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Transcriptional and epigenetic control of gene expression in embryo development
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
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 [en]
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: urn:nbn:se:su:diva-134354ISBN: 978-91-7649-537-7 (print)ISBN: 978-91-7649-538-4 (print)OAI: oai:DiVA.org:su-134354DiVA: diva2:1033053
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
List of papers
1. Initiation of diverse epigenetic states during nuclear programming of the Drosophila body plan
Open this publication in new window or tab >>Initiation of diverse epigenetic states during nuclear programming of the Drosophila body plan
2016 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 113, no 31, 8735-8740 p.Article in journal (Refereed) Published
Abstract [en]

Epigenetic patterns of histone modifications contribute to the maintenance of tissue-specific gene expression. Here, we show that such modifications also accompany the specification of cell identities by the NF-kappa B transcription factor Dorsal in the precellular Drosophila embryo. We provide evidence that the maternal pioneer factor, Zelda, is responsible for establishing poised RNA polymerase at Dorsal target genes before Dorsal-mediated zygotic activation. At the onset of cell specification, Dorsal recruits the CBP/p300 coactivator to the regulatory regions of defined target genes in the presumptive neuroectoderm, resulting in their histone acetylation and transcriptional activation. These genes are inactive in the mesoderm due to transcriptional quenching by the Snail repressor, which precludes recruitment of CBP and prevents histone acetylation. By contrast, inactivation of the same enhancers in the dorsal ectoderm is associated with Polycomb-repressed H3K27me3 chromatin. Thus, the Dorsal morphogen gradient produces three distinct histone signatures including two modes of transcriptional repression, active repression (hypoacetylation), and inactivity (H3K27me3). Whereas histone hypoacetylation is associated with a poised polymerase, H3K27me3 displaces polymerase from chromatin. Our results link different modes of RNA polymerase regulation to separate epigenetic patterns and demonstrate that developmental determinants orchestrate differential chromatin states, providing new insights into the link between epigenetics and developmental patterning.

Keyword
epigenetics, cell specification, Drosophila embryo, Dorsal morphogen, Zelda
National Category
Biological Sciences
Research subject
Developmental Biology
Identifiers
urn:nbn:se:su:diva-133375 (URN)10.1073/pnas.1516450113 (DOI)000380586600057 ()27439862 (PubMedID)
Available from: 2016-09-09 Created: 2016-09-06 Last updated: 2016-10-06Bibliographically approved
2. 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: 2016-10-06Bibliographically approved
3. CBP binding outside of promoters and enhancers in Drosophila melanogaster
Open this publication in new window or tab >>CBP binding outside of promoters and enhancers in Drosophila melanogaster
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2015 (English)In: Epigenetics & Chromatin, ISSN 1756-8935, E-ISSN 1756-8935, Vol. 8, 48Article in journal (Refereed) Published
Abstract [en]

Background: CREB-binding protein (CBP, also known as nejire) is a transcriptional co-activator that is conserved in metazoans. CBP plays an important role in embryonic development and cell differentiation and mutations in CBP can lead to various diseases in humans. In addition, CBP and the related p300 protein have successfully been used to predict enhancers in both humans and flies when they occur with monomethylation of histone H3 on lysine 4 (H3K4me1). Results: Here, we compare CBP chromatin immunoprecipitation sequencing data from Drosophila S2 cells with modENCODE data and show that CBP is bound at genomic sites with a wide range of functions. As expected, we find that CBP is bound at active promoters and enhancers. In addition, we find that the strongest CBP sites in the genome are found at Polycomb response elements embedded in histone H3 lysine 27 trimethylated (H3K27me3) chromatin, where they correlate with binding of the Pho repressive complex. Interestingly, we find that CBP also binds to most insulators in the genome. At a subset of these, CBP may regulate insulating activity, measured as the ability to prevent repressive H3K27 methylation from spreading into adjacent chromatin. Conclusions: We conclude that CBP could be involved in a much wider range of functions than has previously been appreciated, including Polycomb repression and insulator activity. In addition, we discuss the possibility that a common role for CBP at all functional elements may be to regulate interactions between distant chromosomal regions and speculate that CBP is controlling higher order chromatin organization.

Keyword
CBP/p300, Drosophila melanogaster, Chromatin structure, Gene regulation, Insulators, Polycomb response elements
National Category
Biological Sciences
Research subject
Developmental Biology
Identifiers
urn:nbn:se:su:diva-124741 (URN)10.1186/s13072-015-0042-4 (DOI)000365564300001 ()
Available from: 2016-01-09 Created: 2016-01-04 Last updated: 2016-10-06Bibliographically approved
4. CBP regulates promoter-proximal RNA polymerase II
Open this publication in new window or tab >>CBP regulates promoter-proximal RNA polymerase II
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(English)Manuscript (preprint) (Other academic)
National Category
Developmental Biology
Research subject
Developmental Biology
Identifiers
urn:nbn:se:su:diva-134352 (URN)
Available from: 2016-10-05 Created: 2016-10-05 Last updated: 2016-10-06Bibliographically approved

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