Funktionelle Charakterisierung von LINT und anderen dCoREST Komplexen
Zusammenfassung der Projektergebnisse
We have used a combination of biochemical and proteomic approaches to identify and characterise three protein complexes containing the corepressor dCoREST. These complexes are built on a common core consisting of dCoREST and the histone deacetylase dRpd3. Association with complex-specific subunits leads to assembly of LINT, a dLSD1/dCoREST complex or a dG9a/dCoREST complex. We have also uncovered evidence for dCoREST isoform-specific complex formation. Thus, our work has demonstrated that dCoREST forms complexes with several histone modifying activities (deacetylase, demethylase, methyltransferase) and that these associations are in part controlled at the level of dCoREST alternative splicing. We have systematically determined the genomewide binding profiles of all three dCoREST complexes. They bind to a set of sites that are only partially overlapping confirming the view that they act as independent entities. dCoREST complexes are associated with promoters supporting a role in gene regulation. Indeed, RNAi/RNA-seq analysis identified LINT as a major repressor of gene regulation in S2 cells. LINT prevents the expression of cell type-inappropriate genes in macrophage-derived S2 cells. Surprisingly, depletion of signature subunits of the dLSD1/ dCoREST and dG9a/dCoREST complexes did not produce major transcriptome changes indicating that these complexes either exert their gene regulator role only in certain situations or that they function in processes that are not directly related to transcription. The targeted depletion in the male germ line revealed that the dLSD1/dCoREST complex is essential for sperm differentiation and fertility. Indeed, this complex is important for the repression of lineage-inapproriate genes in testes. In summary, our work has identified a network of chromatin associated dCoREST repressor complexes with different histone modifying activities. Interestingly, these complexes appear to act in a cell type-specific fashion: LINT is a major regulator of transcription in macrophage-derived S2 cells whereas dLSD1/dCoREST controls gene expression in testes. Problems and open issues. We had attempted to gain structural insights into LINT complex composition and binding to nucleosomes by reconstituting a recombinant LINT complex. Despite extensive efforts we have been unable to achieve this. In particular, it was impossible to co-express more than two LINT subunits at sufficiently high levels even when using virus-based expression systems. We, therefore, abandoned this line of research. We had also planned to use the wing as a model system to study LINT's role in differentiation. This, however, did not distinguish between the functions of different dCoREST complexes. Therefore, we switched to the male germ line as a model system. This has allowed us to identify an essential and specific role for the dLSD1/ dCoREST complex in spermatogenesis. We do not yet understand the apparent cell type-specific activities of dCoREST complexes that our work has uncovered. Future research will be aimed at identifying the mechanisms underlying this division of labour.
Projektbezogene Publikationen (Auswahl)
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(2009) Monomethylation of Lysine 20 on Histone H4 Facilitates Chromatin Maturation. Mol Cell Biol, 29, 57–67
Scharf, A.N., Meier, K., Seitz, V., Kremmer, E., Brehm, A. and Imhof A.
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LINT, a novel dL(3)mbt-containing complex, represses malignant brain tumour signature genes. PLoS Genet., 2012, 8(5), e 1002676
Meier, K., Mathieu, E.L., Finkernagel, F., Reuter, L.M., Scharfe, M., Doehlemann, G., Jarek, M. and Brehm, A.
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(2014) Chromatin regulation: How complex does it get? Epigenetics, 9(11): 1485-95
Meier, K. and Brehm, A.
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(2017) tBRD-1 and tBRD-2 regulate expression of genes necessary for spermatid differentiation. Biol Open. 15;6(4): 439-448
Theofel, I., Bartkuhn, M., Boettger, T., Gärtner, S.M.K., Kreher, J., Brehm, A. and Rathke, C.
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(2019) Distinct CoREST complexes act in a cell-type-specific manner. Nucleic Acids Res.
Mačinković, I., Theofel, I., Hundertmark, T., Kovač, K., Awe, S., Lenz, J., Forné, I., Lamp, B., Nist, A., Imhof, A., Stiewe, T., Renkawitz-Pohl, R., Rathke, C. and Brehm, A.
