Periodic Reporting for period 4 - PolyDomFormFuncReg (Discovering how polycomb domains form and function in gene regulation)
Período documentado: 2020-12-01 hasta 2021-05-31
The capacity to control gene expression is fundamental to normal cellular function and organismal development. When these processes go wrong this can lead to debilitating human disease. Through studying the mechanisms by which the cell regulates gene expression it has become clear that chromatin, the template on which gene transcription occurs, can profoundly affect gene expression. In studying this aspect of gene expression, it has been shown that post-translational modifications to histones are central to gene regulation by chromatin. The Polycomb repressive proteins form a highly conserved chromatin modifying system in animals. The Polycomb system is known to repress gene expression, but how Polycomb proteins are recruited to target genes and ultimately how they create chromatin environments that are repressive to gene expression in vertebrates remains poorly understood. Furthermore, it has recently been demonstrated that disruption of the Polycomb system can lead to human diseases, including cancer. Therefore, in order to understand how the Polycomb system contributes to disease we first need to understand how it functions in normal contexts. This new knowledge will then provide a basis on which to devise therapeutic interventions to counter Polycomb system pathology in in human disease.
In the context of understanding the basic molecular mechanisms that support Polycomb mediate gene repression in vertebrates, the central aims of this ERC funded programme were to (i) Discover how the KDM2B/PRC1 complex initiates polycomb domain formation, (ii) Discover how Polycomb target sites are selected and polycomb domains formed during normal cell lineage commitment, and (iii) Discover how Polycomb domains regulate gene expression.
In addressing these fundamental aims, we discovered that variant PRC1 complexes and their capacity to deposit and regulate H2AK119ub1 are central to formation of repressive Polycomb chromatin domains. We have shown that PRC1 and H2AK119bu1 are important for how target genes are selected and repressed during cell lineage commitment. Finally, we discovered that the Polycomb system drives gene repression by counteracting RNA Polymerase II binding and transcription burst frequency. Together these discoveries provide a new mechanistic rationale for Polycomb system mediated gene repression and more generally a new understanding of how chromatin can influence gene regulation.
In the context of understanding the basic molecular mechanisms that support Polycomb mediate gene repression in vertebrates, the central aims of this ERC funded programme were to (i) Discover how the KDM2B/PRC1 complex initiates polycomb domain formation, (ii) Discover how Polycomb target sites are selected and polycomb domains formed during normal cell lineage commitment, and (iii) Discover how Polycomb domains regulate gene expression.
In addressing these fundamental aims, we discovered that variant PRC1 complexes and their capacity to deposit and regulate H2AK119ub1 are central to formation of repressive Polycomb chromatin domains. We have shown that PRC1 and H2AK119bu1 are important for how target genes are selected and repressed during cell lineage commitment. Finally, we discovered that the Polycomb system drives gene repression by counteracting RNA Polymerase II binding and transcription burst frequency. Together these discoveries provide a new mechanistic rationale for Polycomb system mediated gene repression and more generally a new understanding of how chromatin can influence gene regulation.
In progressing the aims of this project, the programme discovered in Aim (i) that the KDM2B/PRC1 protein complex is highly active at placing a modification on chromatin (H2AK119ub1) in vitro and that this activity is regulated by a protein called RYBP (Rose NR, et al, 2016). We also discovered that this complex does not rely on the histone demethylase activity of KDM2B to efficiently deposit H2AK119ub1 (Rose NR, et al, 2016 and Turberfield AH, et al, 2019). Finally, we demonstrated that deposition of H2AK119ub1 and its regulation by RYBP are central to Polycomb chromatin domain formation inside cells and for gene repression (Rose NR, et al, 2016 and Blackledge NP, et al, 2020).
In addressing Aim (ii) we mechanistically dissected Polycomb chromatin domain formation in progenitor cells as a prerequisite for understanding how this system selects and forms Polycomb chromatin domains during cell lineage commitment. We discovered a central role for variant PRC1 complexes and H2AK119ub1 in Polycomb chromatin domain formation and gene repression (Fursova NA, et al, 2019) and showed that this is regulated by a deubiquitylase enzyme called BAP1 (Fursova, et al, 2021). Importantly, this repressive activity did not rely on Polycomb chromatin domains limiting access to chromatin as previously proposed (King HW, et al, 2018). These discoveries are currently being built on to continue understanding precisely how these systems support Polycomb chromatin domain formation and gene regulation during cell lineage commitment (Sugishita, H, et al, in press 2021).
Finally, in addressing the goals of Aim (iii) we have developed new cell-based systems where we can rapidly disrupt Polycomb chromatin domains and observe how Polycomb system function and gene expression is regulated in single cells. This allowed us to show that the repressive nature of Polycomb chromatin domains relies on H2AK119ub1 but not Polycomb-dependent three-dimensional chromatin topologies (Rhodes JDP, et al, 2019 and Dobrinic P, et al, 2020). Importantly, using these approaches and single-particle tracking in live cells, we provide new evidence that Polycomb chromatin domains drive gene repression through H2AK119ub1 counteracting RNA Polymerase II binding and transcriptional burst frequency (Huseyin MK and Klose RJ, 2021 and Dobrinic P, et al, 2020).
In addressing Aim (ii) we mechanistically dissected Polycomb chromatin domain formation in progenitor cells as a prerequisite for understanding how this system selects and forms Polycomb chromatin domains during cell lineage commitment. We discovered a central role for variant PRC1 complexes and H2AK119ub1 in Polycomb chromatin domain formation and gene repression (Fursova NA, et al, 2019) and showed that this is regulated by a deubiquitylase enzyme called BAP1 (Fursova, et al, 2021). Importantly, this repressive activity did not rely on Polycomb chromatin domains limiting access to chromatin as previously proposed (King HW, et al, 2018). These discoveries are currently being built on to continue understanding precisely how these systems support Polycomb chromatin domain formation and gene regulation during cell lineage commitment (Sugishita, H, et al, in press 2021).
Finally, in addressing the goals of Aim (iii) we have developed new cell-based systems where we can rapidly disrupt Polycomb chromatin domains and observe how Polycomb system function and gene expression is regulated in single cells. This allowed us to show that the repressive nature of Polycomb chromatin domains relies on H2AK119ub1 but not Polycomb-dependent three-dimensional chromatin topologies (Rhodes JDP, et al, 2019 and Dobrinic P, et al, 2020). Importantly, using these approaches and single-particle tracking in live cells, we provide new evidence that Polycomb chromatin domains drive gene repression through H2AK119ub1 counteracting RNA Polymerase II binding and transcriptional burst frequency (Huseyin MK and Klose RJ, 2021 and Dobrinic P, et al, 2020).
Together these ground-breaking discoveries provide detailed new molecular insight into Polycomb system function and how it regulates gene expression. These conceptual advances provide a platform on which the field can build towards a complete understanding of the Polycomb system in normal development and human disease.