Principal Supervisor: Dr. Maarten Hoogenkamp, Institute of Cancer and Genomic Sciences
Co-supervisor: Dr. Douglas Ward, Institute of Cancer and Genomic Sciences
PhD project title: The role of recruiting epigenetic regulators by the LMO2 complex in making cell fate choices
University of Registration: University of Birmingham
The three germ layers give rise to the approximate 200 different cell types found in mouse and man. The cardiovascular system, including the blood, is derived from the mesoderm. This lineage specification is determined by transcription factor complexes turning genes on and off.
Using in vitro mouse ES cell differentiation, haemangioblasts can be generated, which give rise to blood cells, endothelium, and smooth muscle cells. LMO2 is a transcription factor that does not directly bind DNA, but is a crucial component of DNA binding complexes (reviewed in ref 1). LMO2 is absent in ES cells and starts to become expressed at the haemangioblast stage, where it can form a DNA binding complex with Ldb1, Tal1 and GATA-2, regulating genes that are crucial for haematopoiesis to occur (ref 2). We are interested in understanding how specific genes are turned on or off by this LMO2 complex, thereby regulating cell fate choices. To address this we have performed initial protein pull down assays followed by mass spectrometry, using an anti-LMO2 antibody in haemangioblasts. These experiments indicated the presence of epigenetic regulators interacting with the LMO2 complex. Now we wish to address the following questions:
(1) Which epigenetic regulators associate with the LMO2 complex?
(2) Are there several distinct LMO2 protein complexes and which genes do they regulate?
(3) How do the LMO2 complexes drive cell fate decisions?
In order to answer these questions we will use in vitro ES cell differentiation to generate haemangioblasts. These will be purified on basis of surface Flk-1 (VEGFR), expression. In order to address question (1), we will perform further pull-down assays using antibodies against LMO2 and those raised against the epigenetic regulators identified. After quality control by Western blotting, these samples will be analysed by mass spectrometry. This will tell us which proteins are associated with the LMO2 complexes at this stage of development. For question (2) we will combine genome wide ChIPseq experiments for LMO2 and the epigenetic factors, which will allow us to determine where in the genome the complexes bind and, in combination with RNAseq, which genes are regulated by them. In order to address question (3), we will use an ES cell line where we can bring the expression of proteins of interest under control of a doxycycline inducible promoter. We will induce high expression of the epigenetic regulator at the haemangioblast stage and assess the effect it has on the differentiation into the different cell types.
We think that the answers we will obtain within this project will greatly increase our understanding of how the LMO2 complex regulates the lineage choices of blood cell development.
- Chambers J. and Rabbitts, T.H. (2015) LMO2 at 25 years: a paradigm of chromosomal translocation proteins. Open Biol. 5, 150062
- Stanulovic, V.S., Cauchy, P., Assi, S.A. and Hoogenkamp, M. (2017) LMO2 is required for TAL1 DNA binding activity and initiation of definitive haematopoiesis at the haemangioblast stage. Nucleic Acids Reseach in press, doi: 10.1093/nar/gkx573
BBSRC Strategic Research Priority: Molecules, Cells and Systems
Techniques that will be undertaken during the project:
- The project uses cell culture, in particular mouse ES cell differentiation, as a source of material. Cells will be isolated by magnetic or fluorescence activated cell sorting.
- Protein pull-down assays will be performed, followed by Western blotting, and mass spectrometry is used to identify proteins associated with the LMO2 complex. We will perform genome wide ChIP-seq and RNA-seq to determine which genes are regulated by these LMO2 complexes.
Contact: Dr Maarten Hoogenkamp, Institute of Cancer and Genomic Sciences