In vivo characterization of the lysine-methyltransferases Set7/9 and G9a by conditional mutagenesis in the mouse

Increasing evidence suggests that site-specific lysine methylation of histone and non-histone proteins is fundamentally involved in epigenetic regulation of gene expression during cellular differentiation and tumorigenesis. To study the in vivo relevance of the lysine methyltransferases Set7/9 and G9a, I have generated and characterized two conditional knockout mouse strains. Despite its widely proposed role in transcriptional activation through the methylation of histone H3 lysine 4 (H3K4) and a number of transcription factors, I found that Set7/9 knockout mice develop normally and do not display any overt phenotypic alteration. Since Set7/9 was shown to methylate the tumor suppressor protein p53 and was suggested to be important for its activity, I mainly focused my characterization of the Set7/9 knockout strain towards the proposed impairment of p53 function in these mice. Contrary to all reports, I found that in the absence of Set7/9, the p53 target genes p21WAF¹/CIP¹, Mdm2, Puma and Bax are normally expressed under basal and stressed conditions in different cell types. As a consequence, no functional p53 impairment was detectable upon DNA damage or in response to ectopic oncogene expression in Set7/9⁻/⁻ cells. Hence, my data demonstrates that Set7/9-mediated methylation of p53 represents, if at all, only a minor event in its regulation and does not appreciably control p53 activity in vivo. In the generated conditional G9a knockout strain, I primarily focused my efforts towards describing its role in the hematopoietic system. Mice that conditionally lack G9a expression in the blood, develop normally and can sustain the development of all hematopoietic cell types under homeostatic conditions. Interestingly however, when performing competitive bone marrow transplantation assays, I detected a marked impairment in G9a knockout bone marrow cells in the reconstitution of the hematopoietic system. Consistently, G9a-deficient myeloid and erythroid progenitors are dramatically reduced in their proliferation capacity. My experiments indicate for the first time, that G9a is specifically important for the biology of hematopoietic stem and progenitor cells under stress conditions and its inactivation might represent a promising way to interfere with blood development in pathological and regenerative settings.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat