Principal Supervisor: Rui Monteiro, Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences

Co-supervisor: Professor Ferenc Mueller, Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences

PhD project title: Elucidating the role of cis- and trans- regulation of transcription in the formation of blood stem cells

University of Registration: University of Birmingham

Project outline:

Background

Haematopoietic stem cells (HSCs) arise from specialized haemogenic endothelial cells located in the floor of the embryonic dorsal aorta, the haemogenic endothelium (HE). My lab is interested in learning how lineage fate decisions lead from HE to the HSCs that give rise to all blood cells throughout the life of an organism. Understanding how endothelial and blood stem cells grow and differentiate during embryonic development is critical to inform attempts to tackle cardiovascular and haematological diseases.

The genetic programming underlying cellular differentiation is driven by trans-regulators, transcription factors (TFs) that bind to specific cis-regulatory elements in the DNA (promoters and enhancers) and regulate gene expression. These TFs act as part of larger transcriptional complexes that include epigenetic modifiers whose function is to govern chromatin accessibility and ultimately gene expression that determines cellular fate.

We have recently demonstrated that TGFb is an important player in programming HE to become haematopoietic by regulating the expression of key Notch pathway receptors. This was the first instance where TGFb signalling was directly implicated in the formation of HSCs1. This is summarized in a blog post in The Node here. Following up from this research, we aim to identify key transcriptional regulators of the cellular programing that programmes endothelial cells to give rise to HSCs, including TFs, epigenetic modifiers and cis-regulatory regions. Our approach includes classical genetic analyses, but also the use of Tol2-mediated transgenesis and genome editing tools (CRISPRs/TALENs), transcriptional profiling and epigenetic analysis. Because endothelial and HSC development are very well conserved, we use zebrafish as a model to learn how these processes function in vivo. Zebrafish has become an important resource for biomedical research, helping

to understand human disease and addressing critical questions in regenerative medicine. The lessons we learn from this model can be applied to other vertebrate systems and to human health.

Objectives

1. Uncover the genetic programming downstream of TGFb signalling that leads to HSC formation. Having established that both TGFb1 and TGFb3 inputs are required to make HSCs in the embryo (Monteiro et al, 2016), we have further identified a number of candidate transcriptional regulators that modulate the TGFb response. Here we aim to investigate the role of selected transcriptional regulators in the formation of HSCs in vivo. Functional analysis will be performed by generation of loss and gain-of function mutants using CRISPR/Cas9 genome editing tools. The consequences of these genetic manipulations will be analysed at the cellular level by live imaging in the appropriate transgenic lines, and at the molecular level by transcriptional and epigenetic profiling of specific endothelial and stem cell populations. We expect that this work will help unravel the genetic programming and cellular responses to TGFb in the formation of HSCs in vivo.

2. Genome-wide identification of cis-regulatory elements that drive lineage specification. We have started mapping potential cis-regulatory elements in endothelial cells in the zebrafish embryo by ATAC-seq, a technique that allows the identification of open regions of chromatin2. Here we will profile the in vivo activity of putative tissue-specific enhancers by using transient transgenesis, and then perform functional analysis of selected enhancers using CRISPR/Cas9 genome editing tools. Thus, we will start building the regulatory relationships between the transcriptional regulators and the cis-regulatory elements that drive haemogenic endothelial cells to become haematopoietic.

BBSRC Strategic Research Priority: Molecules, Cells and Systems

Techniques that will be undertaken during the project: