Role of CDYL in translating histone modification patterns for chromatin organization and regulation
Zusammenfassung der Projektergebnisse
While several studies have associated the CDY/L (chromodomain on the Y chromosome / like) protein family with cellular development and differentiation, the molecular working mechanisms of these factors are still obscure. In this project we focused on CDYL1 as it is the major CDY/L protein in human cells. Our work identified three splicing variants of this factor with differential expression in diverse tissues and cell types. The resulting proteins have different properties in interaction with the heterochromatic histone modification H3K9me3. Whereas histone peptides have been the standard in analyzing the binding properties of putative effector proteins of histone modifications, we wanted to study CDYL1 chromatin binding in native context. We therefore generated ‘designer’ oligonucleosomes carrying H3K9me3 based on native chemical ligation. While we confirmed that binding of the N-terminal chromodomain to H3K9me3 is a major determinant of CDYL1 chromatin interaction, our studies pointed also to an important role of the C-terminal ECH-like (enoyl-coenzyme A hydratase) region. We failed to reproduce earlier findings on histone acetyltransferase activity of this domain. Also molecular modeling approaches on the basis of available structures of this class of enzymes indicated an unusual loop blocking the CoA binding site. Instead, we found a major role of the ECH domain in trimerization of CDYL1. Multimerization is not only essential for stable CDYL1/H3K9me3 chromatin binding but is also required to induce CDYL1/oligonucleosome condensation. Biochemical studies identified PRMT5 as novel interaction partner of CDYL1b. We could show that this arginine methyltransferase modifies a single residue (R80) within the linker region of CDYL1 that connects the chromo and ECH-like domains. CDYL1 R80me is not incorporated into the CoREST complex indicating that this post-translational modification regulates the function of the protein. Lastly, experiments in Xenopus laevis point to an important role of CDYL1 in early embryonic development. Up or down regulation of xlCDYL1 causes severe abnormalities of the dorsal-ventral axis. Further studies will clarify the role of H3K9me3 interaction, trimerization and post-translational modification of the protein in cellular development and differentiation. Despite extensive efforts it was so far impossible to generate antibodies specifically recognizing CDYL1 for immunoprecipitation analysis. Therefore, our attempts to characterize the biological role of the factor were limited to surrogate cellular systems of expressing tagged fusion proteins. In lack of specific antibodies we turned to damID to obtain insights into the genome wide distribution of CDYL1. Despite several technical difficulties the preliminary results of this approach appear promising. Since the method allows the comparison of wild type and mutant factors, we are confident to obtain further insights into the role of CDYL multimerization in (hetero-)chromatin association. We wanted to generate mouse conditional knock out strains to investigate the biological role of CDYL1. Unfortunately, the available gene trap failed to interfere with CDYL1 pre-mRNA splicing as predicted. Since the new technology of CRISPR/Cas gene engineering has become highly proficient in recent years, we have restarted our efforts of genetic analysis of CDYL1 using this approach.
Projektbezogene Publikationen (Auswahl)
- (2009) Multimerization and H3K9me3 binding is required for CDYL1b heterochromatin association. J. Biol. Chem. 284:35049-35059
Franz, H., Mosch, K., Soeroes, S., Urlaub, H. and Fischle, W.
- (2010) Epigenetic markers and their crosstalk. Essays Biochem. 48: 45-61
Winter, S., and Fischle, W.
- (2011) HP1 recruits activity-dependent neuroprotective protein to H3K9me3 marked pericentromeric heterochromatin for silencing of major satellite repeats. PLoS One 6:e15894
Mosch, K., Franz, H., Soeroes, S., Singh, P.B. and Fischle W.
