On the formation of chromatin boundaries and the evolution of silencing.
Jasper Rine, Michael Kobor, Josh Barbiarz, Jennifer Gin, Bilge Ozaydin, Jessica Cande, Lenny Teytelman, Jason Zemansky
Molecular and Cell Biology, University of California, 522 Barker Hall, Berkeley, Ca, 94720-3202, USA
Histone variant, H2A.Z, helps form the boundary between silent and active chromatin. How is H2A.Z inserted into chromatin; what determines the location of H2A.Z; and how do boundaries work? We, with the Madhani lab, discovered a protein complex which binds H2A.Z, called SWR1-Com, and deposits H2A.Z into chromatin. SWR1-Com has 13 subunits, 4 are shared with the histone acetyltransferase NuA4. This sharing of subunits is likely important. Double mutants lacking H2A.Z and Eaf1p, a new subunit of NuA4, are synthetically lethal. In vitro, NuA4 acetylates H2A.Z at one position, and we have mutant forms of H2A.Z to define the role of this modification. We moved sequences enriched in H2A.Z in the genome onto plasmids, where these same sequences remain rich in H2A.Z. Hence the localization signals for H2A.Z are discrete and local. Yaf9 protein, shared by SWR1-Com and NuA4, contains a YEATS domain common to proteins associated with leukemias. We defined a protease resistant core containing this domain, have crystals, and are determining its structure. We have explored the evolution of silencing among other genera of Saccharomyces. Surprisingly, there are many Sir protein orthologs among these sequences that are not revealed by the SGD database. Using more directed searches, we found that SIR genes are present in multiple copies in these species, including 3 SIR1 paralogs in one. We will describe studies of sir mutants and mating type interconversion in these species.
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