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Methyltransferase Ash1, histone methylation and their impact on Polycomb repression
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University.
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Antagonistic interactions between Polycomb Group (PcG) and Trithorax Group (TrxG) proteins orchestrate the expression of key developmental genes. Distinct maternally loaded repressors establish the silenced state of these genes in cells where they should not be expressed and later PcG proteins sense whether a target gene is inactive and maintain the repression throughout multiple cell divisions. PcG proteins are targeted to genes by DNA elements called Polycomb Response Elements (PREs). The proteins form two major classes of complexes, namely Polycomb Repressive Complex 1 (PRC1) and Polycomb Repressive Complex 2 (PRC2). Mechanistic details of Polycomb repression are not fully understood, however, tri-methylation of Lysine 27 of histone H3 (H3K27me3) is essential for this process. Using Drosophila cell lines deficient for either PRC1 or PRC2, I investigated the role of H3K27 methylation and the interdependence of PRC1 complexes for their recruitment to PREs. My results indicate that recruitment of PcG complexes to PREs proceed via multiple pathways and that H3K27 methylation is not needed for their targeting. However, the methylation is required to stabilize interactions of PRE-anchored PcG complexes with surrounding chromatin.

TrxG proteins prevent erroneous repression of Polycomb target genes where these genes need to be expressed. Ash1 is a TrxG protein which binds Polycomb target genes when they are transcriptionally active. It contains a SET domain which methylates Lysine 36 of histone H3 (H3K36). In vitro, histone H3 methylated at K36 is a poor substrate for H3K27 methylation by PRC2. This prompted a model where Ash1 counteracts Polycomb repression through H3K36 methylation. However, this model was never tested in vivo and does not consider several experimental observations. First, in the ash1 mutant flies the bulk H3K36me2/H3K36me3 levels remain unchanged. Second, in Drosophila, there are two other H3K36-specific histone methyltransferases, NSD and Set2, which should be capable to inhibit PRC2. Third, Ash1 contains multiple evolutionary conserved domains whose roles have not been investigated. Therefore, I asked whether H3K36 methylation is critical for Ash1 to counteract Polycomb repression in vivo and whether NSD and Set2 proteins contribute to this process. I used flies lacking endogenous histone genes and complemented them with transgenic histone genes where Lysine 36 is replaced by Arginine. In these animals, I assayed erroneous repression of HOX genes as a readout for erroneous Polycomb repression. I used the same readout in the NSD or Set2 mutant flies. I also asked if other conserved domains of Ash1 are essential for its function. In addition to SET and domain, Ash1 contains three AT hook motifs as well as BAH and PHD domains. I genetically complemented ash1 loss of function animals with transgenic Ash1 variants, in each, one domain of Ash1 is deleted. I found that Ash1 is the only H3K36-specific histone methyltransferase which counteracts Polycomb repression in Drosophila. My findings suggest that the model, where Ash1 counteracts PcG repression by inhibiting PRC2 via methylation of H3K36, has to be revised. I also showed that, in vivo, Ash1 acts as a multimer and requires SET, BAH and PHD domains to counteract Polycomb repression.

This work led to two main conclusions. First, trimethylation of H3K27 is not essential for targeting PcG proteins to PREs but acts afterwards to stabilize their interaction with the chromatin of the neighboring genes. Second, while SET domain is essential for Ash1 to oppose Polycomb repression, methylation of H3K36 does not play a central role in the process.

Place, publisher, year, edition, pages
Umeå: Umeå University , 2018. , p. 46
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1986
Keywords [en]
Trithorax Group proteins, Polycomb Group proteins, PRC1, PRC2, PRE, histone methylation, histone ubiquitylation, Ash1, SET domain, H3K36, H3K27, Drosophila
National Category
Genetics Developmental Biology Other Biological Topics
Identifiers
URN: urn:nbn:se:umu:diva-153379ISBN: 978-91-7601-932-0 (print)OAI: oai:DiVA.org:umu-153379DiVA, id: diva2:1264009
Public defence
2018-12-18, A103, Building 6A, Umeå, 13:00 (English)
Opponent
Supervisors
Funder
Knut and Alice Wallenberg FoundationSwedish Research CouncilThe Kempe FoundationsSwedish Cancer SocietyAvailable from: 2018-11-26 Created: 2018-11-19 Last updated: 2018-11-23Bibliographically approved
List of papers
1. Interdependence of PRC1 and PRC2 for recruitment to Polycomb Response Elements
Open this publication in new window or tab >>Interdependence of PRC1 and PRC2 for recruitment to Polycomb Response Elements
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2016 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, no 21, p. 10132-10149Article in journal (Refereed) Published
Abstract [en]

Polycomb Group (PcG) proteins are epigenetic repressors essential for control of development and cell differentiation. They form multiple complexes of which PRC1 and PRC2 are evolutionary conserved and obligatory for repression. The targeting of PRC1 and PRC2 is poorly understood and was proposed to be hierarchical and involve tri-methylation of histone H3 (H3K27me3) and/or monoubiquitylation of histone H2A (H2AK118ub). Here, we present a strict test of this hypothesis using the Drosophila model. We discover that neither H3K27me3 nor H2AK118ub is required for targeting PRC complexes to Polycomb Response Elements (PREs). We find that PRC1 can bind PREs in the absence of PRC2 but at many PREs PRC2 requires PRC1 to be targeted. We show that one role of H3K27me3 is to allow PcG complexes anchored at PREs to interact with surrounding chromatin. In contrast, the bulk of H2AK118ub is unrelated to PcG repression. These findings radically change our view of how PcG repression is targeted and suggest that PRC1 and PRC2 can communicate independently of histone modifications.

Keywords
Epigenetics, Gene regulation, Polycomb repression mechanisms, Polycomb Response Elements
National Category
Genetics Biochemistry and Molecular Biology
Research subject
Genetics; Molecular Biology
Identifiers
urn:nbn:se:umu:diva-128511 (URN)10.1093/nar/gkw701 (DOI)000393979400015 ()27557709 (PubMedID)
Available from: 2016-12-06 Created: 2016-12-06 Last updated: 2018-11-19Bibliographically approved
2. Hierarchical recruitment of Polycomb complexes revisited
Open this publication in new window or tab >>Hierarchical recruitment of Polycomb complexes revisited
2017 (English)In: Nucleus, ISSN 1949-1034, E-ISSN 1949-1042, Vol. 8, no 5, p. 496-505Article in journal (Refereed) Published
Abstract [en]

Polycomb Group (PcG) proteins epigenetically repress key developmental genes and thereby control alternative cell fates. PcG proteins act as complexes that can modify histones and these histone modifications play a role in transmitting the memory of the repressed state as cells divide. Here we consider mainstream models that link histone modifications to hierarchical recruitment of PcG complexes and compare them to results of a direct test of interdependence between PcG complexes for recruitment to Drosophila genes. The direct test indicates that PcG complexes do not rely on histone modifications to recognize their target genes but use them to stabilize the interactions within large chromatin domains. It also shows that multiple strategies are used to coordinate the targeting of PcG complexes to different genes, which may make the repression of these genes more or less robust.

Place, publisher, year, edition, pages
Taylor & Francis Group, 2017
Keywords
Drosophila, epigenetics, Polycomb, Polycomb targeting, Polycomb Response Elements
National Category
Developmental Biology Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-143565 (URN)10.1080/19491034.2017.1363136 (DOI)000418054400008 ()28910569 (PubMedID)
Available from: 2018-01-03 Created: 2018-01-03 Last updated: 2018-11-19Bibliographically approved
3. Does Ash1 counteract Polycomb repression by methylating H3K36?
Open this publication in new window or tab >>Does Ash1 counteract Polycomb repression by methylating H3K36?
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Polycomb repression is critical to maintain cell type specific genome expression programs in a wide range of multicellular animals. Equally important but less studied is the Trithorax group system, which safeguards Polycomb target genes from the repression in cells where they have to remain active. Based on in vitro studies it was proposed that the Trithorax group system acts via methylation of histone H3 at Lysine 4 (H3K4) and Lysine 36 (H3K36) thereby inhibiting histone methyltransferase activity of the Polycomb complexes. This hypothesis is yet to be comprehensively tested in vivo. Here we used the power of the Drosophila model to investigate how the Trithorax group protein Ash1 and the H3K36 methylation counteract Polycomb repression. We show, for the first time, that Ash1 is the only Drosophila H3K36-specific methyltransferase required to prevent excessive Polycomb repression of homeotic genes. Unexpectedly, our experiments revealed no correlation between the extent of H3K36 methylation and the resistance to Polycomb repression. Furthermore, we find that complete substitution of the zygotic histone H3 with a variant in which Lysine 36 is replaced by Arginine does not cause excessive repression of Drosophila homeotic genes. Together with earlier studies, our results suggest that the model, where the Trithorax group proteins methylate histone H3 to inhibit the histone methyltransferase activity of the Polycomb complexes, may need to be reevaluated.

Keywords
Polycomb, H3K36 methylation, HOX genes, Trithorax, Ash1, Drosophila
National Category
Genetics Developmental Biology
Research subject
Molecular Biology; Genetics
Identifiers
urn:nbn:se:umu:diva-153350 (URN)
Funder
Knut and Alice Wallenberg FoundationSwedish Research CouncilThe Kempe Foundations
Available from: 2018-11-19 Created: 2018-11-19 Last updated: 2018-11-20
4. Functional dissection of Drosophila Ash1 domains
Open this publication in new window or tab >>Functional dissection of Drosophila Ash1 domains
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(English)Manuscript (preprint) (Other academic)
Keywords
Ash1, Ash1 domains, PHD, SET, BAH, AT hooks, novel alleles
National Category
Genetics
Research subject
Molecular Biology; Genetics
Identifiers
urn:nbn:se:umu:diva-153352 (URN)
Funder
Swedish Research CouncilKnut and Alice Wallenberg FoundationThe Kempe Foundations
Available from: 2018-11-19 Created: 2018-11-19 Last updated: 2018-11-20

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