Analysis of Histone Deacetylase 1 and 2 function
My laboratory studies the control of gene expression by histone deacetylases (HDAC) 1 and 2 using novel conditional knock-out (cKO) embryonic stem (ES) cell and mouse models. HDAC1 and HDAC2 are chromatin modifying enzymes found in all cells and whose function is generally associated with gene repression via deacetylation of histone tails. In addition to their chromatin modifying role, HDACs have also become the focus of intense research as targets for cancer therapy. Inhibition of HDAC activity in cancer cell lines, using small molecular inhibitors which block the active site, results in cell cycle arrest, differentiation and in some circumstances apoptosis.
Understanding the function of HDAC1/2 in ES cell proliferation and differentiation
To address the unique requirement for HDAC1 in early embryogenesis we have generated conditional knock-out embryonic stem cells in which HDAC1 or HDAC2 genes can be inactivated. Deletion of HDAC1, but not HDAC2, causes a significant reduction in the HDAC activity of Sin3A, NuRD and CoREST co-repressor complexes. ES cells lacking either HDAC1 or HDAC2 were capable of forming embryoid bodies (EBs) which stimulates differentiation into the three primary germ layers. However, HDAC1 deficient EBs were significantly smaller, showed spontaneous rhythmic contraction, and increased expression of both cardiomyocyte and neuronal markers.
HDAC1/2 activity is essential of T-cell development
Our previous work and that of others has established a direct link between HDAC1/2 function and T-cell development. In addition, HDACi's are currently being used in the clinic as a therapy for cutaneous T-cell lymphoma (CTCL). Therefore a greater understanding of individual HDAC enzyme function in T-cells is highly desirable. Analysis of mice with a double deletion of HDAC1 and 2 in developing T cells reveals a significant increase in double-positive(CD4+CD8+) T-cells, indicating a block in development. We are in the process of characterizing these double KO T cells for their maturity, ability to proliferate, undergo apoptosis and gene expression properties.
Techniques and expertise:
- Embryonic stem cell culture - maintenance, differentiation
- Gene targeting for transgenics - construction of targeting vectors, high-through screening
- Analysis of HDAC complexes, western blotting, IPs, HDAC assays
- Analysis of T cells - isolation, characterization by FACs
- Flow cytometry - four colour analysis of lymphocytes
- Maintenance of transgenic mice - genotyping, breeding, analysis of early embryos.