Summary Conserved protein kinases with core cellular functions have been frequently redeployed during metazoan evolution to regulate specialized developmental processes. The Ser/Arg (SR)-rich splicing factor (SRSF) protein kinase (SRPK), which is implicated in splicing regulation, is one such conserved eukaryotic kinase. Surprisingly, we show that SRPK has acquired the capacity to control a neurodevelopmental ubiquitin signaling pathway. In mammalian embryonic stem cells and cultured neurons, SRPK phosphorylates Ser-Arg motifs in RNF12/RLIM, a key developmental E3 ubiquitin ligase that is mutated in an intellectual disability syndrome. Processive phosphorylation by SRPK stimulates RNF12-dependent ubiquitylation of nuclear transcription factor substrates, thereby acting to restrain a neural gene expression program that is aberrantly expressed in intellectual disability. SRPK family genes are also mutated in intellectual disability disorders, and patient-derived SRPK point mutations impair RNF12 phosphorylation. Our data reveal unappreciated functional diversification of SRPK to regulate ubiquitin signaling that ensures correct regulation of neurodevelopmental gene expression.

BackgroundIncreased expression of the cyclooxygenase-2 enzyme (COX2) is one of the main characteristics of gastric cancer (GC), which is a leading cause of death in the world, particularly in Asia and South America. Although the Wnt/β-catenin signaling pathway has been involved in the transcriptional activation of the COX2 gene, the precise mechanism modulating this response is still unknown.Methodology/Principal FindingsHere we studied the transcriptional regulation of the COX2 gene in GC cell lines and assessed whether this phenomenon is modulated by Wnt/β-catenin signaling. We first examined the expression of COX2 mRNA in GC cells and found that there is a differential expression pattern consistent with high levels of nuclear-localized β-catenin. Pharmacological treatment with either lithium or valproic acid and molecular induction with purified canonical Wnt3a significantly enhanced COX2 mRNA expression in a dose- and time-dependent manner. Serial deletion of a 1.6 Kbp COX2 promoter fragment and gain- or loss-of-function experiments allowed us to identify a minimal Wnt/β-catenin responsive region consisting of 0.8 Kbp of the COX2 promoter (pCOX2-0.8), which showed maximal response in gene-reporter assays. The activity of this pCOX2-0.8 promoter region was further confirmed by site-directed mutagenesis and DNA-protein binding assays.Conclusions/SignificanceWe conclude that the pCOX2-0.8 minimal promoter contains a novel functional T-cell factor/lymphoid enhancer factor (TCF/LEF)-response element (TBE Site II; -689/-684) that responds directly to enhanced Wnt/β-catenin signaling and which may be important for the onset/progression of GC.

Development of bone tissue requires maturation of osteoblasts from mesenchymal precursors. BMP2, a member of the TGFbeta superfamily, and the Runx2 (AML3/Cbfa1) transcription factor, a downstream BMP2 effector, are regulatory signals required for osteoblast differentiation. While Runx2 responsive osteogenic gene expression has been functionally linked to alterations in chromatin structure, the factors that govern this chromatin remodeling remain to be identified. Here, we address the role of the SWI/SNF chromatin remodeling enzymes in BMP2-induced, Runx2-dependent development of the osteoblast phenotype. For these studies, we have examined calvarial cells from wild-type (WT) mice and mice that are homozygous for the Runx2 null allele, as well as the C2C12 model of BMP2-induced osteogenesis. By the analysis of microarray data, we find that several components of the SWI/SNF complex are regulated during BMP2-mediated osteoblast differentiation. Brg1 is an essential DNA dependent ATPase subunit of the SWI/SNF complex. Thus, functional studies were carried out using a fibroblast cell line that conditionally expresses a mutant Brg1 protein, which exerts a dominant negative effect on SWI/SNF function. Our findings demonstrate that SWI/SNF is required for BMP2-induced expression of alkaline phosphatase (APase), an early marker reflecting Runx2 control of osteoblast differentiation. In addition, Brg1 is expressed in cells within the developing skeleton of the mouse embryo as well as in osteoblasts ex vivo. Taken together these results support the concept that BMP2-mediated osteogenesis requires Runx2, and demonstrates that initiation of BMP2-induced, Runx2-dependent skeletal gene expression requires SWI/SNF chromatin remodeling complexes.

Here we assess histone modification, chromatin remodeling, and DNA methylation processes that coordinately control the expression of the bone master transcription factor Sp7 (osterix) during mesenchymal lineage commitment in mammalian cells. We find that Sp7 gene silencing is mediated by DNA methyltransferase1/3 (DNMT1/3)-, histone deacetylase 1/2/4 (HDAC1/2/4)-, Setdb1/Suv39h1-, and Ezh1/2-containing complexes. In contrast, Sp7 gene activation involves changes in histone modifications, accompanied by decreased nucleosome enrichment and DNA demethylation mediated by SWI/SNF- and Tet1/Tet2-containing complexes, respectively. Inhibition of DNA methylation triggers changes in the histone modification profile and chromatin-remodeling events leading to Sp7 gene expression. Tet1/Tet2 silencing prevents Sp7 expression during osteoblast differentiation as it impairs DNA demethylation and alters the recruitment of histone methylase (COMPASS)-, histone demethylase (Jmjd2a/Jmjd3)-, and SWI/SNF-containing complexes to the Sp7 promoter. The dissection of these interconnected epigenetic mechanisms that govern Sp7 gene activation reveals a hierarchical process where regulatory components mediating DNA demethylation play a leading role.