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Regulators of chromatin and transcription in Drosophila
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Development of multicellular organisms is achieved by organized temporal and spatial patterns of gene expression leading to cell differentiation. Chromatin regulators control how the DNA is utilized by altering access of proteins to DNA and thereby function as co-factors in transcription. Gene regulation also involves co-factors interacting with transcription factors at regulatory sequences of DNA. In this thesis, we have studied the in vivo role of three co-factors, CBP, dKDM4A and Brakeless, in regulating chromatin and transcription using Drosophila melanogaster. The CREB binding protein (CBP) belongs to histone acetyl transferases (HATs) and facilitates gene activation by many transcription factors. Our work has demonstrated that CBP occupies the genome preferentially together with Rel and Smad proteins controlling dorsal-ventral patterning in the Drosophila embryo. CBP occupancy generally correlates with gene expression but also occurs at silent genes without resulting in histone acetylation. KDM4A belongs to a family of JmjC domain proteins and demethylates H3K36me3, a histone modification enriched in the 3’end of active genes. We generated dKDM4A mutants with a global elevation of H3K36me3 levels and identify mis-regulated genes in first instar larvae. The data indicate that dKDM4A regulates some genes by mechanisms that do not involve H3K36 methylation. Further, over-expression of dKDM4A result in male lethality and globally reduced H3K36me3 levels, indicating impaired dosage compensation of the X-chromosome. Brakeless is a conserved co-factor participating in several important processes during development. We generated mutant brakeless embryos and identify direct genomic targets of Brakeless. To our surprise, Brakeless behaves as a direct activator for some genes but repressor in other cases. We also identify an interaction of Brakeless with the Mediator subunit Med19. In summary, these studies reveal unexpected roles for co-regulators in Drosophila development. The HAT CBP can bind silent genes without leading to histone acetylation. Brakeless has the ability to function both as a direct activator and repressor of transcription.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University , 2013. , 65 p.
Keyword [en]
Transcription, chromatin, co-regulators, CBP, lysine demethylase, KDM4A, Brakeless, Drosophila
National Category
Developmental Biology
Research subject
Developmental Biology
Identifiers
URN: urn:nbn:se:su:diva-87378ISBN: 978-91-7447-646-0 (print)OAI: oai:DiVA.org:su-87378DiVA: diva2:603052
Public defence
2013-03-08, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.

Available from: 2013-02-14 Created: 2013-02-04 Last updated: 2013-03-20Bibliographically approved
List of papers
1. Preferential Genome Targeting of the CBP Co-Activator by Rel and Smad Proteins in Early Drosophila melanogaster Embryos
Open this publication in new window or tab >>Preferential Genome Targeting of the CBP Co-Activator by Rel and Smad Proteins in Early Drosophila melanogaster Embryos
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2012 (English)In: PLOS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 8, no 6, e1002769Article in journal (Refereed) Published
Abstract [en]

CBP and the related p300 protein are widely used transcriptional co-activators in metazoans that interact with multiple transcription factors. Whether CBP/p300 occupies the genome equally with all factors or preferentially binds together with some factors is not known. We therefore compared Drosophila melanogaster CBP (nejire) ChIP-seq peaks with regions bound by 40 different transcription factors in early embryos, and we found high co-occupancy with the Rel-family protein Dorsal. Dorsal is required for CBP occupancy in the embryo, but only at regions where few other factors are present. CBP peaks in mutant embryos lacking nuclear Dorsal are best correlated with TGF-beta/Dpp-signaling and Smad-protein binding. Differences in CBP occupancy in mutant embryos reflect gene expression changes genome-wide, but CBP also occupies some non-expressed genes. The presence of CBP at silent genes does not result in histone acetylation. We find that Polycomb-repressed H3K27me3 chromatin does not preclude CBP binding, but restricts histone acetylation at CBP-bound genomic sites. We conclude that CBP occupancy in Drosophila embryos preferentially overlaps factors controlling dorsoventral patterning and that CBP binds silent genes without causing histone hyperacetylation.

National Category
Developmental Biology
Research subject
Developmental Biology
Identifiers
urn:nbn:se:su:diva-79911 (URN)10.1371/journal.pgen.1002769 (DOI)000305961000033 ()
Note

AuthorCount:6;

Available from: 2012-09-12 Created: 2012-09-11 Last updated: 2017-12-07Bibliographically approved
2. Gene regulation by the lysine demethylase KDM4A in Drosophila
Open this publication in new window or tab >>Gene regulation by the lysine demethylase KDM4A in Drosophila
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2013 (English)In: Developmental Biology, ISSN 0012-1606, E-ISSN 1095-564X, Vol. 373, no 2, 453-463 p.Article in journal (Refereed) Published
Abstract [en]

Lysine methylation of histones is associated with both transcriptionally active chromatin and with silent chromatin, depending on what residue is modified. Histone methyltransferases and demethylases ensure that histone methylations are dynamic and can vary depending on cell cycle- or developmental stage. KDM4A demethylates H3K36me3, a modification enriched in the 3' end of active genes. The genomic targets and the role of KDM4 proteins in development remain largely unknown. We therefore generated KDM4A mutant Drosophila, and identified 99 mis-regulated genes in first instar larvae. Around half of these genes were down-regulated and the other half up-regulated in dKDM4A mutants. Although heterochromatin protein 1a (HP1a) can stimulate dKDM4A demethylase activity in vitro, we find that they antagonize each other in control of dKDM4A-regulated genes. Appropriate expression levels for some dKDM4A-regulated genes rely on the demethylase activity of dKDM4A, whereas others do not. Surprisingly, although highly expressed, many demethylase-dependent and independent genes are devoid of H3K36me3 in wild-type as well as in dKDM4A mutant larvae, suggesting that some of the most strongly affected genes in dKDM4A mutant animals are not regulated by H3K36 methylation. By contrast, dKDM4A over-expression results in a global decrease in H3K36me3 levels and male lethality, which might be caused by impaired dosage compensation. Our results show that a modest increase in global H3K36me3 levels is compatible with viability, fertility, and the expression of most genes, whereas decreased H3K36me3 levels are detrimental in males.

Keyword
Chromatin, Histone methylation, Gene regulation, Drosophila
National Category
Developmental Biology
Research subject
Developmental Biology
Identifiers
urn:nbn:se:su:diva-85852 (URN)10.1016/j.ydbio.2012.11.011 (DOI)000313381200020 ()23195220 (PubMedID)
Available from: 2013-01-09 Created: 2013-01-09 Last updated: 2017-12-06Bibliographically approved
3. Brakeless can directly activate and repress trancription in early Drosophila embryos
Open this publication in new window or tab >>Brakeless can directly activate and repress trancription in early Drosophila embryos
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The Brakeless protein performs many important functions during development in Drosophila, but how it controls gene expression is not understood. We previously showed that Brakeless can function as a transcriptional co-repressor. In this work, we perform transcriptional profiling of brakeless mutant embryos. Unexpectedly, the majority of target genes are down-regulated in brakeless mutants. We demonstrate that genomic regions in close proximity to some of the affected genes are occupied by Brakeless, that over-expression of Brakeless causes a reciprocal effect on expression of these genes, and that the activator function of Brakeless is intact when an activation domain is fused to Brakeless. By contrast, Brakeless repressor function is neutralized by the activation domain. Together, this shows that Brakeless can both repress and activate gene expression. To identify protein interactions that result in gene repression or activation, a yeast two-hybrid screen was performed. We find that the Mediator complex subunit Med19 interacts with an evolutionarily conserved part of Brakeless. Interestingly, down-regulated but not up-regulated Brakeless target genes are also affected in Med19-depleted embryos. Our data provide support for a Brakeless activator function that regulates transcription by interacting with Med19.

National Category
Developmental Biology
Research subject
Developmental Biology
Identifiers
urn:nbn:se:su:diva-87351 (URN)
Funder
Swedish Cancer SocietySwedish Research Council
Available from: 2013-02-04 Created: 2013-02-04 Last updated: 2013-02-05Bibliographically approved

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