Regulators of chromatin and transcription in Drosophila

Development of multicellular organisms is achieved by organized temporal and spatial patterns of gene expression leading to cell differentiation. The DNA is compacted by histone proteins into chromatin in eukaryotic cells. Gene regulation occurs at several steps, for example at the chromatin and transcriptional level. Chromatin regulators control how the DNA is utilized by altering access and recruitment possibilities 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 as a model. 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 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. Together the data indicate a more complex role for CBP in gene regulation influenced by genomic context, signaling and chromatin state than previously thought. 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 that have global elevation of H3K36me3 levels and identify mis-regulated genes in first instar larvae. The expression levels of some genes depend on the demethylase activity of dKDM4A whereas others do not, and many dKDM4A-regulated genes are devoid of H3K36me3. The data indicate that dKDM4A regulates some genes by mechanisms that do not involve H3K36 methylation. Further, overexpression of dKDM4A result in male lethality and globally reduced H3K36me3 levels, indicating impaired dosage compensation of the Xchromosome. 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. The data provide support for a Brakeless activator function that regulates transcription by interacting with 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. List of Publications I Per-Henrik Holmqvist, Ann Boija, Philge Philip, Filip Crona, Per Stenberg and Mattias Mannervik (2012) Preferential genome targeting of the CBP co-activator by Rel and Smad proteins in early Drosophila melanogaster embryos. PLoS Genet. 8(6): e1002769. II Filip Crona, Olle Dahlberg, Lina E. Lundberg, Jan Larsson and Mattias Mannervik (2013) Gene regulation by the lysine demethylase KDM4A in Drosophila. Dev. Biol. 373(2): 453-463. III Filip Crona, Bhumica Singla, Per-Henrik Holmqvist, Helin Norberg, Katrin Fantur and Mattias Mannervik (2013) Brakeless can directly activate and repress transcription in early Drosophila embry-