17-600 Sigma-AldrichChIPAb+ CREB - ChIP Validated Antibody and Primer Set, rabbit monoclonal

Obesity results from numerous, interacting genetic, behavioral, and physiological factors. Adipogenesis is partially regulated by several adipocyte-selective microRNAs (miRNAs) and transcription factors that regulate proliferation and differentiation of human adipose-derived mesenchymal stem cells (hMSCs-Ad). In this study, we examined the roles of adipocyte-selective miRNAs in the differentiation of hMSCs-Ad to adipocytes. Results showed that the levels of miR-148a, miR-26b, miR-30, and miR-199a increased in differentiating hMSCs-Ad. Among these miRNAs, miR-148a exhibited significant effects on increasing PPRE luciferase activity (it represents PPAR-dependent transcription, a major factor in adipogenesis) than others. Furthermore, miR-148a expression levels increased in adipose tissues from obese people and mice fed high-fat diet. miR-148a acted by suppressing its target gene, Wnt1, an endogenous inhibitor of adipogenesis. Ectopic expression of miR-148a accelerated differentiation and partially rescued Wnt1-mediated inhibition of adipogenesis. Knockdown of miR-148a also inhibited adipogenesis. Analysis of the upstream region of miR-148a locus identified a 3 kb region containing a functional cAMP-response element-binding protein (CREB) required for miR-148a expression in hMSCs-Ad. The results suggest that miR-148a is a biomarker of obesity in human subjects and mouse model, which represents a CREB-modulated miRNA that acts to repress Wnt1, thereby promoting adipocyte differentiation.

Brain-derived neurotrophic factor (BDNF) has a crucial role in modulating neural and behavioral plasticity to drugs of abuse. We found a persistent downregulation of exon-specific Bdnf expression in the ventral tegmental area (VTA) in response to chronic opiate exposure, which was mediated by specific epigenetic modifications at the corresponding Bdnf gene promoters. Exposure to chronic morphine increased stalling of RNA polymerase II at these Bdnf promoters in VTA and altered permissive and repressive histone modifications and occupancy of their regulatory proteins at the specific promoters. Furthermore, we found that morphine suppressed binding of phospho-CREB (cAMP response element binding protein) to Bdnf promoters in VTA, which resulted from enrichment of trimethylated H3K27 at the promoters, and that decreased NURR1 (nuclear receptor related-1) expression also contributed to Bdnf repression and associated behavioral plasticity to morphine. Our findings suggest previously unknown epigenetic mechanisms of morphine-induced molecular and behavioral neuroadaptations.

Transcriptional silencing during mitosis is caused by inactivation of critical transcriptional regulators and/or chromatin condensation. Inheritance of gene expression patterns through cell division involves various bookmarking mechanisms. In this report, we have examined the mitotic and post-mitotic expression of the DRA major histocompatibility class II (MHCII) gene in different cell types. During mitosis the constitutively MHCII-expressing B lymphoblastoid cells showed sustained occupancy of the proximal promoter by the cognate enhanceosome and general transcription factors. In contrast, although mitotic epithelial cells were depleted of these proteins irrespectively of their MHCII transcriptional activity, a distal enhancer selectively recruited the PP2A phosphatase via NFY and maintained chromatin accessibility. Based on our data, we propose a novel chromatin anti-condensation role for this element in mitotic bookmarking and timing of post-mitotic transcriptional reactivation.

Although many long non-coding RNAs (lncRNAs) have been discovered, their function and their association with RNAi factors in the nucleus have remained obscure. Here, we identify RNA transcripts that overlap the cyclooxygenase-2 (COX-2) promoter and contain two adjacent binding sites for an endogenous miRNA, miR-589. We find that miR-589 binds the promoter RNA and activates COX-2 transcription. In addition to miR-589, fully complementary duplex RNAs that target the COX-2 promoter transcript activate COX-2 transcription. Activation by small RNA requires RNAi factors argonaute-2 (AGO2) and GW182, but does not require AGO2-mediated cleavage of the promoter RNA. Instead, the promoter RNA functions as a scaffold. Binding of AGO2 protein/small RNA complexes to the promoter RNA triggers gene activation. Gene looping allows interactions between the promoters of COX-2 and phospholipase A2 (PLA2G4A), an adjacent pro-inflammatory pathway gene that produces arachidonic acid, the substrate for COX-2 protein. miR-589 and fully complementary small RNAs regulate both COX-2 and PLA2G4A gene expression, revealing an unexpected connection between key steps of the eicosanoid signaling pathway. The work demonstrates the potential for RNA to coordinate locus-dependent assembly of related genes to form functional operons through cis-looping.

In humans, obesity is associated with long QT, increased frequency of premature ventricular complexes, and sudden cardiac death. The mechanisms of the pro-arrhythmic electrophysiologic remodeling of obesity are poorly understood. We tested the hypothesis that there is decreased expression of voltage-gated potassium channels in the obese heart, leading to long QT. Using implanted telemeters, we found that diet-induced obese (DIO) wild-type mice have impaired cardiac repolarization, demonstrated by long QT, as well as more frequent ventricular ectopy, similar to obese humans. DIO mice have reduced protein and mRNA levels of the potassium channel Kv1.5 caused by a reduction of the transcription factor cyclic AMP response element binding protein (CREB) in DIO hearts. We found that CREB knock-down by siRNA reduces Kv1.5, CREB binds to the Kv1.5 promoter in the heart, and CREB increases transcription of mouse and human Kv1.5 promoters. The reduction in CREB protein during lipotoxicity can be rescued by inhibiting protein kinase D (PKD). Our results identify a mechanism for obesity-induced electrophysiologic remodeling in the heart, namely PKD-induced reduction of CREB, which in turn decreases expression of the potassium channel Kv1.5.

The enhancer pioneer transcription factor FoxA1 is a global mediator of steroid receptor (SR) action in hormone-dependent cancers. In castration-resistant prostate cancer (CRPC), FoxA1 acts as an androgen receptor cofactor to drive G₂ to M-phase cell-cycle transit. Here, we describe a mechanistically distinct SR-independent role for FoxA1 in driving G₁ to S-phase cell-cycle transit in CRPC. By comparing FoxA1 binding sites in prostate cancer cell genomes, we defined a codependent set of FoxA1-MYBL2 and FoxA1-CREB1 binding sites within the regulatory regions of the Cyclin E2 and E2F1 genes that are critical for CRPC growth. Binding at these sites upregulate the Cyclin E2 and Cyclin A2 genes in CRPC but not in earlier stage androgen-dependent prostate cancer, establishing a stage-specific role for this pathway in CRPC growth. Mechanistic investigations indicated that FoxA1, MYBL2, or CREB1 induction of histone H3 acetylation facilitated nucleosome disruption as the basis for codependent transcriptional activation and G₁ to S-phase cell-cycle transit. Our findings establish FoxA1 as a pivotal driver of the cell-cycle in CRPC which promotes G₁ to S-phase transit as well as G₂ to M-phase transit through two distinct mechanisms.

The CREB transcription factor regulates differentiation, survival, and synaptic plasticity. The complement of CREB targets responsible for these responses has not been identified, however. We developed a novel approach to identify CREB targets, termed serial analysis of chromatin occupancy (SACO), by combining chromatin immunoprecipitation (ChIP) with a modification of SAGE. Using a SACO library derived from rat PC12 cells, we identified approximately 41,000 genomic signature tags (GSTs) that mapped to unique genomic loci. CREB binding was confirmed for all loci supported by multiple GSTs. Of the 6302 loci identified by multiple GSTs, 40% were within 2 kb of the transcriptional start of an annotated gene, 49% were within 1 kb of a CpG island, and 72% were within 1 kb of a putative cAMP-response element (CRE). A large fraction of the SACO loci delineated bidirectional promoters and novel antisense transcripts. This study represents the most comprehensive definition of transcription factor binding sites in a metazoan species.

Peutz-Jeghers syndrome is an inherited cancer syndrome that results in a greatly increased risk of developing tumors in those affected. The causative gene is a protein kinase termed LKB1, predicted to function as a tumor suppressor. The mechanism by which LKB1 is regulated in cells is not known. Here, we demonstrate that stimulation of Rat-2 or embryonic stem cells with activators of ERK1/2 or of cAMP-dependent protein kinase induced phosphorylation of endogenously expressed LKB1 at Ser(431). We present pharmacological and genetic evidence that p90(RSK) mediated this phosphorylation in response to agonists that activate ERK1/2 and that cAMP-dependent protein kinase mediated this phosphorylation in response to agonists that activate adenylate cyclase. Ser(431) of LKB1 lies adjacent to a putative prenylation motif, and we demonstrate that full-length LKB1 expressed in 293 cells was prenylated by addition of a farnesyl group to Cys(433). Our data suggest that phosphorylation of LKB1 at Ser(431) does not affect farnesylation and that farnesylation does not affect phosphorylation at Ser(431). Phosphorylation of LKB1 at Ser(431) did not alter the activity of LKB1 to phosphorylate itself or the tumor suppressor protein p53 or alter the amount of LKB1 associated with cell membranes. The reintroduction of wild-type LKB1 into a cancer cell line that lacks LKB1 suppressed growth, but mutants of LKB1 in which Ser(431) was mutated to Ala to prevent phosphorylation of LKB1 were ineffective in inhibiting growth. In contrast, a mutant of LKB1 that cannot be prenylated was still able to suppress the growth of cells.

Prostaglandins are important mediators of activated macrophage functions, and their inducible synthesis is mediated by cyclooxygenase-2 (COX-2). Here, we make use of the murine macrophage cells RAW264 as well as of immortalized macrophages derived from mice deficient for the transcription factor CCAAT enhancer-binding protein beta (C/EBP beta) to explore the molecular mechanisms regulating COX-2 induction in activated macrophages. We demonstrate that lipopolysaccharide-mediated COX-2 mRNA induction is biphasic. The initial phase is independent of de novo protein synthesis, correlates with cAMP-response element-binding protein (CREB) activation, is inhibited by treatments that abolish CREB phosphorylation and reduce NF-kappa B-mediated gene activation, and requires the presence of the transcription factor C/EBP beta. On the other hand, C/EBP delta appears to be essential in addition to C/EBP beta to effect the second phase of COX-2 gene transcription, which is important for maintaining the induced state and requires de novo protein synthesis. Indeed, both phases of COX-2 induction were defective in C/EBP beta-/- macrophages. Moreover, the synthesis of C/EBP delta was increased dramatically by treatment with lipopolysaccharide and, like COX-2 induction, repressed by combined inhibition of the MAPK and of the SAPK2/p38 cascades. Taken together, these data identify CREB, NF-kappa B, and both C/EBP beta and -delta as key factors in coordinately orchestrating transcription from the COX-2 promoter in activated macrophages.

The present study investigated the effects of excitotoxic lesions of the prefrontal cortex (PFC) on dopamine (DA) and excitatory amino acid (EAA) function in the nucleus accumbens core using in vivo microdialysis in freely moving rats. As a postsynaptic marker of neuronal function, the nuclear levels of the transcriptional factor CREB and its active phosphorylated form, CREB-P, were measured in the ventral tegmental area (VTA), and in the core and shell subregions of the nucleus accumbens of sham and lesioned animals. PFC-lesioned animals exhibited a greater locomotor response to novelty and amphetamine administration (125-500 microg/kg i.v.). No change was observed in extracellular levels of glutamate or saturable d-aspartate binding (a marker for the high-affinity EAA transporter) in the nucleus accumbens of PFC-lesioned animals. Extracellular levels of DA were comparable in sham and lesioned animals under tonic conditions, however, following amphetamine administration, DA efflux was significantly attenuated in lesioned animals. No correlation was observed between microdialysate levels of amino acids and the attenuated dopaminergic response to amphetamine in lesioned animals. Further, no effect of the lesion was found on nuclear CREB protein in saline- and amphetamine-treated rats. The density of CREB-P immunoreactive nuclei, while remaining unchanged in the VTA, increased in the nucleus accumbens shell following amphetamine treatment in lesioned animals. The results show that an important modulatory role of the PFC on the behavioural response to novelty and amphetamine is associated with the level of immediate-early gene regulation rather than levels of extracellular DA and amino acids in the ventral striatum.