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.
|Mieczkowski, Jakub; Cook, April; Bowman, Sarah K et al. (2016) MNase titration reveals differences between nucleosome occupancy and chromatin accessibility. Nat Commun 7:11485|
|Ray, Mridula K; Wiskow, Ole; King, Matthew J et al. (2016) CAT7 and cat7l Long Non-coding RNAs Tune Polycomb Repressive Complex 1 Function during Human and Zebrafish Development. J Biol Chem 291:19558-72|
|Yildirim, Ozlem; Kingston, Robert E (2016) Molecular Dissection of Chromatin Maturation via Click Chemistry. Curr Protoc Mol Biol 114:21.33.1-21.33.11|
|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|
|Alpatov, Roman; Lesch, Bluma J; Nakamoto-Kinoshita, Mika et al. (2014) A chromatin-dependent role of the fragile X mental retardation protein FMRP in the DNA damage response. Cell 157:869-81|
|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|
|Fang, Rui; Chen, Fei; Dong, Zhenghong et al. (2013) LSD2/KDM1B and its cofactor NPAC/GLYR1 endow a structural and molecular model for regulation of H3K4 demethylation. Mol Cell 49:558-70|
|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|
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