Periodic Reporting for period 5 - PRCTOERC (Novel Regulatory Principles of Polycomb Repressive Complex 2)
Reporting period: 2021-12-01 to 2022-05-31
Posttranslational modifications of histone proteins have emerged as central regulators of gene expression. Through the factors that install, interpret, and erase them, histone marks control access to the genome, establishing chromatin environments that either support or counteract transcription. The histone methyltransferase Polycomb Repressive Complex 2 (PRC2) is a key mediator of gene repression all throughout development and adulthood. It is often misregulated in disease states such as cancer. The overarching goal of the Action is to advance our understanding of the mechanisms by which Polycomb Repressive Complex (PRC) 2 regulates expression of developmental genes in embryonic stem cells (ESCs) and during differentiation. Specifically, we ask how PRC2 is regulated in a context-dependent manner to achieve both terminal repression as well as poising of genes, keeping them in a plastic state for subsequent activation at the so-called bivalent domains. We hypothesise that the interplay and cooperation of PRC2 with other chromatin regulators and its ability to modify nucleosomes in both symmetric and asymmetric fashion are central to determining whether PRC2 acts to repress or to poise genes. Given its role in development and diseases, insight into the mechanisms underlying PRC2 function will inform future therapeutic approaches for regenerative medicine and for treatment of cancer and other diseases.
Work package 1 of this project examines how PRC2 cooperates with other histone modifiers and chromatin organisers at enhancers and promoters to achieve poising of developmental genes. These studies will enable us to appreciate how the pivotal PRC2 module interfaces with other players in a complex system of chromatin regulators, contributing to a much-needed integrated view of chromatin regulation. In work package 2 we assess how the generation of asymmetric nucleosomes by PRC2 is regulated by PRC2-intrinsic catalytic properties and through interactions with other chromatin modifiers, especially at bivalent domains. Work package 3 aims to generate a systems biology-informed quantitative view of PRC2 recruitment and function at bivalent domains. Together, these studies aim to determine how PRC2 achieves both full repression and transcriptional poising in a context-dependent manner, crucial functions for proper embryonic development.
Through this project, we determined how PRC2 regulates chromatin structure and uncovered novel mechanisms by which the complex controls expression of developmental genes in embryonic stem cells and during differentiation. We set up a novel system for the quantitative study of bivalent domain function and applied it to assess how bivalency achieves poising of developmental genes. We further found a novel level of regulation for the histone acetyltransferase CBP/p300, a key antagonist of PRC2-mediated gene repression. Its TAZ2 domain interacts with DNA, thereby achieving specificity towards H3K27. Together, these findings shed light on how developmental genes are repressed, poised, and activated during embryonic stem cell differentiation.
Work package 1 of this project examines how PRC2 cooperates with other histone modifiers and chromatin organisers at enhancers and promoters to achieve poising of developmental genes. These studies will enable us to appreciate how the pivotal PRC2 module interfaces with other players in a complex system of chromatin regulators, contributing to a much-needed integrated view of chromatin regulation. In work package 2 we assess how the generation of asymmetric nucleosomes by PRC2 is regulated by PRC2-intrinsic catalytic properties and through interactions with other chromatin modifiers, especially at bivalent domains. Work package 3 aims to generate a systems biology-informed quantitative view of PRC2 recruitment and function at bivalent domains. Together, these studies aim to determine how PRC2 achieves both full repression and transcriptional poising in a context-dependent manner, crucial functions for proper embryonic development.
Through this project, we determined how PRC2 regulates chromatin structure and uncovered novel mechanisms by which the complex controls expression of developmental genes in embryonic stem cells and during differentiation. We set up a novel system for the quantitative study of bivalent domain function and applied it to assess how bivalency achieves poising of developmental genes. We further found a novel level of regulation for the histone acetyltransferase CBP/p300, a key antagonist of PRC2-mediated gene repression. Its TAZ2 domain interacts with DNA, thereby achieving specificity towards H3K27. Together, these findings shed light on how developmental genes are repressed, poised, and activated during embryonic stem cell differentiation.
As summarised above, our work generated novel insights into roles of PRC2 in gene repression and poising in embryonic stem cells and during differentiation, especially in the context of the so-called bivalent domains. Two manuscripts are in preparation that will report these currently unpublished findings to the scientific community. We aim to further disseminate them via seminar and conference presentations.
Beyond PRC2, our studies surprisingly revealed a novel DNA binding activity of the TAZ2 domain of the two closely related histone acetyltransferases CBP and p300. TAZ2 binds DNA in a sequence-independent manner, promoting interaction between CBP and nucleosomes, thereby enhancing enzymatic activity and controlling substrate specificity of CBP. We found that TAZ2 is required for efficient H3K27 acetylation, while curbing activity towards off-target acetylation sites. Interfering with TAZ2-mediated DNA binding caused promiscuous acetylation by CBP in ESCs, leading to aberrant gene activation. These findings suggest that concerted action of several HATs that each target specific histone residues is essential to achieve tightly regulated gene activation. A revised manuscript reporting these findings is in preparation (Sheahan et al. bioRxiv 2020) and the findings will further be disseminated through seminar and conference presentations.
Beyond PRC2, our studies surprisingly revealed a novel DNA binding activity of the TAZ2 domain of the two closely related histone acetyltransferases CBP and p300. TAZ2 binds DNA in a sequence-independent manner, promoting interaction between CBP and nucleosomes, thereby enhancing enzymatic activity and controlling substrate specificity of CBP. We found that TAZ2 is required for efficient H3K27 acetylation, while curbing activity towards off-target acetylation sites. Interfering with TAZ2-mediated DNA binding caused promiscuous acetylation by CBP in ESCs, leading to aberrant gene activation. These findings suggest that concerted action of several HATs that each target specific histone residues is essential to achieve tightly regulated gene activation. A revised manuscript reporting these findings is in preparation (Sheahan et al. bioRxiv 2020) and the findings will further be disseminated through seminar and conference presentations.
Our novel, unpublished findings on roles and mechanisms of PRC2 in gene repression and genome organisation, as well as our work on the mechanisms by which bivalent domains poise developmental genes for expression in embryonic stem cells and in differentiation significantly advance the field beyond the current state of the art.
Our finding that the TAZ2 domain of the histone acetyltransferase CBP binds DNA and regulates substrate specificity of this important enzyme significantly advances our understanding of CBP regulation and transcription. These insights further highlight the importance of multivalent interactions, not only with histones and their posttranslational modifications, but also with DNA, in the regulation of chromatin modifiers and effector proteins.
Our finding that the TAZ2 domain of the histone acetyltransferase CBP binds DNA and regulates substrate specificity of this important enzyme significantly advances our understanding of CBP regulation and transcription. These insights further highlight the importance of multivalent interactions, not only with histones and their posttranslational modifications, but also with DNA, in the regulation of chromatin modifiers and effector proteins.