PhD Research Project: Recruitment of epigenetic regulators by the LMO2 complex for making cell fate
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 no directly bind DNA, but is a crucial component of DNA binding complexes. 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. 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) 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.
This studentship starting in October 2017 is funded by the Midlands Integrative Biosciences Training Partnership (MIBTP), a BBSRC funded studentship collaboration between Birmingham, Leicester and Warwick for UK and EU nationals (who have lived in the UK for 3+ years).
Applications are received via the online admissions system. Applicants are advised to check their ability through the MIBTP website and submit a notification form to Warwick. When applying, include the name of the supervisor (Hoogenkamp), state the funding source (MIBTP) and the project title above. More information on: http://www2.warwick.ac.uk/fac/cross_fac/mibtp/about_mibtp/
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