process is the recruitment of Pol II.
That is, C. elegans equalizes X-chromosome-wide gene expression between the sexes by reducing Pol II recruitment to the
promoters of X-linked genes in XX embryos by about half. One of our research directions is to dissect the mechanisms by which
the DCC limits Pol II recruitment.

Our data set also enabled us to analyze starvation-controlled gene regulation in collaboration with Ryan Baugh's lab at Duke
University. We found a new phenomenon of Pol II docking, the stable association of Pol II upstream of the transcription start
sites, and hence sites of pausing. We found that docked Pol II accumulates, without initiating, upstream of inactive growth genes
that are turned off during starvation are activated upon feeding. We found that Pol II pausing occurs at active stress-response
genes that are downregulated upon feeding. Hence, growth and stress genes are controlled by distinct mechanisms to
coordinate gene expression with nutrient availability.

DCC recruitment and binding to X chromosomes.
We showed that many of the DCC recruitment (rex) sites have a DNA motif (called MEX) that is highly enriched on X compared to
autosomes and is essential for DCC binding to a subset of rex sites. However, not all rex sites have this motif. We recently
defined new principles by which the DCC is recruited to X chromosomes, including the identification of a new, essential DCC
binding motif (MEX II) that is enriched on X. We found that MEX II acts in combination with MEX to foster high-affinity binding at
some rex sites but also acts alone at other rex sites to foster stable binding. We demonstrated these DCC binding principles by
using DCC binding assays in vivo and in vitro.

We also showed that SUMOylation of specific DCC subunits is essential for sex-specific assembly and function of the DCC on X.
Depletion of SUMO in vivo severely disrupts DCC binding and causes changes in X-linked gene expression similar to those
caused by deleting the genes that encode DCC subunits. Three DCC subunits undergo SUMOylation, one subunit essential for
DCC loading and two subunits that are integral to the condensin portion of the DCC.

DCC SUMOylation is triggered by the signal that initiates DCC assembly onto X. The initial step of assembly--binding of
X-targeting factors to rex sites--is independent of SUMOylation, but robust binding of the complete complex requires
SUMOylation. One of SUMOylated DCC subunits also participates in condensin complexes essential for chromosome
segregation, but its SUMOylation occurs only in the context of the DCC. Our results reinforce a newly emerging theme in which
multiple proteins of a complex are collectively SUMOylated in response to a specific stimulus, leading to accelerated complex
formation and enhanced function.

Condensin-driven remodeling of X-chromosome topology during dosage compensation.
The three-dimensional organization of a genome plays a critical role in regulating gene expression, yet little is known about the
machinery and mechanisms that determine higher-order chromosome structure. The involvement of bona fide condensin
subunits in dosage compensation together with our observation that the DCC acts at a distance to regulate gene expression
suggested that the DCC might alter the topology of X chromosomes to reduce gene expression chromosome wide.

Using genome-wide chromosome conformation capture techniques (in collaboration with Job Dekker's lab at U. Mass.
Worcester) with single-cell fluorescence in situ hybridization and RNA-seq to compare chromosome structure and gene
expression in wild-type and dosage-compensation-defective embryos, we showed that the DCC remodels X chromosomes of
hermaphrodites into a unique, sex-specific spatial conformation, distinct from autosomes, using its highest-affinity rex sites to
facilitate long-range interactions across X. Dosage-compensated X chromosomes consist of self-interacting domains (~ 1 Mb)
resembling mammalian Topologically Associating Domains (TADs). TADs on X have stronger boundaries and more regular
spacing than those on autosomes. Many TAD boundaries on X coincide with the highest-affinity rex sites, and these boundaries
become diminished or lost in mutants lacking DCC binding, causing the structure of X to resemble that of autosomes. These
results predicted that deletion of an endogenous rex site at a DCC-dependent boundary should disrupt the boundary. As
predicted, Cas9-mediated deletion of a rex site greatly diminished the boundary, further demonstrating the condensin-driven
remodeling of X-chromosome topology during dosage compensation. Thus, condensin acts as a key structural element to
reorganize interphase chromosomes and thereby regulate gene expression. Prior to our work, no molecular trigger or set of
DNA binding sites was known to cause a comparably strong effect on TAD structure in higher eukaryotes. Our understanding of
the topology of dosage-compensated X chromosomes provides fertile ground to decipher the detailed mechanistic relationship
between higher-order chromosome structure and chromosome-wide regulation of gene expression.























Targeted Genome-editing Across Highly Diverged Nematode Species.
Thwarted by the lack of reverse genetic approaches to enable cross-species comparisons of gene function, we established
robust strategies for targeted genome editing across nematode species diverged by 300 MYR. In our initial work, a collaboration
with Sangamo BioSciences, we used engineered nucleases containing fusions between the DNA cleavage domain of the enzyme
FokI and a custom-designed DNA binding domain: either zinc-finger motifs for zinc-finger nucleases or transcription
activator-like effector domains for TALE nucleases (TALENs). In those experiments, we allowed the DNA double-strand breaks to
be repaired imprecisely by non-homologous end joining (NHEJ) to create mutations in precise locations.

We then extended the use of TALENs to achieve precise insertion and deletion of desired sequences by introducing
single-stranded or double-stranded templates to generate precise insertions or deletions through homology directed repair
(HDR), the first demonstration of HDR using ZFNs or TALENs in the nematode community. We then adopted the use of the
CRISPR-associated nuclease Cas9 because of the ease in making RNA guides to program target specificity.

Despite successful application of Cas9 technology, predicting DNA targets and guide RNAs that support efficient genome editing
was problematic. We then devised a strategy for high-frequency genome editing (both NHEJ and HDR) at all targets tested. The
key innovation was designing guide RNAs with a GG motif at the 3' end of their target-specific sequences. This design increased
the frequency of mutagenesis 10-fold. The ease of mutant recovery was further enhanced by combining this efficient guide
design with a co-conversion strategy, in which targets of interest are analyzed in animals exhibiting a dominant phenotype
caused by Cas9-dependent editing of an unrelated target.

Evolution cis-acting Regulatory Sites that Control Dosage Compensation.
Mechanisms that specify sexual fate and compensate for X-chromosome dose have diverged rapidly across species compared to
other developmental processes, making it particularly informative to study these rapidly changing processes over short
evolutionary time scales. Application of our genome editing strategies to C. briggsae revealed that the core dosage
compensation machinery and key components of the genetic hierarchy that controls dosage compensation and sex
determination were conserved across the 30 MYR separation between C. elegans and C. briggsae. In contrast, the set of
cis-acting elements on X that recruit the DCC (rex sites) has diverged, retaining no functional overlap. ChIP-seq analysis defined
the C. briggsae DCC binding sites, and in vivo binding assays confirmed the ability of these sites to recruit the DCC when
detached from X in C. briggsae but not in C. elegans, and vice versa. The evolution of these sites differs dramatically from the
highly conserved DCC binding sites used by equivalently diverged fruit fly species and from the unchanged target sites of
conserved transcription factors that control multiple developmental processes from flies to humans. Hence, the divergence in
DCC binding specificity across nematode species provides a powerful opportunity to understand the path and timing for the
concerted change in hundreds of DNA target sites and the evolution of X chromosomes. We have extended our analysis of DCC
binding specificity to other nematode species and have shown that rex sites have diverged functionally at least three times in 30
MYR of evolutionary history.

Tethering Replicated Chromosomes via Cohesin to Ensure Genome Stability during Meiosis.
Faithful segregation of chromosomes during cell division is essential for genome stability. Accurate chromosome segregation is
required both for the proliferative cell divisions that produce daughter cells during mitosis and the two sequential divisions that
produce haploid sperm and eggs from diploid germline stem cells during meiosis. Approximately 30% of human zygotes have
abnormal chromosome content at conception due to defects in meiosis. Such aneuploidy is a leading cause of miscarriages and
birth defects and arises, in part, from defects in sister chromatid cohesion (SCC). SCC tethers replicated sister chromatids prior
to cell divisions to ensure proper chromosome segregation. In humans, SCC is established in the developing germ cells of a fetus
and must be maintained until ovulation in adults. This long-lived SCC is established and maintained by cohesin complexes,
evolutionarily conserved protein complexes structurally related to condensin (Figure 6).



















Studies in budding yeast showed that mitotic and meiotic cohesins are distinct but differ only in a single subunit called the
kleisin. During yeast meiosis, a single cohesin complex carries out all aspects of SCC. In contrast, our work in nematodes shows
that regulation of meiotic SCC in higher eukaryotes is more complex. We found that multiple functionally specialized cohesin
complexes mediate the establishment and two-step release of SCC that underlies the production of haploid gametes (Figure 6).
The meiotic complexes differ by a single kleisin subunit, and the kleisin influences nearly all aspects of meiotic cohesin function:
the mechanisms for loading cohesins onto chromosomes, for triggering DNA-bound cohesins to become cohesive, and for
releasing cohesins in a temporal- and location-specific manner (Figure 7). One kleisin triggers cohesion just after the
chromosomes replicate, as in yeast. Unexpectedly, the other triggers cohesion in a replication-independent manner, only after
programmed DSBs are made during meiosis to initiate recombination between homologous maternal and paternal
chromosomes. Thus, break-induced cohesion is essential for tethering replicated meiotic chromosomes. Later, recombination
stimulates separase-independent removal of the two different cohesin complexes from reciprocal chromosomal territories
flanking the crossover site. This region-specific removal likely underlies the two-step separation of homologs and sisters.
Unexpectedly, one cohesin complex also performs cohesion-independent functions in synaptonemal complex assembly. Our
findings establish a new model for cohesin function in meiosis: the choreographed actions of multiple cohesins, endowed with
unexpectedly specialized functions by their kleisins, underlie the stepwise separation of homologous chromosomes and then
sister chromatids required for reduction of genome copy number. This model diverges significantly from that in yeast but likely
applies to plants and mammals, which utilize similar meiotic kleisins.

Meiotic Chromosome Structures Constrain and Respond to Designation of Crossover Sites.
Crossover recombination events between homologous chromosomes are required to form chiasmata, temporary connections
between homologues that ensure their proper segregation at meiosis I. Despite this requirement for crossovers and an excess
of the double-strand DNA breaks that are the initiating events for meiotic recombination, most organisms make very few
crossovers per chromosome pair. Moreover, crossovers tend to inhibit the formation of other crossovers nearby on the same
chromosome pair, a poorly understood phenomenon known as crossover interference. We showed (in collaboration with the
Villenueve lab at Stanford) that the synaptonemal complex, a meiosis-specific structure that assembles between aligned
homologous chromosomes, both constrains and is altered by crossover recombination events. Partial depletion of the
synaptonemal complex central region proteins attenuates crossover interference, increasing crossovers and reducing the
effective distance over which interference operates, indicating that synaptonemal complex proteins limit crossovers. Moreover,
we showed that crossovers are associated with a local 0.4-0.5-micrometre increase in chromosome axis length. We proposed
that meiotic crossover regulation operates as a self-limiting system in which meiotic chromosome structures establish an
environment that promotes crossover formation, which in turn alters chromosome structure to inhibit other crossovers at
additional sites.

Ironically, the effect of depleting condensin I or condensin II on increasing crossovers appears to occur by a different
mechanism, because the sites of extra crossovers are not marked by the same molecular markers as the crossovers created by
reducing the synaptonemal complex. We are investigating the crossover pathway employed to achieve these extra,
non-interfering crossovers in condensin mutants.
Figure 6. Multiple cohesin complexes tether
meiotic chromosomes. (Click to enlarge.)
Figure 7. Divergent kleisin subunits of cohesin specify distinct
mechanisms that tether and release meiotic chromosomes. 
Figure 1. The X:A sex determining signal. (Click to enlarge.)
Figure 2. Model depicting the molecular antagonism between
XSEs and ASEs that determines sex.  (Click to enlarge.)
Figure 3. Biochemically distinct condensin complexes with interchangeable subunits control chromosome structure
throughout C. elegans development. (Click to enlarge.)
Figure 5. DCC modulates spatial organization of X chromosomes. (Click to enlarge.)
Figure 4. DPY-21 is a chromatin regulator that is harnessed during development for different biological functions. DPY-21 crystal structure and biochemical activity revealed a novel H4K20me2 Jumonji C demethylase. 
In somatic cells, DPY-21 enriches H4K20me1 on X chromosomes to repress gene expression.  H4K20me1 enrichment controls the higher-order structure of X chromosomes.  In germ cells, DPY-21 enriches H4K20me1
on autosomes in a DCC-independent manner to compact autosomes. (Click to enlarge.)