Our research focuses on understanding the mechanisms by which entire regions of the genome are rendered inaccessible to transcription and recombination. The silencing of genomic domains involves interactions between silencers and repressor proteins. The silencers recruit repressor protein complexes composed of the Sir proteins, which interact with histones in nucleosomes to form a chromatin domain that is both inaccessible and inert to various cellular processes. Thus, in an effort to understand in molecular detail the mechanism by which silencing is effected, we are currently investigating Sir proteins and their interactions with histones.
Histone variants and cell cycle progression
Dhillon
The several histone variants are expressed to varying degrees and differ in localization; moreover, their expression is regulated, suggesting a role in gene regulation. We isolated a histone variant Htz1p as a suppressor of a Sir1p mutant and are currently investigating the many roles of this protein in the cell. Cells lacking the protein are hypersensitive to a variety of drugs, such as hydroxyurea (HU). While cells lacking Htz1 are unable to grow on media containing sublethal amounts of HU, the protein is not involved in mediating the checkpoint response that is normally triggered when cells are exposed to higher doses of the drug. We have uncovered genetic interactions between Htz1 and proteins involved in replication checkpoint control, and our analyses suggest that cells lacking the protein exhibit increased defects during S-phase. In particular, cells are slower to progress through S-phase, and the timing of replication of both early- and late-replicating loci is delayed. In addition, we show that the cells are delayed in progression through the cell cycle. Strikingly, we also find that transcription of specific cell cycle–regulated genes is delayed in htz1 cells. Our results led us to suggest that this histone variant is required for an open chromatin state that facilitates activation of specific genes during the cell cycle. We also concluded that delays in the expression of these genes results in delays in origin firing and S-phase progression, and DNA damage during replication.
Kamakaka RT, Biggins S. Histone variants: deviants? Genes Dev 2005;19:295-310.
Characterization of Sir protein complexes involved in silencing
Kotomura, Haldar
How Sir proteins function to form silenced domains is our principal biochemical problem. Genetic studies have revealed that distinct combinations of the Sir protein complexes repress multiple loci. Silencing at all the loci requires Sir2p, which possesses histone deacetylase activity. In addition, Sir2p is the only Sir protein to have additional homologues in yeast (Hst1-4p) and the only Sir protein conserved throughout evolution. We are reconstituting Sir2p-containing protein complexes to characterize the function of each component within the complexes.
We have begun studies on the reconstitution of silenced chromatin by using the Sir2p-containing protein complexes and histones in nucleosomes. We are performing in vitro binding studies with purified recombinant Sir proteins with positioned nucleosomes. Our long-term plans call for studies on the regulation of the enzymes within the cell and in vitro studies aimed toward the eventual development of specific enzyme inhibitors that may have therapeutic value. Depending on whether it is possible to mimic exactly the silenced state in vitro, our studies will provide an important index of our current understanding of transcriptional silencing, particularly given that mechanisms are rarely established by genetic means and usually require biochemical tests.
A complete understanding of transcriptional repression requires the analysis of several unrelated loci in different and distinct systems so that salient principles of repression can be distinguished from organism- and locus-specific variation. Silencing of chromatin domains in S. pombe bears many similarities to heterochromatin formation and position-effect variegation in other eukaryotes such as S. cerevisiae and Drosophila. We are identifying functional homologues in S. pombe of the various S. cerevisiae genes that affect repression and will then purify and characterize protein complexes containing these proteins; our work will be coupled with mechanistic studies on nucleosomal binding in S. pombe.
Gangadharan S, Ghidelli S, Kamakaka RT. Purification of Sir2 proteins from yeast. Methods Enzymol 2004;377:234-254.
Chromatin domains in silencing
Oki, Valenzuela, Gangadharan
We are interested in understanding the mechanism by which silenced chromatin domains are restricted to specific regions along the DNA fiber. Eukaryotic chromosomes are organized into discrete domains delimited by domain boundaries. We have demonstrated that a specific t-RNA gene mediates barrier functions at HMR (Donze et al., Genes Dev 1999;13:698). In addition, we have identified the proteins that are required to prevent the spread of heterochromatin into neighboring euchromatin (Donze and Kamakaka, EMBO J 2000;20:520). Our results suggest that barrier activity may arise from an underlying competition between chromatin-remodeling and silencing activities at the interface between euchromatin and heterochromatin. In our ongoing studies of yeast barriers, we used genetic screens to isolate other DNA elements from yeast that act as barrier elements.
We also performed a systematic genome-wide screen for proteins that could block the spread of silencing in yeast. The analysis identified numerous proteins with efficient silencing-blocking activities; some of the proteins are known to be involved in chromatin dynamics. We isolated subunits of Swi/Snf, mediator, and TFIID as well as subunits of the Sas-I, SAGA, NuA3, NuA4, Spt10p, Rad6p, and Dot1p complexes as barrier proteins. We demonstrated that histone acetylation as well as chromatin remodeling occurred at both the synthetic barrier and the native boundaries of the silenced domains and correlated with a block in the spread of silencing (Oki et al., 2004).
Further mapping analysis in strains in which the native barrier elements were mutated indicate that the interface between active and silenced chromatin is a junction of opposing activities with competition between activities that aid and activities that prevent the spread of silencing. Our data suggest that several overlapping mechanisms are involved to delimit silenced and active domains in vivo (Oki and Kamakaka, 2005).
We are also analyzing a novel form of gene repression mediated by the dominant mutant SUM1-1. Sum1p normally functions as a mitotic repressor of meiotic genes, but SUM1-1 is a neomorphic allele that can repress the MATa1 genes at HMR. SUM1-1 spreads across a large region of DNA, and the repression of MATa1 is not localized but rather occurs throughout the region. Our analyses indicate that such repression is specific to the MATa1 gene, given that URA3 or ADE2 at HMR is not stably repressed owing to an inability to inherit the repressed state.
Dhillon N, Oki M, Kamakaka RT. H2A.Z functions to regulate progression through the cell cycle. 2005 (in press).
Oki M, Kamakaka RT. Barrier function at HMR. Mol Cell 2005;19:707-716.
Oki M, Valenzuela L, Chiba T, Ito T, Kamakaka RT. Barrier proteins remodel and modify chromatin to restrict silenced domains. Mol Cell Biol 2004;24:1956-1967.
Valenzuela L, Gangadharan S, Kamakaka RT. SUM1-1 is a promoter-specific long-range repressor. Genetics 2005 [Epub ahead of print].
For further information, contact rohinton@helix.nih.gov.