Functional characterisation of variant dMi-2 nucleosome remodeling complexes in epigenome regulation
Biochemistry
Developmental Biology
Cell Biology
Final Report Abstract
The ATP-dependent nucleosome remodeler CHD4 plays important roles in differentiation, DNA repair and other cellular processes. The molecular mechanisms of how ATP-dependent remodelers in general and CHD4 in particular contribute to these processes is not well understood. The Drosophila CHD4 homolog, dMi-2, exists in two biochemically separable protein complexes, dNuRD and dMec. Accordingly, we had hypothesized that dNuRD and dMec bind to different genomic regions and regulate different sets of genes. We have determined the genomewide binding sites of both complexes using high throughput sequencing approaches (ChIP-seq). We have found that both complexes occupy the same genomic sites. This surprising result does not support our hypothesis. Rather it suggests that dNuRD and dMec associate on chromatin and possibly form a common macromolecular assembly when bound to their target regions. dMi-2 and the transcriptional regulator U-shaped (Ush) cooperate to regulate gene expression during hematopoiesis. In order to elucidate the mechanism of dMi-2/Ush-mediated chromatin regulation we have established an inducible protein degradation system in Drosophila S2 cells (deGradFP). This allows us to deplete deplete Ush within a few hours after induction and follow the resulting changes to chromatin structure and gene expression in a time resolved manner. In summary, our studies have resulted in a revised model of the dNuRD/dMec interplay and have set the stage for a kinetic analysis of chromatin and gene regulation.
