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Preferential Genome Targeting of the CBP Co-Activator by Rel and Smad Proteins in Early Drosophila melanogaster Embryos
Stockholm University, Faculty of Science, The Wenner-Gren Institute.
Stockholm University, Faculty of Science, The Wenner-Gren Institute.
Stockholm University, Faculty of Science, The Wenner-Gren Institute.
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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.

Place, publisher, year, edition, pages
2012. Vol. 8, no 6, e1002769
National Category
Developmental Biology
Research subject
Developmental Biology
Identifiers
URN: urn:nbn:se:su:diva-79911DOI: 10.1371/journal.pgen.1002769ISI: 000305961000033OAI: oai:DiVA.org:su-79911DiVA: diva2:551707
Note

AuthorCount:6;

Available from: 2012-09-12 Created: 2012-09-11 Last updated: 2017-12-07Bibliographically approved
In thesis
1. Regulators of chromatin and transcription in Drosophila
Open this publication in new window or tab >>Regulators of chromatin and transcription in Drosophila
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
Transcription, chromatin, co-regulators, CBP, lysine demethylase, KDM4A, Brakeless, Drosophila
National Category
Developmental Biology
Research subject
Developmental Biology
Identifiers
urn:nbn:se:su:diva-87378 (URN)978-91-7447-646-0 (ISBN)
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
2. Transcriptional and epigenetic control of gene expression in embryo development
Open this publication in new window or tab >>Transcriptional and epigenetic control of gene expression in embryo development
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

During cell specification, temporal and spatially restricted gene expression programs are set up, forming different cell types and ultimately a multicellular organism. In this thesis, we have studied the molecular mechanisms by which sequence specific transcription factors and coactivators regulate RNA polymerase II (Pol II) transcription to establish specific gene expression programs and what epigenetic patterns that follows.

We found that the transcription factor Dorsal is responsible for establishing discrete epigenetic patterns in the presumptive mesoderm, neuroectoderm and dorsal ectoderm, during early Drosophila embryo development. In addition, these different chromatin states can be linked to distinct modes of Pol II regulation. Our results provide novel insights into how gene regulatory networks form an epigenetic landscape and how their coordinated actions specify cell identity.

CBP/p300 is a widely used co-activator and histone acetyltransferase (HAT) involved in transcriptional activation. We discovered that CBP occupies the genome preferentially together with Dorsal, and has a specific role during development in coordinating the dorsal-ventral axis of the Drosophila embryo. While CBP generally correlates with gene activation we also found CBP in H3K27me3 repressed chromatin.

Previous studies have shown that CBP has an important role at transcriptional enhancers. We provide evidence that the regulatory role of CBP does not stop at enhancers, but is extended to many genomic regions. CBP binds to insulators and regulates their activity by acetylating histones to prevent spreading of H3K27me3. We further discovered that CBP has a direct regulatory role at promoters. Using a highly potent CBP inhibitor in combination with ChIP and PRO-seq we found that CBP regulates promoter proximal pausing of Pol II. CBP promotes Pol II recruitment to promoters via a direct interaction with TFIIB, and promotes transcriptional elongation by acetylating the first nucleosome. CBP is regulating Pol II activity of nearly all expressed genes, however, either recruitment or release of Pol II is the rate-limiting step affected by CBP.

Taken together, these results reveal mechanistic insights into cell specification and transcriptional control during development.

 

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, 2016. 72 p.
Keyword
Epigenetics, Cell specification, Drosophila embryo, Dorsal morphogen, CBP/p300, Gene regulation, Chromatin, Promoter proximal Pol II pausing
National Category
Developmental Biology
Research subject
Developmental Biology
Identifiers
urn:nbn:se:su:diva-134354 (URN)978-91-7649-537-7 (ISBN)978-91-7649-538-4 (ISBN)
Public defence
2016-11-25, Nordenskiöldsalen, Geovetenskapens hus, Svante Arrhenius väg 12, 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 4: Manuscript.

 

Available from: 2016-11-01 Created: 2016-10-05 Last updated: 2016-10-24Bibliographically approved

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