Research in this laboratory is devoted to understanding the mechanisms by which entire regions of the genome are rendered inaccessible to transcription and recombination. The laboratory uses genetic analysis, coupled with biochemical fractionation and reconstitution experiments, to explore the issues of genome accessibility. Silencing of genomic domains requires a complex series of interactions between inactivation centers called silencers and numerous repressor proteins. The silencers recruit repressor protein complexes composed of the Sir proteins, which then interact with histones in nucleosomes to form a chromatin domain, a domain that is both inaccessible and inert to various cellular processes. We are currently focusing on these proteins and their interactions with the histones to understand, in molecular detail, the mechanism by which silencing is effected.

Histone Variants and Cell Cycle Progression
Dhillon
It has been previously demonstrated that RAP1, ORC, and SIR1 are necessary to establish the silent state. Sir1p acts as an intermediary mol-ecule between the proteins bound to the silencer element (Rap1p and Orc) and the proteins in-volved in maintaining the repressed state: Sir2, Sir3, Sir4, and the histones.

The role of histone variants in gene expression is poorly understood. To understand the function of Sir1 in silencing in S. cerevisiae, we generated yeast cells with mutations in this protein that were unable to silence even after Sir1 was recruited to the silencer, presumably owing to a weakened interaction between Sir1p and the other proteins involved in silencing. Overproduction of the histone variant Htz1p in the sir1 mutant could compensate for its weakened interaction (Dhillon and Kamakaka, 2000). We are currently interested in understanding the many roles of this protein in the cell. Classical molecular genetic and biochemical experiments are under way to identify the domains of Htz1 involved in cell cycle progression, determine the response to drugs such as hydroxyurea and benomyl, and identify the proteins that interact with Htz1 to function in the cell.

Identification and Characterization of Sir Protein Complexes Involved in Silencing
Gangadharan
Our main goal is to determine how Sir proteins function to form silenced domains. Genetic studies have revealed that distinct combinations of the Sir protein complexes repress multiple loci. Silencing at these loci requires Sir2p, which possesses histone deacetylase activity. In addition, Sir2p is the only Sir protein to have additional homologs in yeast (Hst1-4p) and the only Sir protein conserved throughout evolution. We are purifying Sir2p-containing protein complexes from yeast cells to identify and characterize individual components within these complexes and their associated enzymatic activities. We have also initiated studies on the reconstitution of silenced chromatin by using these purified complexes and histones in nucleosomes (Ghidelli et al., 2001). Our ability to mimic the silenced state in vitro will be an important gauge of our current understanding of transcriptional silencing.

We will employ reverse genetics to determine the function of any novel polypeptides associated with these complexes. We will also perform experiments to analyze the nature of the interactions between these Sir protein complexes and histones in nucleosomes. This analysis will involve binding studies with purified Sir complexes and positioned nucleosomes in arrays, followed by DNaseI foot-printing and protein-protein cross-linking and sedimentation analyses. Long-term goals call for studies on the regulation of these enzymes within the cell and in vitro studies aimed toward the eventual development of specific inhibitors for these enzymes that may have therapeutic value.

Role of Nuclear Architecture in Silencing
Oki, Valenzuela; in collaboration with Ito
We are also 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 that are delimited by domain boundaries. We have demonstrated that a specific tRNA gene mediates barrier functions at the HMR locus. The proteins that are required to prevent the spread of heterochromatin into neighboring euchromatin have also been identified (Donze and Kamakaka, 2000). Our results suggest that barrier activity may arise from an underlying competition between chromatin remodeling and silencing at the interface of euchromatin and heterochromatin. In our ongoing studies on yeast barriers, we have used genetic screens both to isolate other DNA elements from yeast and other eukaryotes that act as barriers and to identify additional proteins from yeast and other eukaryotes that possess barrier activity. These studies will help delineate general principles of barrier activity in all eukaryotes. In addition, given that other transcriptionally inactive genes such as the SMK1 loci in S. cerevisiae are also packaged into repressive chromatin domains, we can address whether the repressors that function at this locus are restricted by the presence of specific barriers and whether these elements function by similar mechanisms.

Analysis of Sir2p in Other Eukaryotes
Haldar
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 shows many similarities to heterochromatin formation and position effect variegation in other eukaryotes such as S. cerevisiae and Drosophila. Functional homologs of the various S. cerevisiae genes that affect repression are being identified in S. pombe to allow the purification and characterization of protein complexes containing such proteins. This work will be coupled with mechanistic studies on nucleosomal binding in this organism.