For development of complex organisms to occur properly, the genes that determine cell type must be regulated both spatially and temporally. One key aspect of this regulation is the effective lineage-specific repression of master regulatory genes that must be maintained in an ?off? status. This problem has been studied since the 1940s in Drosophila, which has led to the identification of a group of genes called the Polycomb- Group (PcG) after the founding member, the Polycomb gene. The PcG encodes proteins that form complexes that direct a repressed state. The central repressive complex in this family is called Polycomb Repressive Complex 1 (PRC1), and a second complex that methylates histones and helps target PcG function is called PRC2. PRC1 and PRC2 are targeted to appropriate locations by Polycomb Response Elements (PREs), which are usually about 1 kb in length and nucleosome depleted. This application describes experiments designed to understand function of the PcG system, with a focus on the role of nucleosome compaction, PRE function and the ability of non-coding RNAs to regulate this system. Three areas are addressed in three specific Aims in this application.
Aim 1 examines the ability of a mammalian Polycomb protein called Cbx2 to compact nucleosomes in vitro and in cell culture. It also describes approaches to crystallize proteins involved in creating a compacted state with the goal of defining these mechanisms for Polycomb at this high level of resolution.
Aim 2 explores the location and function of PREs in the human and mouse HOX clusters. Nucleosome depletion is used as one method to identify potential PRE sequences, which are then tested for function using reporter constructs. The hypothesis that the Jarid2 protein is generally involved in mammalian PRE function is tested using these PRE sequences.
Aim 3 describes the development of a new technology to map the binding sites for ncRNAs. This technology will be used to test the hypothesis that specific ncRNAs are involved in targeting PcG function to specific loci.
We describe experiments to characterize the several aspects of the complex gene system, called the Polycomb-Group, that silences genes to maintain proper cell identity in mammals. We will characterize the protein domain and the molecular mechanism that creates a compacted chromatin structure perhaps causal to silencing, will identify DNA elements that target this system to specific genes, and will develop techniques to determine where non-coding RNAs involved in regulation function on the genome.
|Bowman, Sarah K; Deaton, Aimee M; Domingues, Heber et al. (2014) H3K27 modifications define segmental regulatory domains in the Drosophila bithorax complex. Elife 3:e02833|
|West, Jason A; Davis, Christopher P; Sunwoo, Hongjae et al. (2014) The long noncoding RNAs NEAT1 and MALAT1 bind active chromatin sites. Mol Cell 55:791-802|
|West, Jason A; Cook, April; Alver, Burak H et al. (2014) Nucleosomal occupancy changes locally over key regulatory regions during cell differentiation and reprogramming. Nat Commun 5:4719|
|Simon, Matthew D; Pinter, Stefan F; Fang, Rui et al. (2013) High-resolution Xist binding maps reveal two-step spreading during X-chromosome inactivation. Nature 504:465-9|
|Simon, Jeffrey A; Kingston, Robert E (2013) Occupying chromatin: Polycomb mechanisms for getting to genomic targets, stopping transcriptional traffic, and staying put. Mol Cell 49:808-24|
|Woo, Caroline J; Kharchenko, Peter V; Daheron, Laurence et al. (2013) Variable requirements for DNA-binding proteins at polycomb-dependent repressive regions in human HOX clusters. Mol Cell Biol 33:3274-85|
|Armache, Karim-Jean; Garlick, Joseph D; Canzio, Daniele et al. (2011) Structural basis of silencing: Sir3 BAH domain in complex with a nucleosome at 3.0 A resolution. Science 334:977-82|
|Grau, Daniel J; Chapman, Brad A; Garlick, Joe D et al. (2011) Compaction of chromatin by diverse Polycomb group proteins requires localized regions of high charge. Genes Dev 25:2210-21|
|Simon, Matthew D; Wang, Charlotte I; Kharchenko, Peter V et al. (2011) The genomic binding sites of a noncoding RNA. Proc Natl Acad Sci U S A 108:20497-502|
|Woo, Caroline J; Kharchenko, Peter V; Daheron, Laurence et al. (2010) A region of the human HOXD cluster that confers polycomb-group responsiveness. Cell 140:99-110|
Showing the most recent 10 out of 17 publications