Using genome-wide data to model signalling-responsive gene regulatory mechanisms in blood development

Maytum, Alexander Iain (2023). Using genome-wide data to model signalling-responsive gene regulatory mechanisms in blood development. University of Birmingham. Ph.D.

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Abstract

The control of gene expression driving developmental haematopoiesis crucially depends on distal cis-regulatory elements such as enhancers which directly interact with promoters in the nucleus. However, no global experiments have been conducted which identify the cell type and cell stage-specific activity of enhancers in a chromatin context. It is through these elements that lineage specific transcription factors orchestrate cell fate decisions and direct haematopoietic lineage development emerging from the mesoderm. The roles of transcriptional regulators are beginning to be understood, however, it is still unclear how the myriad of extracellular signals modulate their activity. In this work, we report a global method which enables the identification of thousands tissue-specifically active cisregulatory elements able to stimulate a minimal promoter in cells representing five stages of haematopoietic specification derived from embryonic stem cells. Using serum-free differentiation culture, we demonstrate that our method can identify signalling-responsive enhancer elements and we highlight that it can be adapted to any embryonic stem cell differentiation system generating different cell types. We demonstrate that thousands of cell stage-specific sets of cis-elements are responsive to cytokine signals terminating at signalling-responsive transcription factors. Integrating these data with chromatin accessibility and single cell RNA-Seq data provided important new insights into the regulatory dynamics of the gene regulatory network transitions driving haematopoiesis. Our work identified the cytokine signalling-responsive transcription factors mediating responsiveness of enhancers at each developmental stage. We validated enhancers for Sparc, Pxn, Hspg2, Cdh5, Dlk1 and Mrpl15 as being signalling responsive to VEGF. We found that the cytokine VEGF is a crucial factor that regulates the balance between endothelial and haematopoietic development and our scRNA-seq analysis revealed that in the presence of VEGF Sox17 fails to be downregulated and Runx1 fails to be upregulated in the haemogenic endothelium and progenitor cells. For two Runx1 enhancers (the +23kb and +3.7kb) we studied the transcription factors motifs mediating the responsiveness of the enhancers to VEGF by mutation of these sites. Taken together, our work generated an important novel resource for future studies of haematopoietic differentiation and provides insights into how and where in the genome extrinsic signals program the cell type-specific chromatin landscape driving this process.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):