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Defining the Hierarchical Regulation of BMP Enhancers in Early Drosophila Development.

Pinheiro, Marco

[Thesis]. Manchester, UK: The University of Manchester; 2017.

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Abstract

Higher-order regulatory interactions between enhancer elements and target gene promoters have been implicated in the coordination and regulation of transcription in a spatio-temporal manner. Within development, the graded activity of enhancers controls transcriptional programs necessary to establish cell fates and tissue patterning. How enhancer promoter interactions form and dynamically change throughout development remains largely unknown. The aim of this thesis is to further characterise BMP enhancers during development. Using ChIP data, an enrichment of architectural binding proteins (ABPs) with enhancers regulated by the BMP pathway was identified. Analysis of chromatin signatures revealed a correlation with the active histone marks, H3K27ac and H3K36me3, over BMP enhancers enriched for the ABP BEAF32. BEAF32 mutants show disrupted expression of BMP target genes and altered tissue fates defined by the BMP pathway. Consequently, the role of BEAF32 genome-wide was considered, revealing interactions with factors associated with enhancers and promoters, in addition to a correlation with RNA Polymerase II (RNAPII) pausing at promoter regions. This suggests a possible role for BEAF32 in bridging enhancer promoter interactions and releasing paused RNAPII. Based on the prevalence of BEAF32 at some enhancer sites and interaction with CBP, eRNAs were identified within the Drosophila embryo, utilising available GRO-seq data and GroHMM. eRNA expression correlates to accessible enhancer states regardless of chromatin composition, with transcribed enhancers revealing interactions with active promoters, supporting correlations to transcriptional activation. Chromatin architecture of BMP targets were lastly considered using Capture-C against BMP regulated promoters, revealing multiple regulatory interactions including contacts with enhancers regulated along the Dorso-ventral (DV) axis and additional BMP promoters, with dynamic interactions between enhancers and promoters. Overall the presented data suggest that BMP promoters are dynamically regulated by distal enhancers, with a plausible role for BEAF32 in mediating enhancer promoter interactions, to co-ordinate transcription programs used to pattern dorsal tissues in the Drosophila embryo.