The Ash1L methyltransferase is essential for normal hematopoiesis in the mouse

Histones have essential functions in the regulation of gene expression through epigenetic modifications of their N-terminal tails. Acetylation, methylation, phosphorylation, ubiquitination and other post-translational histone modifications constitute a complex spectrum of changes referred to as the "histone code" that promotes activation or induces repression of gene transcription. In Drosophila, Polycomb and Trithorax group proteins are histone methyltransferases that catalyze repressive (H3K27) and permissive (H3K4) histone 3 lysine methylation, respectively. In mammals, the Polycomb gene Bmi1 is essential for preventing premature lineage specification of hematopoietic stem cells and multipotent progenitors, and as such it is necessary to sustain the long-term self-renewal potential of blood-forming stem cells. The Mixed Lineage Leukemia (MLL) gene is the mammalian homologue of the Drosophila Trithorax (Trx) gene. MLL is required for the normal emergence of hematopoietic stem cells during embryogenesis and their homeostasis during adult life. In addition, MLL fusion proteins are involved in leukemic transformation. These examples illustrate the importance of identifying histone methyltransferases with critical non-redundant function in hematopoietic stem cells and other somatic stem cell populations. In this context, I discovered a novel essential hematopoietic function for Ash1L, the mammalian homologue of the fly Absent, small, or homeotic discs 1 (Ash1) gene. In the fly, Ash1 is a SET domain containing protein that was identified as a Trithorax family member and has been reported to have H3K4, H4K9, and H4K20 methyltransferase activity. In mammals, Ash1L’s biochemical effects are still debated, with studies suggesting H3K4 or H3K36 methyltransferase activity, both associated with transcriptional activation. To investigate the role of Ash1L in vivo, I studied mice with profoundly deficient Ash1L expression as a result of a "gene trap" strategy. These mice display various non-hematopoietic phenotypes including growth retardation and infertility. In the hematopoietic system, Ash1L deficiency resulted in the near complete exhaustion of the stem cell pool in young adult mice. While neonates displayed normal numbers of hematopoietic stem cells, lack of Ash1L impaired their normal repopulation capacity in bone marrow competitive transplantation experiments resulting in a profound tri-lineage hematopoietic defect. These findings indicate a critical cell-autonomous requirement for Ash1L in the maintenance of hematopoietic stem cells. To further understand the biological basis of Ash1L’s action in hematopoietic cells, it is essential to characterize its biochemical activity. For this purpose, I generated expression vectors that will allow me to purify the functional Ash1L domains and associated protein partners via immunoprecipitation or in-vivo biotinylation with the BirA enzyme. In addition, I generated retroviral vectors that will allow me to perform overexpression and rescue assays as a basis for Ash1L structure-function studies in hematopoietic tissues. By combining this approach with a detailed study of Ash1L-deficient hematopoietic stem cells, I anticipate that I will be able to understand the unique function of this protein in the hematopoietic system and in other mammalian tissues