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Brakeless can directly activate and repress trancription in early Drosophila embryos
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. (Mannervik)
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. (Mannervik)
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. (Mannervik)
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. (Mannervik)
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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: urn:nbn:se:su:diva-87351OAI: oai:DiVA.org:su-87351DiVA: diva2:602945
Funder
Swedish Cancer SocietySwedish Research Council
Available from: 2013-02-04 Created: 2013-02-04 Last updated: 2013-02-05Bibliographically 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

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