The C1/RIPE3b1 (؊118/؊107 bp) binding factor regulates pancreatic-␤-cell-specific and glucose-regulated transcription of the insulin gene. In the present study, the C1/RIPE3b1 activator from mouse ␤TC-3 cell nuclear extracts was purified by DNA affinity chromatography and two-dimensional gel electrophoresis. C1/RIPE3b1 binding activity was found in the roughly 46-kDa fraction at pH 7.0 and pH 4.5, and each contained N-and C-terminal peptides to mouse MafA as determined by peptide mass mapping and tandem spectrometry. MafA was detected in the C1/RIPE3b1 binding complex by using MafA peptide-specific antisera. In addition, MafA was shown to bind within the enhancer region (؊340/؊91 bp) of the endogenous insulin gene in ␤TC-3 cells in the chromatin immunoprecipitation assay. These results strongly suggested that MafA was the ␤-cell-enriched component of the RIPE3b1 activator. However, reverse transcription-PCR analysis demonstrated that mouse islets express not only MafA but also other members of the large Maf family, specifically c-Maf and MafB. Furthermore, immunohistochemical studies revealed that at least MafA and MafB were present within the nuclei of islet ␤ cells and not within pancreas acinar cells. Because MafA, MafB, and c-Maf were each capable of specifically binding to and activating insulin C1 element-mediated expression, our results suggest that all of these factors play a role in islet ␤-cell function.Insulin is an essential regulator of metabolism. This hormone, which is synthesized by the ␤ cells of the islets of Langerhans, increases the storage of glucose, fatty acids, and amino acids through its actions in liver, adipose tissue, and muscle. Experiments performed in vivo with transgenic animals have established that the cis-acting elements controlling ␤-cell-selective expression are located within the insulin enhancer region, which is found between nucleotides Ϫ340 and Ϫ91 relative to the transcription start site. Several key control elements within the enhancer have been identified, including C2 (Ϫ317/ Ϫ311 bp), A3 (Ϫ201/Ϫ196 bp), C1 (Ϫ118/Ϫ107 bp), and E1 (Ϫ100/Ϫ91 bp) (37,60,67). Mutations that decrease the binding affinity of the A3, C1, and E1 activators also reduce glucose-regulated transcription (37,60,67).The activator of insulin C2-element stimulated transcription is Pax6 (61). Proteins in the Pax family all contain a paired box bipartite DNA-binding domain, although Pax6 also has a homeodomain. The Pdx-1 homeodomain protein (formerly known as IPF-1, STF-1, and IDX-1) is the regulator of A3 elementactivated expression (46,48,49,50), whereas the E1 activator is a heterodimer composed of proteins in the basic helix-loophelix family that are enriched in islets (i.e., BETA2 [42]) and generally distributed (i.e., HEB [51] and E2A [2,10,17,65]). In the adult pancreas, Pax6 (61) and BETA2 (42) are found in all islet cell types, whereas Pdx-1 appears to be found only in ␤ cells, a subset of islet ␦ cells (48,49), and exocrine acinar cells (71,80). These transcription factors are necessary for ma...

SummaryIn Arabidopsis, two floral homeotic genes APETALA2 (AP2) and AGAMOUS (AG) specify the identities of perianth and reproductive organs, respectively, in flower development. The two genes act antagonistically to restrict each other to their proper domains of action within the floral meristem. In addition to AG, which antagonizes AP2, miR172, a microRNA, serves as a negative regulator of AP2. In this study, we showed that AG and miR172 have distinct functions in flower development and that they largely act independently in the negative regulation of AP2. We uncovered functions of miR172-mediated repression of AP2 in the regulation of floral stem cells and in the delineation of the expression domain of another class of floral homeotic genes. Given the antiquity of miR172 in land plants, our findings have implications for the recruitment of a microRNA in the building of a flower in evolution.

The islet-enriched MafA, PDX-1, and BETA2 activators contribute to both ␤ cell-specific and glucose-responsive insulin gene transcription. To investigate how these factors impart activation, their combined impact upon insulin enhancer-driven expression was first examined in non-␤ cell line transfection assays. Individual expression of PDX-1 and BETA2 led to little or no activation, whereas MafA alone did so modestly. MafA together with PDX-1 or BETA2 produced synergistic activation, with even higher insulin promoter activity found when all three proteins were present. Stimulation was attenuated upon compromising either MafA transactivation or DNA-binding activity. MafA interacted with endogenous PDX-1 and BETA2 in coimmunoprecipitation and in vitro GST pull-down assays, suggesting that regulation involved direct binding. Dominant-negative acting and small interfering RNAs of MafA also profoundly reduced insulin promoter activity in ␤ cell lines. In addition, MafA was induced in parallel with insulin mRNA expression in glucose-stimulated rat islets. Insulin mRNA levels were also elevated in rat islets by adenoviral-mediated expression of MafA. Collectively, these results suggest that MafA plays a key role in coordinating and controlling the level of insulin gene expression in islet ␤ cells.Insulin is selectively expressed in the pancreatic ␤ cells of the islet of Langerhans. Restricted expression is due to a unique combination of factors that stimulate through conserved enhancer region sequences located approximately between nucleotides Ϫ340 and Ϫ90 relative to the transcription start site (1-4). Detailed analysis has revealed that activation is primarily controlled by PAX6, PDX-1, MafA, and BETA2 binding to the C2 (Ϫ317 to Ϫ311 bp), A3 (Ϫ201 to Ϫ196 bp), C1 (Ϫ126 to Ϫ101 bp), and E1 (Ϫ100 to Ϫ91 bp) elements, respectively (5, 6). These distinct factors are enriched in islet cells, with BETA2 (7, 8) and PAX6 (9) present in all islet cell types, PDX-1 in ␤ and a subset of ␦ cells

T-helper 17 (Th17) cells have important functions in adaptor immunity and have also been implicated in inflammatory disorders. The bromodomain and extraterminal domain (BET) family proteins regulate gene transcription during lineage-specific differentiation of naïve CD4 T cells to produce mature T-helper cells. Inhibition of acetyl-lysine binding of the BET proteins by pan-BET bromodomain (BrD) inhibitors, such as JQ1, broadly affects differentiation of Th17, Th1, and Th2 cells that have distinct immune functions, thus limiting their therapeutic potential. Whether these BET proteins represent viable new epigenetic drug targets for inflammatory disorders has remained an unanswered question. In this study, we report that selective inhibition of the first bromodomain of BET proteins with our newly designed small molecule MS402 inhibits primarily Th17 cell differentiation with a little or almost no effect on Th1 or Th2 and Treg cells. MS402 preferentially renders Brd4 binding to Th17 signature gene loci over those of housekeeping genes and reduces Brd4 recruitment of p-TEFb to phosphorylate and activate RNA polymerase II for transcription elongation. We further show that MS402 prevents and ameliorates T-cell transfer-induced colitis in mice by blocking Th17 cell overdevelopment. Thus, selective pharmacological modulation of individual bromodomains likely represents a strategy for treatment of inflammatory bowel diseases.

SUMMARYThe establishment of organ boundaries is a fundamental process for proper morphogenesis in multicellular organisms. In plants, the shoot meristem repetitively forms organ primordia from its periphery, and boundary cells are generated between them to separate their cellular fates. The genes CUP-SHAPED COTYLEDON1 (CUC1) and CUC2, which encode plant-specific NAC transcription factors, play central roles in establishment of the shoot organ boundaries in Arabidopsis thaliana. Here we show that CUC1 protein activates expression of LIGHT-DEPENDENT SHORT HYPOCOTYLS 4 (LSH4) and its homolog LSH3 in shoot organ boundary cells. Both genes encode nuclear proteins of the Arabidopsis LSH1 and Oryza G1 (ALOG) family, the members of which are widely conserved in land plants. Expression of LSH4 and LSH3 is detected in the boundary cells of various shoot organs, such as cotyledons, leaves and floral organs, and requires the activity of CUC1 and CUC2. Experiments using the glucocorticoid receptor system indicate that transcription of LSH4 and LSH3 is directly up-regulated by CUC1. Constitutive expression of LSH4 in the shoot apex causes inhibition of leaf growth in the vegetative phase, and formation of extra shoots or shoot organs within a flower in the reproductive phase. Together, our results indicate that CUC1 directly activates transcription of the nuclear factor genes LSH4 and LSH3, which may suppress organ differentiation in the boundary region.

Background: MG53 is a membrane repair gene whose role in wound healing has not been studied. Results: Topical administration of MG53 protein facilitates wound healing and reduces scar formation. Conclusion: This study establishes MG53 as facilitator of injury repair and inhibitor of myofibroblast differentiation during wound healing. Significance: MG53 has therapeutic benefits in treating wounds and fibrotic diseases.

Consensus-binding sites for many transcription factors are relatively non-selective and found at high frequency within the genome. This raises the possibility that factors that are capable of binding to a cis-acting element in vitro and regulating transcription from a transiently transfected plasmid, which would not have higher order chromatin structure, may not occupy this site within the endogenous gene. Closed chromatin structure and competition from another DNA-binding protein with similar nucleotide specificity are two possible mechanisms by which a transcription factor may be excluded from a potential binding site in vivo. Multiple transcription factors, including Pdx-1, BETA-2, and Pax6, have been implicated in expression of the insulin gene in pancreatic ␤ cells. In this study, the chromatin immunoprecipitation assay has been used to show that these factors do, in fact, bind to insulin control region sequences in intact ␤ cells. In addition, another key islet-enriched transcription factor, Nkx2.2, was found to occupy this region using the chromatin immunoprecipitation assay. In vitro DNA-binding and transient transfection assays defined how Nkx2.2 affected insulin gene expression. Pdx-1 was also shown to bind within a region of the endogenous islet amyloid polypeptide, pax-4, and glucokinase genes that were associated with control in vitro. Because Pdx-1 does not regulate gene transcription in isolation, these sequences were examined for occupancy by the other insulin transcriptional regulators. BETA-2, Pax6, and Nkx2.2 were also found to bind to amyloid polypeptide, glucokinase, and pax-4 control sequences in vivo. These studies reveal the broad application of the Pdx-1, BETA-2, Pax6, and Nkx2.2 transcription factors in regulating expression of genes selectively expressed in islet ␤ cells. Transcription of the insulin (INS)1 gene is restricted to pancreatic islet ␤ cells. The 5Ј-flanking sequences within 350 base pairs of the transcription start site contain the binding sites for the factors that control cell type-specific expression (1-4). An extensive series of in vitro gel shift and cell transfection experiments indicates that regulation is mediated by islet-enriched DNA-binding transcription factors, including Pdx-1 (5-7), BE-TA-2 (8), and Pax6 (9). Strikingly, each of these factors is also a key regulator of pancreas development. Thus, homozygous loss of Pdx-1 leads to pancreatic agenesis in mice (10, 11) and humans (12), whereas there are severe defects in islet cell formation in BETA-2 (i.e. ␤, ␣, and ␦ cells; Ref. 13) and Pax6 (i.e. ␣ cells; Ref. 14) null mice.Islet cell development and function is also affected by other islet-enriched transcription factors (15,16). For example, insulin is not made in ␤ cells of mice lacking Nkx2.2 (17). Interestingly, this appears to be a rather specific effect as other ␤ cell-enriched markers continue to be expressed (e.g. Pdx-1 and islet amyloid polypeptide (IAPP). Nkx2.2 and insulin are also co-expressed during embryogenesis, although Nkx2.2 expression is no...

SummaryLight regulates plant growth and development through a network of endogenous factors. By screening Arabidopsis activation-tagged lines, we isolated a dominant mutant (light-dependent short hypocotyls 1-D (lsh1-D)) that showed hypersensitive responses to continuous red (cR), far-red (cFR) and blue (cB) light and cloned the corresponding gene, LSH1. LSH1 encodes a nuclear protein of a novel gene family that has homologues in Arabidopsis and rice. The effects of the lsh1-D mutation were tested in a series of photoreceptor mutant backgrounds. The hypersensitivity to cFR and cB light conferred by lsh1-D was abolished in a phyA null background (phyA-201), and the hypersensitivity to cR and cFR light conferred by lsh1-D was much reduced in the phytochrome-chromophore synthetic mutant, hy1-1 (long hypocotyl 1). These results indicate that LSH1 is functionally dependent on phytochrome to mediate light regulation of seedling development.