Myocyte enhancer factor 2 (MEF2) transcription factors play pivotal roles in striated muscle, neuron, and lymphocyte gene expression and are targets of stressand calcium-mediated signaling. All MEF2 gene products have a common DNA binding and dimerization domain, but MEF2 transcripts are alternatively spliced among coding exons to produce splicing isoforms. In vertebrate MEF2A, -C, and -D, a splice versus no-splice option gives forms that include or exclude a short domain that we designate . We show that mRNAs containing  are expressed predominantly in striated muscle and brain and that splicing to include  is induced during myocyte differentiation. MEF2 ؉ isoforms are more robust than ؊ forms in activating MEF2-responsive reporters despite similar expression levels. One-hybrid transcription assays using Gal4-MEF2 fusions show similar distinctions in the transactivation produced by ؉ versus ؊ isoforms in all cell types tested, including myocytes.  function is position-independent and exists in all MEF2 splicing variant contexts. The activity is not due to cis effects on MEF2 DNA binding or dimerization nor are established transcription factor or coactivator interactions involved. Each MEF2  domain contains multiple acidic residues, mutation of which abolishes function. Despite a location between the p38 MAPK docking domain and Thr phosphoacceptors of MEF2A and MEF2C, inclusion of  does not influence responses of these factors to this signaling pathway. Thus, a conserved pattern of alternative splicing in vertebrate MEF2 genes generates an acidic activation domain in MEF2 proteins selectively in tissues where MEF2 target genes are highly expressed. Myocyte enhancer factor 2 (MEF2)1 proteins are members of the MADS (MCM1, agamous, deficiens, serum response factor)-box family of transcriptional regulators (1-3). MEF2 was originally recognized as a sequence-specific DNA-binding activity at conserved elements in the promoters of various genes encoding muscle structural proteins and as products of cDNAs encoding proteins related to serum response factor (4, 5). Four distinct vertebrate genes encoding MEF2 forms were subsequently recognized, MEF2A, MEF2B, MEF2C, and MEF2D (6 -9). Initial studies of MEF2 largely considered a role in myogenesis and muscle structural protein expression, but a wider province is now appreciated. Thus, MEF2 target genes include those encoding transporters and metabolic enzymes of striated muscle (10 -13) and effectors of stress signaling in various cell types (14, 15). In addition, MEF2 proteins interact directly with neuron-specific transcription factors (16) and play a critical role in differentiation and programmed cell death in this cell type (17-24). Finally, critical roles for MEF2 factors in leukocyte functions have been established, including T lymphocyte apoptosis (25, 26) and activation (27), maintenance of Epstein-Barr virus latency in B cells (28), and macrophage activation (29).The four MEF2 genes are differentially expressed spatially and temporally during development...
Rationale Wnt signaling regulates key aspects of diabetic vascular disease. Objective We generated SM22-Cre;LRP6(fl/fl);LDLR-/- mice to determine contributions of Wnt co-receptor LRP6 in the vascular smooth muscle lineage (VSM) of male LDLR-null mice, a background susceptible to diet (HFD) - induced diabetic arteriosclerosis. Methods and Results As compared to LRP6(fl/fl);LDLR-/- controls, SM22-Cre;LRP6(fl/fl);LDLR-/- (LRP6-VKO) siblings exhibited increased aortic calcification on HFD without changes in fasting glucose, lipids, or body composition. Pulse wave velocity (index of arterial stiffness) was also increased. Vascular calcification paralleled enhanced aortic osteochondrogenic programs and circulating osteopontin (OPN), a matricellular regulator of arteriosclerosis. Survey of ligands and Frizzled (Fzd) receptor profiles in LRP6-VKO revealed upregulation of canonical and noncanonical Wnts alongside Fzd10. Fzd10 stimulated noncanonical signaling and OPN promoter activity via an USF-activated cognate inhibited by LRP6. RNAi revealed that USF1 but not USF2 supports OPN expression in LRP6-VKO VSM, and immunoprecipitation confirmed increased USF1 association with OPN chromatin. ML141, an antagonist of cdc42/Rac1 noncanonical signaling, inhibited USF1 activation, osteochondrogenic programs, alkaline phosphatase, and VSM calcification. Mass spectrometry identified LRP6 binding to protein arginine methyltransferase (PRMT) - 1, and nuclear asymmetric dimethylarginine modification was increased with LRP6-VKO. RNAi demonstrated that PRMT1 inhibits OPN and TNAP while PRMT4 supports expression. USF1 complexes containing the H3R17Me2a signature of PRMT4 are increased with LRP6-VKO. Jmjd6, a demethylase downregulated with LRP6 deficiency, inhibits OPN and TNAP expression, USF1:H3R17Me2a complex formation and transactivation. Conclusions LRP6 restrains VSM noncanonical signals that promote osteochondrogenic differentiation, mediated in part via USF1- and arginine methylation – dependent relays.
Nuclear respiratory factors NRF1 and NRF2 regulate the expression of nuclear genes encoding heme biosynthetic enzymes, proteins required for mitochondrial genome transcription and protein import, and numerous respiratory chain subunits. NRFs thereby coordinate the expression of nuclear and mitochondrial genes relevant to mitochondrial biogenesis and respiration. Only two of the nuclear-encoded respiratory chain subunits have evolutionarily conserved tissue-specific forms: the cytochrome c oxidase (COX) subunits VIa and VIIa heart/muscle (H) and ubiquitous (L) isoforms. We used genome comparisons to conclude that the promoter regions of COX6A H and COX7A H lack NRF sites but have conserved myocyte enhancer factor 2 (MEF2) elements. We show that MEF2A mRNA is induced with forced expression of NRF1 and that the MEF2A 5-regulatory region contains an evolutionarily conserved canonical element that binds endogenous NRF1 in chromatin immunoprecipitation (ChIP) assays. NRF1 regulates MEF2A promoter-reporters according to overexpression, RNA interference underexpression, and promoter element mutation studies. As there are four mammalian MEF2 isotypes, we used an isoform-specific antibody in ChIP to confirm MEF2A binding to the COX6A H promoter. These findings support a role for MEF2A as an intermediary in coordinating respiratory chain subunit expression in heart and muscle through a NRF1 3 MEF2A 3 COX H transcriptional cascade. MEF2A also bound the MEF2A and PPARGC1A promoters in ChIP, placing it within a feedback loop with PGC1␣ in controlling NRF1 activity. Interruption of this cascade and loop may account for striated muscle mitochondrial defects in mef2a null mice. Our findings also account for the previously described indirect regulation by NRF1 of other MEF2 targets in muscle such as GLUT4. The electron transport chain (ETC)4 consists of four multisubunit enzyme complexes within the inner mitochondrial (mito) membrane. These act in concert to transfer electrons from succinate or NADH to molecular oxygen while pumping protons from the matrix to the intermembranous space, establishing the electrochemical gradient required for oxidative phosphorylation (OXPHOS) (1). Nuclear genes encode all of the components of complex II, but the other complexes have subunits encoded by both mito (ETC mito ) and nuclear (ETC nucl ) genes (1, 2). Appropriate ETC subunit stoichiometry requires the coordinate expression of genes on the two genomes and an accounting for a variable number of mito genomes per cell (2, 3). This is orchestrated by the nuclear respiratory (transcription) factors, NRF1 and NRF2 (2-5). These structurally unrelated factors, encoded by nuclear genes, regulate the transcription of TFAM, TFB1M, and TFB2M, nuclear genes of the mito transcription factor Tfam (mtTFA) (6) and Tfbm specificity factors (7). Tfam and Tfbm proteins are imported into mito where they direct transcription from both heavy and light strands of mito DNA (mtDNA). These transcripts are processed to yield the various ETC mito mRNAs, as well as rRN...
When fed high-fat diets, male LDLR−/− mice develop obesity, hyperlipidemia, hyperglycemia, and arteriosclerotic calcification. An osteogenic Msx-Wnt regulatory program is concomitantly upregulated in the vasculature. To better understand the mechanisms of diabetic arteriosclerosis, we generated SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR−/− mice, assessing the impact of Msx1+Msx2 gene deletion in vascular myofibroblast and smooth muscle cells. Aortic Msx2 and Msx1 were decreased by 95% and 34% in SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR−/− animals versus Msx1(fl/fl);Msx2(fl/fl);LDLR−/− controls, respectively. Aortic calcium was reduced by 31%, and pulse wave velocity, an index of stiffness, was decreased in SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR−/− mice vs. controls. Fasting blood glucose and lipids did not differ, yet SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR−/− siblings became more obese. Aortic adventitial myofibroblasts from SM22-Cre;Msx1(fl/fl);Msx2(fl/fl);LDLR−/− mice exhibited reduced osteogenic gene expression and mineralizing potential with concomitant reduction in multiple Wnt genes. Sonic hedgehog (Shh) and Sca1, markers of aortic osteogenic progenitors, were also reduced, paralleling a 78% reduction in alkaline phosphatase (TNAP)–positive adventitial myofibroblasts. RNA interference revealed that although Msx1+Msx2 supports TNAP and Wnt7b expression, Msx1 selectively maintains Shh and Msx2 sustains Wnt2, Wnt5a, and Sca1 expression in aortic adventitial myofibroblast cultures. Thus, Msx1 and Msx2 support vascular mineralization by directing the osteogenic programming of aortic progenitors in diabetic arteriosclerosis.
MEF2 (myocyte enhancer factor 2) proteins are a small family of transcription factors that play pivotal roles in striated muscle differentiation, development, and metabolism, in neuron survival and synaptic formation, and in lymphocyte selection and activation. Products of the four mammalian MEF2 genes, MEF2A, MEF2B, MEF2C, and MEF2D, are expressed with overlapping but distinct temporospatial patterns. Toward analysis of MEF2A functions and the determinants of its regulated expression, we have mapped and begun studies of the transcriptional control regions of this gene. Heterogeneous 5 -untranslated regions of MEF2A mRNAs result from use of alternative promoters and splicing patterns. The two closely approximated TATA-less promoters are ϳ65 kb upstream of the exon containing the sole initiation codon. Ribonuclease protection and primer extension assays show that each promoter is active in various adult tissues. A canonical MEF2 site overlies the major promoter 1 transcription start site. This element specifically binds MEF2 factors, including endogenous nuclear MEF2A according to chromatin immunoprecipitation studies, and is critical to MEF2A transcription in myocytes. The site exerts reciprocal control of the alternative promoters, silencing promoter 1 and activating promoter 2 under some conditions. Erk5 and p38 MAPK signaling stimulate MEF2A expression by activating both promoters from the MEF2 element. MEF2A transcription is therefore subject to positive or negative regulation by its protein products, depending on signaling activities that influence MEF2 factor trans-activity. The sole MEF2 gene of the cephalochordate amphioxus has a similar regulatory region structure, suggesting that this mode of autoregulatory control is conserved among higher metazoan MEF2 genes.
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