Function in adult mice [21]. The loss of cardiac function in Asxl2-/- hearts is correlated with de-repression of myosin heavy chain (-MHC), the fetal kind of MHC that has reduced ATPase activity than the adult alpha form [21]. We showed that ASXL2 plus the PRC2 core component EZH2 co-localized to numerous conserved regions within the MHC promoter. This, along with our earlier PPARβ/δ list observation that the level of bulk H3K27me3 is drastically decreased in Asxl2-/hearts, led us to hypothesize that ASXL2 and PRC2 could act together to regulate the expression of -MHC along with other target genes. To investigate this hypothesis, we very first sought to identify further targets of ASXL2 inside the murine heart. We performed a microarray analysis on 1-month-old wild-type and Asxl2-/hearts and Kinesin-7/CENP-E Purity & Documentation identified 753 genes which are either induced or repressed more than two fold in Asxl2-/- hearts (Table S1). The mis-expression of these genes is unlikely a secondary impact due to cardiac tension, for the reason that ventricular function is largely regular in Asxl2-/- hearts at this early stage [21]. We chose to examine three genes, in addition to -MHC, in far more detail: Secreted frizzled-related protein 2 (Sfrp2); Actin, alpha 1, skeletal muscle (Acta1); and G protein-coupled receptor kinase 5 (Grk5). Initial, query on the Broad Institute ChIP-seq database revealed that the promoters of those genes are enriched for PRC2 elements and H3K27me3 in embryonic stem (ES) cells (Fig. S1). This suggests that these loci contain regulatory components necessary to recruit PcG activity. Hence, they are great candidates as PcG target genes in not just ES cells but in addition in differentiated cells/tissues, like the heart. In fact, Sfrp2 has been shown to be a PcG target in human embryonic fibroblasts [22]. Second, all three genes have already been implicated in congenital or acquired heart diseases/conditions in human and/or mouse [23?6], suggesting that an understanding of their regulation could be clinically crucial. Working with real-time RT-PCR, we confirmed that Sfrp2, Acta1 and Grk5 are de-PLOS One | plosone.orgRequirement for Asxl2 in PRC2 BindingFigure 2. ASXL2 is expected for the repression of choose cardiac genes. The mRNA levels of Sfrp2 (A), Acta1 (B), and Grk5 (C) in wild-type and Asxl2-/- hearts had been analyzed by real-time RT-PCR. Each and every column shown would be the imply value of data generated from three independent samples. p0.01; Error bar: typical deviation.doi: 10.1371/journal.pone.0073983.grepressed in Asxl2-/- hearts by 4.six, five.eight, and five.9 folds, respectively (Figure two).ASXL2 and PRC2 components co-localize at select target lociGenome-wide research have consistently identified PRC2 elements to be enriched at chromatin regions close to the transcription start out sites (TSSs) of target genes [27?4]. To decide no matter if Sfrp2, Acta1 and Grk5 are directly repressed by ASXL2 and PRC2, we examined enrichment of ASXL2 and PRC2 components at these loci by ChIP-qPCR assays, focusing on regions amongst -2 kb and +2 kb of the TSS. For each locus, we chosen 2-3 genomic websites which might be conserved in between mouse, rat and human (Figure 3A ). ASXL2 was enriched at most of these web pages (Figure 3D ). The majority of the ASXL2-enriched internet sites also exhibited enrichment of PRC2 core elements EZH2 and SUZ12 (Figure 3G ). To investigate the distribution of ASXL2 along target loci, we chosen a series of conserved web pages within the gene bodies of Sfrp2 and Grk5 and examined the amount of ASXL2 enrichment by ChIP-qPCR assays. For both genes, ASXL2 was most hi.
