Recognition of post-translational histone modifications by antibodies and epigenetic reading domains

Molecular epigenetics is described as the study of heritable changes in gene function without alterations in DNA sequence. Epigenetic phenomena are regulated by three interconnected players: DNA methylation, post-translational histone modifications and non-coding RNAs, which are involved in the regulation of many cellular processes including cellular differentiation and transcription. Post-translational histone modifications either directly modulate chromatin structure or they can serve as binding signals for reading domains, which recognize the modifications and mediate most of the biological functions of these modifications. In the present PhD work, we have introduced Celluspots peptide arrays as a tool for the initial screening of putative reading domains interacting with a diverse set of post-translational histone modifications in one experiment in competition. With this tool, we studied the binding specificities of antibodies and reading domains with known as well as unknown primary targets. Furthermore, we studied differences and similarities in substrate recognition of two histone lysine methyltransferases, MLL3 and MLL1, from the same protein family. Celluspots peptide arrays were first validated with antibodies directed towards post-translational histone tail modifications. In this study, we found that most of the antibodies bound well to the modification they had been raised for, but some failed. Some antibodies showed high cross-reactivity and most of the antibodies were inhibited by additional modifications close to the primary one. Furthermore, the comparison of the specificity profiles of antibodies, which had been raised for the same modification, revealed that the binding profiles sometimes differed greatly. Therefore, we did not only validate the method with this approach, but we further introduced Celluspots peptide arrays as a good tool for the quality control of epigenetic antibodies. Additionally, we applied Celluspots peptide arrays for the investigation of the specificity of the interaction of reading domains with known substrate specificity with histone peptides. The results that we obtained with this approach agreed with literature concerning the primary targets of the reading domains, but we also obtained previously unknown information concerning the influence of secondary modifications for the binding affinity to the primary targets of these reading domains. After validation of Celluspots peptide arrays, we screened approximately 20 reading domain candidates and proceeded with the most promising candidates for further analysis. One such candidate is a human polycomb group protein, which was shown to associate with the core components of the Polycomb repressive complex 2. The Polycomb repressive complex 2 is the major histone 3 lysine 27 methyltransferase-containing complex, which tri-methylates this lysine residue. Tri-methylated histone 3 lysine 27 is a post-translational modification, which is associated with transcriptional repression of developmental genes, especially HOX genes. We found that this polycomb group protein recognizes this histone tail modification in a histone variant specific manner. This histone variant is thought to be exclusively expressed in the mammalian testis and we propose that this polycomb group protein is involved in targeting the complex to this histone variant tri-methylated at lysine 27 in the human testis. The histone lysine methyltransferases of the mixed lineage leukemia (MLL) protein family mono-, di- and tri-methylate histone 3 lysine 4. We studied the substrate specificity for two members of this family, MLL3 and MLL1, and revealed that the preferred substrate sequences are different. With these differences in substrate recognition we searched for non-histone targets and found similar, but also different non-histone targets at the peptide level. This is the first time that differences in substrate recognition was observed for MLL methyltransferases, which do not