Mammalian gene silencing is established through methylation of histones and DNA, although the order in which these modifications occur remains contentious. Using the human β-globin locus as a model, we demonstrate that symmetric methylation of histone H4 arginine 3 (H4R3me2s) by the protein arginine methyltransferase PRMT5 is required for subsequent DNA methylation. H4R3me2s serves as a direct binding target for the DNA methyltransferase DNMT3A, which interacts through the ADD domain containing the PHD motif. Loss of the H4R3me2s mark through short hairpin RNA–mediated knockdown of PRMT5 leads to reduced DNMT3A binding, loss of DNA methylation and gene activation. In primary erythroid progenitors from adult bone marrow, H4R3me2s marks the inactive methylated globin genes coincident with localization of PRMT5. Our findings define DNMT3A as both a reader and a writer of repressive epigenetic marks, thereby directly linking histone and DNA methylation in gene silencing.

Despite its prevalence, the molecular basis of squamous cell carcinoma (SCC) remains poorly understood. Here, we identify the developmental transcription factor Grhl3 as a potent tumor suppressor of SCC in mice, and demonstrate that targeting of Grhl3 by a miR-21-dependent proto-oncogenic network underpins SCC in humans. Deletion of Grhl3 in adult epidermis evokes loss of expression of PTEN, a direct GRHL3 target, resulting in aggressive SCC induced by activation of PI3K/AKT/mTOR signaling. Restoration of Pten expression completely abrogates SCC formation. Reduced levels of GRHL3 and PTEN are evident in human skin, and head and neck SCC, associated with increased expression of miR-21, which targets both tumor suppressors. Our data define the GRHL3-PTEN axis as a critical tumor suppressor pathway in SCC.

The proteins of the trithorax and Polycomb groups maintain the differential expression pattern of homeotic genes established by the early embryonic patterning system during development. These proteins generate stable and heritable chromatin structures by acting via particular chromosomal memory elements. We established a transgenic assay system showing that the Polycomb group response elements bxd and Mcp confer epigenetic inheritance throughout development. With previously published data for the Fab7 cellular memory module, we confirmed the cellular memory function of Polycomb group response elements. In Drosophila melanogaster, several of these memory elements are located in the large intergenic regulatory regions of the homeotic bithorax complex. Using a transgene assay, we showed that transcription through a memory element correlated with the relief of silencing imposed by the Polycomb group proteins and established an epigenetically heritable active chromatin mode. A memory element remodeled by the process of transcription was able to maintain active expression of a reporter gene throughout development. Thus, transcription appears to reset and change epigenetic marks at chromosomal memory elements regulated by the Polycomb and trithorax proteins. Interestingly, in the bithorax complex of D. melanogaster, the segment-specific expression of noncoding intergenic transcripts during embryogenesis seems to fulfill this switching role for memory elements regulating the homeotic genes.Developmental decisions resulting in differential gene expression patterns need to be maintained over many rounds of cell divisions. The proteins of the Polycomb group (PcG) and trithorax group (trxG) lock determined gene expression states by generating higher-order chromatin structures which are stable and heritable. The members of the PcG protein family form silencing complexes and maintain the repressed state, whereas the conversely acting trxG proteins promote transcription and maintain the active and open chromatin mode (for a recent review, see reference 13).Well-known targets for this type of regulation, termed cellular memory, are the homeotic gene clusters whose products determine axial cellular identities in multicellular organisms. In Drosophila melanogaster, the bithorax complex (BX-C) contains the three homeotic genes Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B). Parasegment-specific cell identity of parts of the thorax and abdomen is achieved by differential and tightly controlled expression of each homeotic gene along the anterior-posterior axis of the developing fly (21,35).The bithorax complex is organized into nine large cis regulatory units (abx/bx, bxd/pbx, and iab-2 to iab-8), which are responsible for the correct parasegment-specific expression patterns of the three homeotic genes. Embryonically acting maternal and segmentation genes set the parasegment-specific expression pattern of homeotic genes. In the cis regulatory sequences, chromosomal elements such as bithoraxoid (bxd),

SUMMARY The mammalian PCP pathway regulates diverse developmental processes requiring coordinated cellular movement, including neural tube closure and cochlear stereociliary orientation. Here, we show that epidermal wound repair is regulated by PCP signaling. Mice carrying mutant alleles of PCP genes Vangl2, Celsr1, PTK7, and Scrb1, and the transcription factor Grhl3, interact genetically, exhibiting failed wound healing, neural tube defects and disordered cochlear polarity. Using phylogenetic analysis, ChIP, and gene expression in Grhl3−/− mice, we identified RhoGEF19, a homologue of a RhoA activator involved in PCP signaling in Xenopus, as a direct target of GRHL3. Knockdown of Grhl3 or RhoGEF19 in keratinocytes induced defects in actin polymerisation, cellular polarity and wound healing, and re-expression of RhoGEF19 rescued these defects in Grhl3-kd cells. These results define a role for Grhl3 in PCP signaling, and broadly implicate this pathway in epidermal repair.