This proposal investigates molecular aspects of epigenetics. Epigenetics encompasses changes in gene expression profiles that occur without alterations in the genomic DNA sequence of a cell. This arises from the dynamic processes that structure regions of chromosomal DNA through a range of compaction in eukaryotes. The altered pattern of gene expression is pivotal to cellular differentiation and development and is inherited by daughter cells thereby maintaining the integrity, specifications, and functions for a given cell type. Aberrancies in this epigentic process gives rise to perturbations that are also inherited and disruptive to normal cellular properties. The histone proteins that package DNA into chromatin are subject to post- translational modifications that give rise to different chromatin structures. While euchromatin has a relaxed structure permissive to transcription, constitutive heterochromatin is densely packed and inaccessible. The focus of this proposal is facultative chromatin that is repressive, but can be altered in its properties and become active.
The aims are to identify the molecular mechanisms controling the formation of facultative heterochromatin and the epigenetic parameters that ensure its propagation through cell divisions. We focus on three major activities that impact facultative heterochromatin formation: SirT1, L3MBTL1 (L1) and L3MBTL2 (L2) that comprise defined multiprotein complexes through which their role is facilitated. SirT1 is a NAD+ histone deacetylase that targets histone H4-lysine 16 (H4K16) for deacetylation in vivo, and interacts with histone H1b and the histone lysine methyltransferase Suv39h1. Both L1 and L2 contain MBT domains, bind to nucleosomes and associate with HP1 gamma. L1 also associates with histone H1 and binds to two repressive histone marks. L2 associates with three ring finger proteins and E2F6 and catalyzes monoubiquitination of histone H2AK119. Thus all three, SirT1, L1, and L2 exhibit activites on histone residues associated with facultative heterochromatin. We will identify the target genes for each (genome- wide ChIP on ChIP experiments), their localization on target genes as a function of gene expression and as a function of specific histone modifications using highly specific antibodies (immunofluoresecnce and ChIP analyses), and the role of histone H1 and other interacting factors in chromatin compaction analyses (including sucrose gradient sedimentation together with electron microscopy). ? ? ?
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