The interplay of transcription and chromatin regulation during embryonic development

Abstract

A fascinating aspect of multicellular development is the production of diverse cell types from an identical DNA genome, which depends on the ability to express different complements of genes across space and time. This thesis tries to address how the genome, within the chromatin structure that envelops it, is regulated to direct this complex process. The importance of chromatin regulation to the establishment and maintenance of transcriptional states is being rapidly revealed and understanding how chromatin-modifiers influence gene activity during complex developmental processes such as embryogenesis is an important goal. This thesis explores the interplay between chromatin regulation and transcriptional states during development.

The transcriptional impacts of chromatin-modifying complexes such as the p300/CBP coactivator and Polycomb group (PcG) protein repressors are well known but deciphering how these complexes interact to fine-tune the transcriptional output is not well characterized. At repressive PcG-bound chromatin in metazoans, p300/CBP often occupies but is enzymatically silent. The work in this thesis uncovers a novel role of p300/CBP that maintains PcG mediated silencing through the deployment of a subset of known functions that support RNA Polymerase II (Pol II) recruitment at silent PcG sites. This finding highlights the importance of context-dependent chromatin-modifying complex activities in establishing transcriptional states.

To explore the interplay between chromatin and transcriptional states during organismal development, we comprehensively assessed the dorsoventral (DV) patterning axis during early Drosophila embryogenesis. DV patterning is a model gene regulatory network for studying the formation of differential transcriptional programs. Here, we resolved the relationship between tissue-specific chromatin and transcriptional states using genome-wide approaches that reveal new aspects of the DV network. We find that release of paused Pol II from promoters, not recruitment, is the key molecular switch for regulatory cues to drive tissue-specific transcription. We identify an active chromatin state that correlates with the transcription of DV regulated genes and demonstrate that tissue-specific recruitment of the pause releasing P-TEFb complex is a key transcriptional effector linked to the chromatin state. We also infer the transcriptional burst dynamics of DV genes to model how chromatin regulatory signals direct the transcriptional response.

We uncover a novel function of the GAGA transcription factor (GAF) as a propagator of transcriptional states across cell divisions during embryogenesis by bookmarking regulatory sequences during mitosis. A novel ChIP-seq variant that profiles embryos specifically in mitosis reveals the genomic sites bound by GAF at this cell cycle stage. Live imaging of transcription in vivo across cell divisions reveals GAF is important for rapid postmitotic activation and is involved in transcriptional memory, whereby descendants of transcriptionally active nuclei undergo stronger or more rapid transcription.

Overall, the results reveal the complex interactions between chromatin-modifiers that govern transcriptional effects and highlight the importance of regulation across DNA regulatory sequences, the transcription factors they recruit and the surrounding chromatin environment in orchestrating complex developmental processes.

Keywords: transcription, chromatin, chromatin-modifiers, p300/CBP, Polycomb repression, promoter proximal Pol II pausing.