Unraveling the mechanism of DOT1A-mediated replication regulation in Trypanosoma brucei
Cell Biology
Final Report Abstract
In this project, we wanted to decipher how H3K76 methylation mediates the control of replication initiation. As mentioned above, we wanted specifically answer the following questions: 1. Which factors bind to mono- and di-methylated H3K76 in a nucleosomal context and how do they influence replication initiation? 2. Do known replication factors bind chromatin in an H3K76me1/2-dependent manner? 3. How is DOT1A recruited to specific sites in the genome to promote replication initiation? We were not able to find any H3K76me-binding factors due to substantial technical problems and we did not succeed in identifying DOT1A-interacting proteins with the suggested tandem affinity purification protocol. However, we identified a previously unknown chromatin remodeling complex, which most likely act on the same chromatin domain as DOT1A. Further characterization revealed that this SWR1 remodeling complex is important for transcription initiation and that RNAi-mediated depletion of SWR1 leads to massive chromatin condensation. Interestingly, in contrast to conserved SWR1 complexes in other eukaryotes, the trypanosome remodeler complex contains a putative SET-domain histone methyltransferase. There are a few experiments necessary before this project will be suitable for publication. We epitope-tagged the YEATS family member (Tb927.10.11690) and the YL1 nuclear protein (Tb927.11.5830) already and performed co-IP experiments to confirm the conserved (modular) structure of this remodeler complex. Mass spectrometry analysis are pending. We also generated RNAi-cell lines to deplete these components of the complex and histone H2A.Z. We will monitor growth, protein expression and the luciferase reporter in these cell lines. Furthermore, we will analyze them by TEM. Additionally, we will perform chromatin immunoprecipitation with H2A.Z in the SWR1-depleted cell line. With these experiments we want to confirm that this SWR1 complex is responsible for incorporation of histone H2A.Z into transcription start site (TSS) chromatin and that depletion of components of the SWR1 complex abolishes transcription initiation and causes massive chromatin condensation. In a new project, we want to investigate the function of the SWR1-associated putative histone methyltransferase. A potential function of the SWR1 complex in replication regulation will be difficult to investigate due to the early and severe transcription block and the massive changes in chromatin structure. However, we plan to investigate the molecular mechanisms that are responsible for the observed global chromatin condensation. The SRW1 RNAi cell line is the perfect tool to decipher the molecular machinery, which is necessary to facilitate nuclear processes that require a dynamic chromatin structure.
