Transcription regulation across levels of chromatin organization

Sammanfattning: With advancements in high-throughput sequencing and high-resolution microscopy techniques, the significance of genome organization in transcription regulation is rapidly unveiling. Nonetheless, we are yet far from completely understanding this key relationship. In this thesis, I utilize some of the cutting-edge high-throughput sequencing techniques along with the power of Drosophila as a model to understand some of the molecular mechanisms active at various levels of chromatin organization that influence gene expression. At the level of DNA sequence, our analysis revealed occupancy of CBP/p300, a transcription co-activator and histone acetyltransferase at insulator regions genome wide, we also identified a novel role of CBP as a barrier to heterochromatin spreading at these insulator regions.To gain insight into transcription regulation at level of nucleosomes, we perturbed histone modifications either chemically, by inhibiting histone deacetylases (HDACs), or genetically, by creating Drosophila mutants for the H3K14 residue.  We found that the immediate transcriptional response to HDAC inhibition is only up-regulation (96 genes). Moreover, our results suggest that histone acetylation stimulates transcription by releasing promoter-proximal paused Pol II into elongation. In another study we discovered that acetylation of histone 3 lysine 14, H3K14ac, decorates some genes that lack canonical histone marks, thereby constituting a unique chromatin state. Further, Drosophila mutants expressing only H3K14R histones revealed that this residue is vital for expression of these genes, for animal survival and for developmental patterning.To further understand gene regulation during tissue specification in early embryo development, we utilized dorsoventral (DV) patterning as a model along with PRO-seq, ATAC-seq and ChIP-seq. We identified zygotically transcribed DV patterning genes that are spatially and temporally resolved. Most interestingly, the DV patterning genes are all highly paused and promoter proximal paused Pol II is released into active elongation in a tissue-specific manner. Finally our single cell RNA-seq (scRNA-seq) and HiC data in DV mutants revealed that differential regulation of gene expression occurs independently of differences in higher-order chromatin organization.Collectively, we provide evidence that transcription is being modulated at various levels of chromatin organization and our results suggest that histone modifications but not higher order chromatin organization influence transcriptional output.