The long-term goal of our research is to investigate the molecular mechanisms of chromatin dynamics for understanding in molecular detail the fundamental questions of how transcription, DNA replication, and DNA repair take place within the context of highly compacted chromatin, and how mis-regulation of chromatin causes human diseases such as cancer. The overall objective of this proposed research is to determine how the deposition of the conserved histone variant H2A.Z is regulated by chromatin remodeling factors in mammalian cells. H2A.Z is deposited within nucleosomes that flank gene promoters, and plays essential roles in gene expression, genome stability, and proper embryonic stem cell (ESC) differentiation. Furthermore, mis- regulation of H2A.Z deposition is linked to cancer and cardiac hypertrophy. In yeast, SWR1, one of the well- characterized members of the SWR1/INO80 subfamily of remodeling enzymes, has a unique dimer exchange activity to remove H2A/H2B dimers from a nucleosome and replace them with H2A.Z/H2B dimers. The p400 and SRCAP chromatin remodeling enzymes are mammalian homologs of yeast SWR1 that are thought to be responsible for H2A.Z deposition. Interestingly, in lung cancer cells where H2A.Z is upregulated, suppression of p400 does not affect H2A.Z deposition while suppression of SRCAP leads to a decrease in H2A.Z deposition. Moreover, although p400 is required for maintenance of ESC identity such as self-renewal and pluripetency, H2A.Z is required for ESC differentiation, but not for maintenance of ESC identity. These observations suggest cell-type specific, distinct functions of p400 and SRCAP. Our overall strategy in this proposal is to exploit a powerful combination of biochemical and biophysical techniques, and genomics in ES cells to dissect the molecular mechanisms by which p400 and SRCAP regulate H2A.Z deposition and define the distinct biochemical and biological functions of these remodeling enzymes. This proposal has two specific aims.
In Aim 1, we will dissect the mechanisms of H2A.Z deposition by the p400 and SRCAP remodeling complexes. The molecular mechanisms by which p400 and SRCAP catalyzes H2A.Z deposition are largely unknown, mainly due to the limited protein availability, as p400 and SRCAP form large multi-protein complexes. To address this, we have reconstituted the p400 and SRCAP complexes from individual, recombinant subunits using the Multibac baculovirus expression system. We will define the detailed kinetic rates and substrate specificities of the p400 and SRCAP complexes in the dimer exchange reactions. We will employ various dimer exchange assays including FRET-based assays. Furthermore, we will exploit state-of- the-art EM analysis of the p400 and SRCAP complexes to dissect the structural and functional relationship of these complexes. We will also explore the functions of p400 and SRCAP in mouse embryonic stem cells (ESCs). We will investigate how suppression of p400 and/or SRCAP alters the epigenetic landscape of H2A.Z and affects ESC identity and differentiation.
In Aim 2, we will investigate how H2A.Z deposition is regulated by subunits of the p400 and SRCAP complexes and histone acetylations. We will define the role of different subunits of the p400 and SRCAP complexes in the dimer exchange reaction, focusing initially on the conserved RUBVL1/2 subunits. We will dissect how RUVBL1/2 govern the assemblies and functions of the p400 and SRCAP complexes using in vitro reconstitution system. We will also investigate how the ATPase activity of RUVLBL1/2 contributes to the dimer exchange activities of these complexes. In addition, the Tip60 histone acetyltransferase is a component of the p400 complex. We will investigate how the dimer exchange activity of p400 coordinates with the histone acetylation by Tip60. Furthermore, we recently identified a novel functional interaction between SWR1 and H3-K56Ac that regulates H2A.Z dynamics in yeast. We will test the hypothesis that the H3-K56Ac regulates the dimer exchange activities of p400 and SRCAP in mammalian cells.
Epigenetic alterations of chromatin structure promote a number of developmental disorders and diseases such as cancer. This proposed studies on key chromatin regulators essential for proper development and dysfunctional cardiac hypertrophy and cancer will provide mechanistic insight into how diseases arise, and lead to new strategies for treatment and drug development.