Howard Hughes Professor and Professor of Genetics, Genomics and DevelopmentLab Homepage: http://mcb.berkeley.edu/labs/rine/
The research in my lab is centered on the epigenetic mechanisms by which the information in a genome is expressed in a manner that is stable and heritable through cell division. This issue has led us to discoveries on how specialized domains of chromatin structure are established, maintained and inherited. Our studies use the yeast Saccharomyces cerevisiae and the powerful genetic, genomic and proteomic approaches that are so facile in this organism to study the nature of the chromatin in specialized domains of the yeast genome, how it is assembled and modified, and how these modifications affect its expression, replication and repair. Recently the lab has started a new emphasis focused on exploring the functional consequences of human genetic and epigenetic variation with the goal of understanding and reducing the incidence of one of the most common birth defects.
The most prominent epigenetic mechanism in Saccharomyces involves the silencing of genes controlling cell type. The a and alpha cell types of yeast are controlled by transposable genes that are alleles of the mating type locus (MAT). These alleles are complex blocks of DNA encoding regulatory proteins whose synthesis controls the expression of genes that specify the identity of different cell types of yeast. In addition to MAT, the a and alpha genes are also located at two additional loci, known as HML and HMR, where they are transcriptionally silenced. Transcriptional silencing requires a complex series of interactions between proteins, regulatory sites, and origins of replication that result in the establishment of inactive chromatin in these regions. This repression is one of the best examples in biology to learn changes in chromatin structure have been shown to cause changes in gene expression.
We have focused our efforts on the SIR genes, which encode proteins required for this position-dependent regulation, and on the silencers, which are sites required for the silencing of flanking sequences. Repression mediated by the SIR genes involves the assembly of inactive states of gene expression at HML and HMR. These states once established, are epigenetically inherited, and thus offer the most genetically tractable opportunity to study the inheritance of states of chromatin structure. Remarkably, the silencer also functions as a chromosomal origin of DNA replication, and we have discovered that ORC, the eukaryotic replication initiator, plays a critical role in silencing.
Silencing the HML and HMR loci requires passage of cells through the cell cycle. We have shown that Sir proteins can bind to silencers and spread through these regions, binding to nucleosome tails, in cells that are in G1, but reduction in transcription does not occur in these cells until they reach G2. We proved that DNA replication per se is not the cell-cycle event required for silencing, and are narrowing in on identifying the requirement. By studying silencing kinetically with single-cell resolution we have discovered that multiple cell cycles are required to fully silence these loci, even though the structural hallmarks of heterochromatin, are laid down in the first cell cycle. Our goal is a complete description of the cell-cycle-dependent steps involved in making silenced chromatin.
One hallmark of silenced chromatin is the presence of boundary elements that block its spreading into, and inactivation of, neighboring euchromatic genes. One important element of a hetrochromatin boundary is nucleosomes containing the histone H2A variant known as H2A.Z. We discovered the SWR1 complex, consisting of 13 different polypeptides, whose role is to deposit H2A.Z into nucleosomes at specific positions in the genome, including heterochromatin boundaries. We would like to learn how the SWR1 Complex achieves this locus-specific deposition of a specialized histone. Once at a boundary, we discovered that H2A.Z enjoys the attention of two different histone acetyltransferases, which decorate it at four different positions. These acetylations are required for its boundary function, and our recent work has identified the bromodomain proteins that read this code and block heterochromatin spreading.
Amazingly, a single amino acid change in a particular repressor can restore heterochromatin in cells lacking the usual heterochromatin components. This discovery implies that heterochromatin may be easy to evolve, and indeed the mechanisms of heterochromatin silencing are very different between Saccharomyces and the distantly related Schizzosaccharomyces pombe. We have embarked on a computational and functional exploration of how heterochromatin evolved by exploiting the fantastic insights that can come from the dozens of sequenced genomes from closely and less-closely related yeast species. Although in their early days, these studies are already revealing crucial steps in the evolution of this type of chromatin structure and have revealed how silencing has shaped the genetic organization of the genome.
The newest project in the lab is focused on human genetic variation. The defining genetic challenge of this century will be understanding the significance of the 3 to 6 million DNA sequence differences between any two people. Many of these differences will be neutral polymorphisms, others will be biologically important and medically significant. There are hundreds of human proteins that can functionally replace their human orthologs when human cDNAs are expressed in a yeast strain in which the corresponding yeast gene has been removed. In this way, yeast can be used to systematically investigate variation in the protein coding sequences of human genes, free of the complications presented by studying the different mixtures of variants found in any two humans. Our earliest studies have focused on enzymes of folate metabolism due to the central role that folate plays in reducing the incidence of neural tube defects, one of the most common forms of birth defects. To our surprise, we found that approximately half of the nonsynonymous changes found in one of these enzymes from a random sample of humans dramatically affect the function of this enzyme. Remarkably, in most of these cases, the defect in the enzyme's function can be largely suppressed by increasing the concentration of the enzyme's cofactor. If these observations apply broadly, it should be possible to identify those individuals most at risk, and at least in some cases, it may be possible to reduce their risk with appropriate cofactor supplementation.
Nine percent of humans are homozygous for a mutation in folate biosynthesis that reduces the synthesis of the methyl-donor for DNA methylation. We hypothesize that homozygotes for this mutation may have a high frequency of epialleles at those positions of the human genome that are most sensitive to a reduction in the level of methyl donor for DNA methylation. We hypothesize that these people may be particularly prone to changes in their epigenetic program. Our goal is to find the most labile epigenetic marks in the human genome and determine if these people exhibit elevated variation at these positions and what consequences that might have.
E.A. Osborne, S. Duderoit, and J. Rine. The establishment of gene silencing at single cell resolution. Nature Genetics. In Press.
J.E. Gallagher, J.E. Babiarz, L. Teytelman, K.H. Wolfe, and J. Rine. Elaboration, diversification and regulation of the Sir1 family of silencing proteins in Saccharomyces. Genetics. 2009; 181: 1477-91.
L. Teytelman, M.B. Eisen, and J. Rine. Silent but not static: acccelerated base-pair substitution in silenced chromatin of budding yeasts. PLos Genetics. 2008; 4: e1000247.
O.A. Zill and J. Rine. Interspecies variation reveals a conserved repressor of alpha-specific genes in Saccharomyces yeasts. Genes Dev. 2008; 22: 1704-16.
N.J. Marini, J. Gin, J. Ziegle, K.H. Keho, D. Ginzinger, D.A. Gilbert, and J. Rine. The prevalence of folate-remedial MTHFR enzyme variants in humans. Proc Natl Acad Sci USA. 2008; 105: 8055-60.
J.A. Mayfield and J. Rine. The genetic basis of variation in susceptibility to infection with Histoplasma capsulatum in the mouse. Genes Immun. 2007; 8: 468-74.
H. Jeong, I. Herskowitz, D.L. Kroetz and J. Rine. Function-altering SNPs in the human multidrug transporter gene ABCB1 using Saccharomyces-based assay. PLoS Genetics. 2007; 3: e39.
M.W. Neff and J. Rine. A fetching model organism. Cell. 2006; 124: 229-31.
A. Kirchmaier and J. Rine. Cell cycle requirements in assembling silent chromatin in Saccharomyces cerevisiae. Mol Cell Biol. 2006; 26: 852-62.
J. Babiarz, J. Halley and J. Rine Telomeric heterochromatin boundaries require NuA4-dependent acetylation of histone variant H2A.Z in Saccharomyces cerevisiae. Genes Dev. 2006; 20: 700-10.
J. Rine. Cell biology. Twists in the tale of the aging yeast. Science. 2005; 310:1124-5
P.Lynch, H. Fraser, E. Sevastopolis, J. Rine and L. Rusche. Sum1p, the origin recognition complex, and the spreading of a promoter-specific repressor in Saccharomyces cerevisiae. Mol Cell Biol. 2005; 25: 5920-32.
B. Suter, A. Tong, M. Chang, L.Yu G.Brown, C. Boone and J. Rine. The origin recognition complex links replication, sister chromatid cohesion and transcriptional silencing in Saccharomyces cerevisiae. Genetics. 2004; 167: 579-91.
L. Pillus and J. Rine. SIR1 and the origin of epigenetic states in Saccharomyces cerevisiae. Cold Spring Harb Symp Quant Biol. 2004; 69: 259-65.
M. Kobor, S. Venkatasubrahmanyam, M. Meneghini, J. Gin, J. Jennings, A. Link, H. Madhani and J. Rine. A protein complex containing the conserved Swi2/Snf2-related ATPase Swr1p deposits histone variant H2A.Z into euchromatin. PLoS Biol. 2004; 2: E131.
Last Updated 2009-06-11