Characterization of mammalian Asx-like genes and role of Asx-like-1 in development and hematopoiesis

Polycomb Group (PcG) chromatin regulatory proteins maintain repression of Hox genes during development, leading to posterior homeotic transformations in the absence of gene product. Conversely, trithorax Group (trxG) proteins maintain activation of Hox genes and trxG null mutants exhibit anterior transformations. Mutations in some PcG genes, termed Enhancers of trx and Pc (ETP), enhance both trxG and PcG gene mutations, result in both posterior and anterior transformations and mediate both silencing and activation of Hox genes. The Additional sex combs (Asx) gene of Drosophila belongs to the ETP group. I have identified and characterized three mammalian orthologues of Asx in mice and humans, named Asx-like-1, Asx-like-2, and Asx-like-3. Primary sequence conservation between ASX and mammalian ASX-like proteins is limited to an N-terminal nuclear receptor binding motif containing region (termed the ASXH domain), and a C-terminal region containing a PHD zinc finger that mediates interaction with the SET domain of the TRITHORAX/MLL protein in Drosophila and mammals respectively. Murine Asxl1 and Asxl2 are expressed ubiquitously in adult tissues and undifferentiated embryonic stem (ES) cells. Asxl1 is expressed selectively in hematopoietic cells. To assess the role of Asxl1 in mouse development and hematopoiesis, I generated Asxl1 deficient mice by targeted mutagenesis of ES cells. Homozygous Asxl1-/- mice exhibit partial perinatal lethality. Surviving adult Asxl1-/- mice fail to thrive, and exhibit cell autonomous reductions in thymopoiesis and B-cell differentiation whereas myeloid lineage differentiation is largely unaffected. Asxl1-/- newborn mice show bidirectional homeotic transformations of the axial skeleton, indicating that Asxl1 is a true functional homologue of Asx. To determine if Asxl1 is a conserved murine ETP gene, I generated compound mutants for Asxl1 and the PcG gene M33, a homologue of the Polycomb gene in Drosophila. Compound Asxl1;M33 mutants exhibit enhanced lethality, and more severe and highly penetrant axial skeletal homeotic transformations as compared to single Asxl1 and M33 mutants. These results are consistent with classification of Asxl1 as the first described ETP gene in mice. Further analysis of the mammalian Asx-like gene family will provide insight into the interacting regulatory mechanisms governing maintenance of gene expression states through cell differentiation and development.Science, Faculty ofZoology, Department ofGraduat