The transcriptional programme controlled by RUNX1 during blood development

Abstract

The RUNX1 transcription factor is indispensable for the establishment of blooddevelopment. RUNX1 mediates the generation of blood progenitors from specialisedendothelial cells with haemogenic potential (termed haemogenic endothelium (HE)),through a process known as the endothelial-to-haematopoietic transition (EHT). Theultimate consequence of EHT is the activation of the haematopoietic programme andthe concomitant down-regulation of endothelial genes. However, due to the rare andtransient nature of the HE, little is known about the initial role of RUNX1 within thispopulation. In this thesis, I used mouse embryonic stem cells as a model to explorehow RUNX1 directs endothelial cells into the haematopoietic fate. To unravel themolecular mechanisms regulated by RUNX1 at the onset of EHT, I employed a highlysensitive DamID methodology to profile RUNX1-binding targets in HE. DamID is amethylation-based tagging technique where the E.coli Dam methyltransferase is fusedto DNA-binding proteins and stably marks the DNA in close proximity of theprotein's binding site. To detect genome-wide RUNX1 targets in HE, I coupledDamID with high-throughput sequencing and supported the development of asequencing analysis pipeline specific for DamID-seq data. I then complemented theRUNX1-binding profile with transcriptional targets regulated by RUNX1 at the samestage. Integration of the binding and transcriptome data in HE revealed an unexpectedearly role for RUNX1 in positively regulating cell adhesion and cell migration, whichcorrelates with the formation of HE clusters in vitro prior to blood emergence.Interestingly, I found that RUNX1 regulates components of the integrin-signallingpathway, which is known to mediate these functions. RUNX1 is also required for themaintenance of the adult haematopoietic system. To better understand the role ofRUNX1 in adult haematopoiesis, I utilised an in vivo DamID system to identifyRUNX1-binding sites in various haematopoietic populations. Despite the low numberof cells in most of these populations, the DamID technique successfully determinedtheir RUNX1-binding signature. Correlation of the RUNX1-binding targets withexpression data revealed again that RUNX1 does not exclusively regulatehaematopoietic genes. Overall, my findings suggest a novel role for RUNX1 inpositively regulating cell adhesion- and migration- associated genes, which precedesits more established function in the up-regulation of the haematopoietic programmeand the down-regulation of endothelial genes.

Date of Award	20 Feb 2015
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Georges Lacaud (Main Supervisor)