Significant advances have been made in the discovery of genes affecting bone mineral density (BMD); however, our understanding of its genetic basis remains incomplete. In the current study, genome-wide association (GWA) and co-expression network analysis were used in the recently described Hybrid Mouse Diversity Panel (HMDP) to identify and functionally characterize novel BMD genes. In the HMDP, a GWA of total body, spinal, and femoral BMD revealed four significant associations (−log10P>5.39) affecting at least one BMD trait on chromosomes (Chrs.) 7, 11, 12, and 17. The associations implicated a total of 163 genes with each association harboring between 14 and 112 genes. This list was reduced to 26 functional candidates by identifying those genes that were regulated by local eQTL in bone or harbored potentially functional non-synonymous (NS) SNPs. This analysis revealed that the most significant BMD SNP on Chr. 12 was a NS SNP in the additional sex combs like-2 (Asxl2) gene that was predicted to be functional. The involvement of Asxl2 in the regulation of bone mass was confirmed by the observation that Asxl2 knockout mice had reduced BMD. To begin to unravel the mechanism through which Asxl2 influenced BMD, a gene co-expression network was created using cortical bone gene expression microarray data from the HMDP strains. Asxl2 was identified as a member of a co-expression module enriched for genes involved in the differentiation of myeloid cells. In bone, osteoclasts are bone-resorbing cells of myeloid origin, suggesting that Asxl2 may play a role in osteoclast differentiation. In agreement, the knockdown of Asxl2 in bone marrow macrophages impaired their ability to form osteoclasts. This study identifies a new regulator of BMD and osteoclastogenesis and highlights the power of GWA and systems genetics in the mouse for dissecting complex genetic traits.

BackgroundPolycomb-group (PcG) and trithorax-group (trxG) proteins regulate histone methylation to establish repressive and active chromatin configurations at target loci, respectively. These chromatin configurations are passed on from mother to daughter cells, thereby causing heritable changes in gene expression. The activities of PcG and trxG proteins are regulated by a special class of proteins known as Enhancers of trithorax and Polycomb (ETP). The Drosophila gene Additional sex combs (Asx) encodes an ETP protein and mutations in Asx enhance both PcG and trxG mutant phenotypes. The mouse and human genomes each contain three Asx homologues, Asx-like 1, 2, and 3. In order to understand the functions of mammalian Asx-like (Asxl) proteins, we generated an Asxl2 mutant mouse from a gene-trap ES cell line.Methodology/Principal FindingsWe show that the Asxl2 gene trap is expressed at high levels in specific tissues including the heart, the axial skeleton, the neocortex, the retina, spermatogonia and developing oocytes. The gene trap mutation is partially embryonic lethal and approximately half of homozygous animals die before birth. Homozygotes that survive embryogenesis are significantly smaller than controls and have a shortened life span. Asxl2−/− mice display both posterior transformations and anterior transformation in the axial skeleton, suggesting that the loss of Asxl2 disrupts the activities of both PcG and trxG proteins. The PcG-associated histone modification, trimethylation of histone H3 lysine 27, is reduced in Asxl2−/− heart. Necropsy and histological analysis show that mutant mice have enlarged hearts and may have impaired heart function.Conclusions/SignificanceOur results suggest that murine Asxl2 has conserved ETP function and plays dual roles in the promotion of PcG and trxG activity. We have also revealed an unexpected role for Asxl2 in the heart, suggesting that the PcG/trxG system may be involved in the regulation of cardiac function.

Polycomb Group (PcG) proteins are epigenetic repressors of gene expression. The Drosophila Additional sex combs (Asx) gene and its mammalian homologs exhibit PcG function in genetic assays; however, the mechanism by which Asx family proteins mediate gene repression is not well understood. ASXL2, one of three mammalian homologs for Asx, is highly expressed in the mammalian heart and is required for the maintenance of cardiac function. We have previously shown that Asxl2 deficiency results in a reduction in the bulk level of histone H3 lysine 27 trimethylation (H3K27me3), a repressive mark generated by the Polycomb Repressive Complex 2 (PRC2). Here we identify several ASXL2 target genes in the heart and investigate the mechanism by which ASXL2 facilitates their repression. We show that the Asxl2-deficient heart is defective in converting H3K27me2 to H3K27me3 and in removing ubiquitin from mono-ubiquitinated histone H2A. ASXL2 and PRC2 interact in the adult heart and co-localize to target promoters. ASXL2 is required for the binding of PRC2 and for the enrichment of H3K27me3 at target promoters. These results add a new perspective to our understanding of the mechanisms that regulate PcG activity and gene repression.

During development and differentiation, cell type-specific chromatin configurations are set up to facilitate cell type-specific gene expression. Defects in the establishment or the maintenance of the correct chromatin configuration have been associated with diseases ranging from leukemia to muscular dystrophy. The heart expresses many chromatin factors, and we are only beginning to understand their roles in heart development and function. We have previously shown that the chromatin regulator Asxl2 is highly expressed in the murine heart both during development and adulthood. In the absence of Asxl2, there is a significant reduction in trimethylation of histone H3 lysine 27 (H3K27), a histone mark associated with lineage-specific silencing of developmental genes. Here we present evidence that Asxl2 is required for the long-term maintenance of ventricular function and for the maintenance of normal cardiac gene expression. Asxl2−/− hearts displayed progressive deterioration of ventricular function. By 10 months of age, there was ~37% reduction in fractional shortening in Asxl2−/− hearts compared to wild-type. Analysis of the expression of myofibril proteins suggests that Asxl2 is required for the repression of βMHC. Asxl2−/− hearts did not exhibit hypertrophy, suggesting that the de-repression of β-MHC was not the result of hypertrophic response. Instead, Asxl2 and the histone methyltansferase Ezh2 co-localize to β-MHC promoter, suggesting that Asxl2 directly represses β-MHC. Interrogation of the CardioGenomics database revealed that ASXL2 is down-regulated in the hearts of patients with ischemic or idiopathic dilated cardiomyopathy. We propose that chromatin factors like Asxl2 function in the adult heart to regulate cell type- and stage-specific patterns of gene expression, and the disruption of such regulation may be involved in the etiology and/or development of certain forms of human heart disease.