Polycomb repressive complex 1 ( PRC1 ) is an essential chromatin-based repressor of gene transcription. However, how PRC1 engages with chromatin to identify its target genes and achieve gene repression remains poorly defined, representing a major hurdle to our understanding of Polycomb system function. Here we use genome engineering and single particle tracking to dissect how PRC1 binds to chromatin in live mouse embryonic stem cells. We reveal that PRC1 is highly dynamic, with only a small fraction stably interacting with chromatin. By integrating subunit-specific dynamics, chromatin binding, and abundance measurements, we discover that PRC1 exhibits surprisingly low occupancy at target sites. Furthermore, we employ perturbation approaches to uncover how specific components of PRC1 define its kinetics and chromatin binding. Together, these discoveries provide a quantitative understanding of chromatin binding by PRC1 in live cells, and suggests that chromatin modification, as opposed to PRC1 complex occupancy, is central to gene repression. Bạn đang xem : Management of temporomandibular disorders and occlusion
Eukaryotic DNA is wrapped around histones to form nucleosomes and chromatin that organise and package the genome within the confines of the nucleus. In addition to this packaging role, chromatin and its post-translational modification can also profoundly influence gene transcription ( Atlasi and Stunnenberg, 2017 ; Kouzarides, 2007 ; Musselman et al., 2012 ). Therefore, significant effort has been placed on studying how chromatin-modifying enzymes regulate gene expression. However, in many cases, the mechanisms that enable these enzymes to bind chromatin and identify their appropriate target sites remains poorly understood .
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Chromatin-based regulation of gene transcription is typified by the Polycomb repressive system, which is essential for normal gene regulation during animal development (Blackledge et al., 2015; Di Croce and Helin, 2013; Schuettengruber et al., 2017; Simon and Kingston, 2009; Vidal, 2019; Voigt et al., 2013). This system is comprised of two central histone-modifying protein complexes, Polycomb repressive complex 1 (PRC1) and PRC2. PRC1 is an E3 ubiquitin ligase that mono-ubiquitylates histone H2A on lysine 119 (H2AK119ub1) (de Napoles et al., 2004; H. Wang et al., 2004). PRC2 is a methyltransferase that methylates histone H3 at lysine 27 (H3K27me1/2/3) (Cao et al., 2002; Czermin et al., 2002; Kuzmichev et al., 2002; Müller et al., 2002). Polycomb complexes bind to their target genes and create Polycomb chromatin domains that are characterised by enrichment of H2AK119ub1, H3K27me3, and the Polycomb repressive complexes themselves (Boyer et al., 2006; Bracken et al., 2006; Lee et al., 2006; Mikkelsen et al., 2007). Once formed, Polycomb chromatin domains are thought to create structural effects on chromatin that repress transcription by limiting access of transcriptional regulators to gene promoters (Eskeland et al., 2010; Francis et al., 2004, 2001; Grau et al., 2011; Isono et al., 2013; King et al., 2002; Lau et al., 2017; Schoenfelder et al., 2015; Shao et al., 1999). However, our understanding of the mechanisms that enable Polycomb complexes to recognise and bind to target sites in vivo remains rudimentary and the extent to which structural effects underpin gene repression remains unclear.
Xem thêm : Cách Backup Win 7 Cứu Hộ Máy Tính Không Cần, Khôi Phục Và Backup Windows 7A series of mechanisms, identified mostly from in vitro biochemical experiments, have been proposed to explain how individual PRC1 complexes bind to chromatin ( Blackledge et al., năm ngoái ; Schuettengruber et al., 2017 ; Simon and Kingston, 2013 ). The contributions of these mechanisms to chromatin binding in vivo have primarily been examined by ensemble fixation-based approaches like chromatin immunoprecipitation coupled with massively parallel sequencing ( ChIP-seq ). This has provided a static snapshot of PRC1 complex distribution throughout the genome and perturbation experiments have provided some information about the mechanisms that enable specific PRC1 complexes to bind chromatin. However, ChIP-seq is blind to kinetics and cannot directly compare and quantitate the chromatin binding activities of individual PRC1 complexes. As such, we currently lack a quantitative Mã Sản Phẩm to describe chromatin binding and the function of PRC1 in live cells. This represents a major conceptual gap in our understanding of the Polycomb system and its role in gene regulation .To overcome this, here we combine genome editing and single particle tracking ( SPT ) to quantify and dissect chromatin binding of PRC1 in live mouse embryonic stem cells ( ESCs ). This reveals that PRC1 is highly dynamic, with a small number of molecules displaying stable binding to chromatin. By quantifying absolute PRC1 complex numbers and integrating genomic information, we estimate maximum target site occupancy and discover that most PRC1 target genes are sparsely bound by PRC1 complexes. In dissecting the mechanisms that underpin chromatin binding by PRC1, we discover that interaction between its catalytic core and the nucleosome contributes little to chromatin binding, indicating that the observed binding behaviours of PRC1 must be defined by auxiliary subunits that are specific to individual PRC1 complexes. By systematically characterising chromatin binding and occupancy by individual PRC1 complexes, we reveal how distinct chromatin binding modalities are related to the activity and function of PRC1. Furthermore, using genetic perturbation approaches, we dissect the contribution of canonical and variant PRC1-specific targeting mechanisms to their chromatin-binding activities. Together, these new discoveries provide a quantitative understanding of chromatin binding by PRC1 complexes in vivo and have important implications for our understanding of PRC1-mediated gene repression.