This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.We have dissected specialized assemblies on the Saccharomyces genome that help define and preserve the boundaries that separate silent and active chromatin. These assemblies contain characteristic stretches of DNA that flank particular regions of silent chromatin, as well as five distinctively modified histones and a set of protein complexes. The complexes consist of at least fifteen chromatin-associated proteins, including DNA pol ?, the Isw2-Itc1 and Top2 chromatin re-modeling proteins, the Sas3-Spt16 chromatin modifying complex, and Yta7, a bromodomain-containing AAA ATPase. We show that these complexes are important for the faithful maintenance of boundaries, as disruption of the complexes results in specific, anomalous alterations of the silent and active epigenetic states. This study was published this year in the J. of Cell Biology (J. Cell Biol., 2005, 169, 35-47). We are currently following up on numerous findings from the above mentioned work. We are 1) re-examining the in vivo DNA binding sites of these boundary chromatin complexes with high resolution DNA microarrays, 2) performing a detailed biochemical analysis of the bromodomain of Yta7 in order to determine how it interacts with histones, 3) performing single cell assays to determine how the Isw2-Itc1 proteins regulate a boundary chromatin region surrounding the arsenic response locus.
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