In this project we identify and characterize transcriptional coregulators. Coregulators are proteins that themselves do not bind to DNA, but that facilitate communication between sequence-specific transcription factors and the basal RNA polymerase machinery. One function of coregulators is to modify the structure of chromatin, by acetylating (coactivators) or deacetylating (corepressors) histones.

 
Transcriptional coregulators associate with DNA bound transcription factors
Transcriptional coregulators associate with DNA bound transcription factors (activators or repressors) and promote or prevent binding of RNA polymerase II to the transcription start site.
 

 

A number of histone acetyltransferases (HATs) and deacetylases (HDACs) are conserved between organisms as diverse as yeast and human. We are characterizing the role of histone modifying proteins in vivo, using the Drosophila embryo as our “test tube”. We have also isolated novel gene regulators from a genetic screen for mutants that disrupt embryonic pattern formation. Using genetic tools, transgenic embryos, and whole-mount in situ hybridizations in combination with in vitro experiments, we can obtain an understanding of the molecular functions of these proteins in development. Given that coregulators are deregulated in some cancers, these studies will also contribute to an understanding of how altered functions of coregulators can cause uncontrolled cell proliferation and cancer.

Many coregulators modify the chromatin structure
Many coregulators modify the chromatin structure by for example acetylation or deacetylation of lysine residues in histones. In most cases, histone acetylation facilitates transcription, whereas histone deacetylation inhibits transcription.
 

   

The CBP coactivator

The p300 and CBP co-activators are histone acetyltransferases and central regulators of transcription in metazoans.. We have shown that CBP controls signaling by the TGF-ß molecule Dpp in the Drosophila embryo by multiple mechanisms (Lilja et al. 2003). An in vivo structure-function analysis of CBP showed that site-specifically altered CBP cDNA transgenes could rescue the gene expression defects observed in CBP mutant early embryos (Lilja et al. 2007). We found that the acetyltransferase activity of CBP is necessary for some processes during development, but is dispensable for expression of genes in the Dpp pathway in early embryos. The genomic occupancy of p300/CBP detected by ChIP-seq experiments can be used to identify transcriptional enhancers (e.g. Negre et al. 2011). However, our studies in Drosophila embryos suggest that there is a preference for some transcription factors in directing CBP to the genome (Holmqvist et al. 2012). Although p300/CBP occupancy in general correlates with gene activation, they can also be found at silent genomic regions, which does not result in histone acetylation. Polycomb-mediated H3K27me3 is associated with repression, but does not preclude p300/CBP binding. An antagonism between H3K27ac and H3K27me3 indicates that p300/CBP may be involved in switching between repressed and active chromatin states (Holmqvist and Mannervik 2013). We have performed ChIP-seq studies of CBP in Drosophila S2 cells (Philip et al. 2015), and are currently studying a link between promoter-proximal RNA polymerase pausing and genomic occupancy of CBP.

  

Genes that are silenced
A) Genes that are silenced by Polycomb-mediated H3K27me3 (K27me) can be occupied by p300/CBP. Association of p300/CBP with silent or poised transcriptional enhancers (with or without H3K27me3) does not result in histone acetylation. B) At active genes, p300/CBP can acetylate histones on H3K27 (K27ac) and H3K18 (not shown), acetylate transcription factors (Ac), function as a scaffolds for recruiting other proteins, or help establish a preinitiation complex by interactions with TFIIB and hypophosphorylated RNA polymerase II.

  

An Ebi-HDAC3 corepressor complex

The Snail repressor protein is required for mesoderm formation through its downregulation of neuroectoderm-specific genes. We found that in Drosophila embryos lacking the maternal contribution of the Ebi protein, Snail target genes are de-repressed leading to mis-expression of neuroectoderm specific genes in the presumptive mesoderm. The Ebi protein is the homolog of mammalian TBL1 that associates with HDAC3 and the co-repressors SMRT and NcoR. We have shown that the first 40 amino acids in Snail bind to Ebi in vitro. Interestingly, the first 40 amino acids contain a motif conserved in all insect Snail-related proteins. Importantly, the Ebi interaction region constitutes a potent repression domain. When fused to a heterologous DNA-binding domain, Snail 1-40 represses transcription in Drosophila tissue-culture cells and in transgenic Drosophila embryos. Ectopic expression of full-length Snail and a mutant Snail lacking the Ebi interaction domain in transgenic embryos demonstrated that the Ebi interaction domain is essential for Snail function in vivo. Together, these results show that Ebi is a corepressor required for Snail activity.

We found that Ebi associates with HDAC3 in tissue-culture cells, and that knock down of HDAC3 by RNAi or inhibition of HDAC activity by drug treatment impairs Snail-meditated repression. This shows that histone deacetylation is part of the mechanism by which Snail represses transcription (Qi et al. 2008).

Brakeless, Atrophin and neurodegeneration

To find novel factors required for gene regulation in the Drosophila embryo, we have analyzed mutants isolated in a screen for maternal factors required for embryo patterning performed in the Nüsslein-Volhard laboratory in Tübingen (Luschnig et al. 2004). In my laboratory, we studied 15 mutants that cause segmentation defects and examined gene expression patterns in mutant embryos. One mutant disrupts the brakeless gene. Brakeless is a nuclear protein of previously unknown function. We found that Brakeless is a novel co-repressor required for function of the Tailless repressor.

 

Tailless and some other nuclear receptors also interact with the co-repressor Atrophin, the homolog of Atrophin-1 that causes the human neurodegenerative disease dentatorubral-pallidoluysian atrophy (DRPLA). We could show that Brakeless and Atrophin interact in vitro, and propose that they act together as a co-repressor complex in many developmental contexts. (Haecker et al. 2007). We found that Brakeless can activate some genes and repress others, suggesting that it may switch between a co-activator and co-repressor function (Crona et al. 2015). To achieve a genome-wide view of Atrophin function, and address how the activities of other transcriptional regulators are integrated with those of Atrophin to control transcriptional output, we have performed ChIP-seq experiments in Drosophila S2 cells. This shows that Atrophin, Brakeless, and CBP function together in gene regulation genome-wide.

 
Three co-regulators Atrophin (Atro), Brakeless (Bks) and CBP interact on chromatin
Our work addresses how the three co-regulators Atrophin (Atro), Brakeless (Bks) and CBP interact on chromatin to activate and repress transcription.
 

 

To investigate the role of these co-regulators in the transcriptional misregulation that causes the neurodegenerative disorder DRPLA, we will perform ChIP-seq in human cells and use a fly model of DRPLA.

 

References

Philip P, Boija A, Vaid R, Churcher AM, Meyers DJ, Cole PA, Mannervik M, Stenberg P. (2015) CBP binding outside of promoters and enhancers in Drosophila melanogaster. Epigenetics & Chromatin. 8:48.

Crona F, Holmqvist PH, Tang M, Singla B, Vakifahmetoglu-Norberg H, Fantur K, Mannervik M. (2015) The Brakeless co-regulator can directly activate and repress transcription in early Drosophila embryos. Dev Biol. 407(1):173-81.

Holmqvist, P.H., & Mannervik, M. (2013). Genomic occupancy of the transcriptional co-activators p300 and CBP. Transcription. 4(1):18-23

Holmqvist, P.H., Boija, A., Philip, P., Crona, F., Stenberg, P., & Mannervik, M.

(2012). Preferential genome targeting of the CBP co-activator by Rel and Smad proteins in early Drosophila melanogaster embryos. PLoS Genetics. 8(6):e1002769

Nègre, N., Brown, C.D., Ma, L., Bristow, C.A., Miller, S.W., Wagner, U., Kheradpour, P., Eaton, M.L., Loriaux, P., Sealfon, R., Li, Z., Ishii, H., Spokony, R.F., Chen, J., Hwang, L., Cheng, C., Auburn, R.P., Davis, M.B., Domanus, M., Shah, P.K., Morrison, C.A., Zieba, J., Suchy, S., Senderowicz, L., Victorsen, A., Bild, N.A., Grundstad, A.J., Hanley, D., MacAlpine, D.M., Mannervik, M., Venken, K., Bellen, H., White, R., Gerstein, M., Russell, S., Grossman, R.L., Ren, B., Posakony, J.W., Kellis, M., & White, K.P. (2011). A cis-regulatory map of the Drosophila genome. Nature. 471(7339):527-31

Dai, Q., Bergman, M., Aihara, H., Nibu, Y., & Mannervik, M. (2008). Drosophila Ebi mediates Snail-dependent transcriptional repression through HDAC3-induced histone deacetylation. EMBO J. 27(6):898-909

Haecker, A., Qi, D., Lilja, T., Moussian, B., Andrioli, L.P., Luschnig, S., & Mannervik, M. (2007). Drosophila Brakeless Interacts with Atrophin and is Required for Tailless-Mediated Transcriptional Repression in Early Embryos. PLoS Biology. 5, 1298-1308

Lilja, T., Aihara, H., Stabell, M., Nibu, Y., & Mannervik, M. (2007). The acetyltransferase activity of Drosophila CBP is dispensable for regulation of the Dpp pathway in the early embryo. Dev Biol. 305(2):650-8

Luschnig, S., et al. (2004). An F1 genetic screen for maternal-effect mutations affecting embryonic pattern formation in Drosophila melanogaster. Genetics. 167(1): 325-42

Lilja, T., Qi, D., Stabell, M., & Mannervik, M. (2003). The CBP coactivator functions both upstream and downstream of Dpp/Screw signaling in the early Drosophila embryo. Dev Biol. 262(2):294-302