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Chronic exposure to elevated levels of glucose and fatty acids leads to dysfunction of pancreatic β-cells by mechanisms that are only partly understood. The transcription factor peroxisome proliferator-activated receptor α (PPARα) is an important regulator of genes involved in fatty acid metabolism and has been shown to protect against lipid-induced β-cell dysfunction. We and others have previously shown that expression of the PPARα gene in β-cells is rapidly repressed by glucose. Here we show that the PPARα gene is transcribed from five alternative transcription start sites, resulting in three alternative first exons that are spliced to exon 2. Expression of all PPARα transcripts is repressed by glucose both in insulinoma cells and in isolated pancreatic islets. The observation that the dynamics of glucose repression of PPARα transcription are very similar to those of glucose activation of target genes by the carbohydrate response element-binding protein (ChREBP) prompted us to investigate the potential role of ChREBP in the regulation of PPARα expression. We show that a constitutively active ChREBP lacking the N-terminal domain efficiently represses PPARα expression in insulinoma cells and in rodent and human islets. In addition, we demonstrate that siRNA-mediated knockdown of ChREBP abrogates glucose repression of PPARα expression as well as induction of well established ChREBP target genes in insulinoma cells. In conclusion, this work shows that ChREBP is a critical and direct mediator of glucose repression of PPARα gene expression in pancreatic β-cells, suggesting that ChREBP may be important for glucose suppression of the fatty acid oxidation capacity of β-cells.

Pin1 is a unique regulator, which catalyzes the conversion of a specific phospho-Ser/Thr-Pro-containing motif in target proteins. Herein, we identified CRTC2 as a Pin1-binding protein by overexpressing Pin1 with Myc and FLAG tags in mouse livers and subsequent purification of the complex containing Pin1. The association between Pin1 and CRTC2 was observed not only in overexpression experiments but also endogenously in the mouse liver. Interestingly, Ser(136) in the nuclear localization signal of CRTC2 was shown to be involved in the association with Pin1. Pin1 overexpression in HepG2 cells attenuated forskolin- induced nuclear localization of CRTC2 and cAMP-responsive element (CRE) transcriptional activity, whereas gene knockdown of Pin1 by siRNA enhanced both. Pin1 also associated with CRTC1, leading to their cytosol localization, essentially similar to the action of CRTC2. Furthermore, it was shown that CRTC2 associated with Pin1 did not bind to CREB. Taken together, these observations indicate the association of Pin1 with CRTC2 to decrease the nuclear CBP.CRTC.CREB complex. Indeed, adenoviral gene transfer of Pin1 into diabetic mice improved hyperglycemia in conjunction with normalizing phosphoenolpyruvate carboxykinase mRNA expression levels, which is regulated by CRE transcriptional activity. In conclusion, Pin1 regulates CRE transcriptional activity, by associating with CRTC1 or CRTC2.

Ligands of the aryl hydrocarbon receptor (AHR), a transcription factor mediating the effects of dioxin, favor Th17 differentiation and exacerbate autoimmunity in mice. We investigated how AHR ligands affected human T-cell polarization. We found that the high affinity and stable AHR-ligand dioxin as well as the natural AHR-ligand 6-formylinolo[3,2-b] carbazole induced the downstream AHR-target cytochrome P450A1, and without affecting IFN-gamma, they enhanced IL-22 while simultaneously decreasing IL-17A production by CD4(+) T cells. The specific AHR-inhibitor CH-223191 abolished these effects. Furthermore, blockade of IL-23 and IL-1, important for Th17 expansion, profoundly decreased IL-17A but not IL-22 production. AHR agonists reduced the expression of the Th17 master transcription factor retinoic acid-related orphan receptor C (RORC), without affecting T-bet, GATA-3 and Foxp3. They also decreased the expression of the IL-23 receptor. Importantly, AHR-ligation did not only decrease the number of Th17 cells but also primed naïve CD4(+) T cells to produce IL-22 without IL-17 and IFN-gamma. Furthermore, IL-22 single producers did not express CD161, which distinguished them from the CD161(+) Th17 cells. Hence, our data provide compelling evidence that AHR activation participates in shaping human CD4(+) T-cell polarization favoring the emergence of a distinct subset of IL-22-producing cells that are independent from the Th17 lineage.

Nucleotide excision repair (NER) requires the coordinated sequential assembly and actions of the involved proteins at sites of DNA damage. Following damage recognition, dual incision 5' to the lesion by ERCC1-XPF and 3' to the lesion by XPG leads to the removal of a lesion-containing oligonucleotide of about 30 nucleotides. The resulting single-stranded DNA (ssDNA) gap on the undamaged strand is filled in by DNA repair synthesis. Here, we have asked how dual incision and repair synthesis are coordinated in human cells to avoid the exposure of potentially harmful ssDNA intermediates. Using catalytically inactive mutants of ERCC1-XPF and XPG, we show that the 5' incision by ERCC1-XPF precedes the 3' incision by XPG and that the initiation of repair synthesis does not require the catalytic activity of XPG. We propose that a defined order of dual incision and repair synthesis exists in human cells in the form of a 'cut-patch-cut-patch' mechanism. This mechanism may aid the smooth progression through the NER pathway and contribute to genome integrity.

The BCL6 transcriptional repressor is the most commonly involved oncogene in diffuse large B-cell lymphomas (DLBCLs). Constitutive expression of BCL6 mediates lymphomagenesis through aberrant proliferation, survival, and differentiation blockade. Binding of BCL6 to the SMRT/N-CoR corepressors mediates the BCL6 survival effect in DLBCL. Although the basis for differentiation blockade is unknown in DLBCL, recent data suggest that BCL6 binding to the MTA3 corepressor might be involved. We report that BCL6 and MTA3 are coexpressed in normal germinal center B cells and DLBCL. Depletion of MTA3 in DLBCL cells induced a differentiation-related BCL6 target gene (PRDM1), but not target genes involved in survival. Accordingly, MTA3 and PRDM1 expression are mutually exclusive in germinal center B cells. We performed chromatin immunoprecipitation (ChIP)–on-chip mapping of the PRDM1 locus, identifying a novel BCL6 binding site on intron 3 of the PRDM1 gene, and show that BCL6 recruits MTA3 to this site. In DLBCL cells, MTA3 depletion induced plasmacytic differentiation but did not decrease viability of DLBCL cells. However, MTA3 depletion synergized with a specific BCL6 inhibitor that blocks SMRT/N-CoR binding to decrease DLBCL viability. Taken together, these results show that BCL6 regulates distinct transcriptional programs through the SMRT/N-CoR and MTA3 corepressors, respectively, and provides a basis for combinatorial therapeutic targeting of BCL6.

Peroxisome proliferator-activated receptors (PPARs) alpha,beta/delta and gamma are fatty acid sensitive transcription factors that have been implicated in colorectal cancer. To better understand their role, we studied the expression levels of all PPAR-isoforms and transcriptional partners such as the retinoid X receptor alpha (RXRalpha) and PPARgamma-coactivator-1 (PGC-1) by means of real-time PCR in 17 patients with colon cancer. While a heterogeneous pattern was observed for the expression level of the PPAR-isoforms alpha,beta/delta and gamma, the coactivator PGC-1 was significantly decreased in 15 of 17 tumors. Taken together our data suggest that the transcriptional activity of PPARgamma may not only be decreased by mutation but also by downregulation of the coactivator PGC-1 of PPARgamma.

Six monogenic forms of maturity-onset diabetes of the young (MODY) have been identified to date. Except for MODY2 (glucokinase), all other MODY subtypes have been linked to transcription factors. We have established a MODY3 transgenic model through the beta-cell-targeted expression of dominant-negative HNF-1alpha either constitutively (rat insulin II promoter) or conditionally (Tet-On system). The animals display either overt diabetes or glucose intolerance. Decreased insulin secretion and reduced pancreatic insulin content contribute to the hyperglycemic state. The conditional approach in INS-1 cells helped to define new molecular targets of hepatocyte nuclear factor (HNF)-1alpha. In the cellular system, nutrient-induced insulin secretion was abolished because of impaired glucose metabolism. Conditional suppression of HNF-4alpha, the MODY1 gene, showed a similar phenotype in INS-1 cells to HNF-1alpha. The existence of a regulatory circuit between HNF-4alpha and HNF-1alpha is confirmed in these cell models. The MODY4 gene, IPF-1 (insulin promoter factor-1)/PDX-1 (pancreas duodenum homeobox-1), controls not only the transcription of insulin but also expression of enzymes involved in its processing. Suppression of Pdx-1 function in INS-1 cells does not alter glucose metabolism but rather inhibits insulin release by impairing steps distal to the generation of mitochondrial coupling factors. The presented experimental models are important tools for the elucidation of the beta-cell pathogenesis in MODY syndromes.

PMCID: PMC3935450.-- et al., Type 2 diabetes affects over 300 million people, causing severe complications and premature death, yet the underlying molecular mechanisms are largely unknown. Pancreatic islet dysfunction is central in type 2 diabetes pathogenesis, and understanding islet genome regulation could therefore provide valuable mechanistic insights. We have now mapped and examined the function of human islet cis-regulatory networks. We identify genomic sequences that are targeted by islet transcription factors to drive islet-specific gene activity and show that most such sequences reside in clusters of enhancers that form physical three-dimensional chromatin domains. We find that sequence variants associated with type 2 diabetes and fasting glycemia are enriched in these clustered islet enhancers and identify trait-associated variants that disrupt DNA binding and islet enhancer activity. Our studies illustrate how islet transcription factors interact functionally with the epigenome and provide systematic evidence that the dysregulation of islet enhancers is relevant to the mechanisms underlying type 2 diabetes., We thank the DIAGRAM and MAGIC consortia, the Singapore Prospective Study Program, the Singapore Consortium of Cohort Studies, the Singapore Indian Eye Study, the Singapore Malay Eye Study and Y.Y. Teo, E.S. Tai, T.Y. Wong, W.Y. Lim and X. Wang (National University of Singapore; funded by the National Medical Research Council of Singapore, Singapore Translational Researcher Award schemes, the Biomedical Research Council of Singapore and the National Research Foundation (NRF) Fellowship scheme). This work was carried out in part at the Centre Esther Koplowitz. This work was funded by grants from a European Foundation for the Study of Diabetes Lilly fellowship (L. Pasquali), the Ministerio de Economía y Competitividad (SAF2011-27086 to J.F., BFU2010-14839 and CSD2007-00008 to J.L.G.S.), the Innovative Medicines Initiative (DIRECT to M.I.M. and J.F.), the Andalusian Government (CVI-3488 to J.L.G.S.), the Biology of Liver and Pancreatic Development and Disease Marie Curie Initial Training Network (F.M. and J.F.), the Wellcome Trust (090532, 98381 and 090367 to M.I.M., 095101 to A.L.G., 101033 to J.F.), Juvenile Diabetes Research Foundation (31-2012-783 to T.B., F.P. and L. Piemonti) and Framework Programme 7 (HEALTH-F4-2007-201413 to M.I.M.).

SummaryFunctional characterization of a gene often requires the discovery of the full spectrum of its associated phenotypes. Mutations in the human GLI3 gene have been identified in Greig cepalopolysyndactyly, Pallister-Hall syndrome (PHS), and postaxial polydactyly type-A (PAP-A). We studied the involvement of GLI3 in additional phenotypes of digital abnormalities in one family (UR003) with preaxial polydactyly type-IV (PPD-IV), three families (UR014, UR015, and UR016) with dominant PAP-A/B (with PPD-A and -B in the same family), and one family with PHS. Linkage analysis showed no recombination with GLI3-linked polymorphisms. Family UR003 had a 1-nt frameshift insertion, resulting in a truncated protein of 1,245 amino acids. A frameshift mutation due to a 1-nt deletion was found in family UR014, resulting in a truncated protein of 1,280 amino acids. Family UR015 had a nonsense mutation, R643X, and family UR016 had a missense mutation, G727R, in a highly conserved amino acid of domain 3. The patient with PHS had a nonsense mutation, E1147X. These results add two phenotypes to the phenotypic spectrum caused by GLI3 mutations: the combined PAP-A/B and PPD-IV. These mutations do not support the suggested association between the mutations in GLI3 and the resulting phenotypes. We propose that all phenotypes associated with GLI3 mutations be called “GLI3 morphopathies,” since the phenotypic borders of the resulting syndromes are not well defined and there is no apparent genotype-phenotype correlation.

The AIRE gene (autoimmune regulator), coding for a putative transcriptional regulatory factor, is mutated in autoimmune-polyendocrinopathy-candidiasis ectodermal dystrophy (APECED). We have investigated the expression of the AIRE gene by mRNA in situ hybridization and immunohistochemistry in various human tissues. Here we show that AIRE is expressed in distinct cells in thymus medulla, and also in rare cells in lymph node paracortex and medulla, and in spleen and fetal liver, but not in the target organs of autoimmune destruction. Double immunofluorescence studies revealed that in thymus medulla both epithelial (cytokeratin positive) and non-epithelial cells expressed AIRE. Subcellularly, AIRE was localised in nuclear dots in thymus and lymph node and also in transfected cells. The cellular localisation of AIRE and its nuclear localisation, compatible with its predicted protein domains, suggest that AIRE may regulate the mechanisms involved in the induction and maintenance of immune tolerance.

All pancreatic endocrine cell types arise from a common endocrine precursor cell population, yet the molecular mechanisms that establish and maintain the unique gene expression programs of each endocrine cell lineage have remained largely elusive. Such knowledge would improve our ability to correctly program or reprogram cells to adopt specific endocrine fates. Here, we show that the transcription factor Nkx6.1 is both necessary and sufficient to specify insulin-producing beta cells. Heritable expression of Nkx6.1 in endocrine precursors of mice is sufficient to respecify non-beta endocrine precursors towards the beta cell lineage, while endocrine precursor- or beta cell-specific inactivation of Nkx6.1 converts beta cells to alternative endocrine lineages. Remaining insulin+ cells in conditional Nkx6.1 mutants fail to express the beta cell transcription factors Pdx1 and MafA and ectopically express genes found in non-beta endocrine cells. By showing that Nkx6.1 binds to and represses the alpha cell determinant Arx, we identify Arx as a direct target of Nkx6.1. Moreover, we demonstrate that Nkx6.1 and the Arx activator Isl1 regulate Arx transcription antagonistically, thus establishing competition between Isl1 and Nkx6.1 as a critical mechanism for determining alpha versus beta cell identity. Our findings establish Nkx6.1 as a beta cell programming factor and demonstrate that repression of alternative lineage programs is a fundamental principle by which beta cells are specified and maintained. Given the lack of Nkx6.1 expression and aberrant activation of non-beta endocrine hormones in human embryonic stem cell (hESC)–derived insulin+ cells, our study has significant implications for developing cell replacement therapies., Author Summary Diabetes is a disease caused by the loss or dysfunction of insulin-producing beta cells in the pancreas. Recent studies suggest that modification of the beta cells' differentiation state is among the earliest events marking the progressive failure of beta cells in diabetes. Currently, very little is known about the factors that instruct cells to adopt beta cell characteristics and maintain the differentiated state of beta cells. We have discovered that a single transcription factor can instruct precursor cells of other endocrine cell types to change their identity and differentiate into beta cells. Conversely, inactivation of the transcription factor in endocrine precursors prevents their differentiation into beta cells and results in excess production of other endocrine cell types. When the factor is specifically inactivated in beta cells, beta cells lose their identity and adopt characteristics of other endocrine cell types, similar to what is seen in animal models of diabetes. Thus, we have identified a single factor that is both sufficient to program beta cells and necessary for maintaining their differentiated state. This factor could be an important target for diabetes therapy and could help reprogram other cell types into beta cells.

ADA (alteration/deficiency in activation) 3 is a conserved component of several transcriptional adaptor and HAT (histone acetyltransferase) complexes that regulate RNA polymerase IImediated gene expression. Within the HAT complexes ADA3 is associated with ADA2 and the HAT GCN5 (general control non-repressed 5). ADA3 plays roles in diverse cellular processes and also in malignancies by modulating GCN5 catalytic activity and/or by interactions with other regulators. To gain a better understanding of ADA3 function, we used a yeast two-hybrid approach to screen a human fetal cDNA library for proteins that interacted with hADA3 (human ADA3). We identified three novel hADA3-interacting partners, a transcriptional regulator, AATF (apoptosis-antagonizing transcription factor), and regulatory subunits of the PP1 (protein phosphatase 1) and PP2A (protein phosphatase 2A) [PPP1R7 (PP1 regulatory subunit 7) and PPP2R5D (PP2A 56 kDa regulatory subunit δ isoform) respectively]. Analysis of truncated versions of hADA3 indicated that the C-terminal ADA2-interacting domain was not required for these interactions. Fluorescent microscopy analysis and co-immunoprecipitation provided support for the co-localization and interaction of hADA3 with these proteins in human cells. Expression of the interacting proteins altered expression of an hADA3-regulated reporter gene, suggesting functional consequences for the interactions. The detected interactions of hADA3 might extend the spectrum of mechanisms by which ADA3 can contribute to the regulation of gene expression and shed light on processes mediated by these newly identified ADA3 partners. © 2013 Biochemical Society.

Brown adipose tissue (BAT) uses the chemical energy of lipids and glucose to produce heat, a function that can be induced by cold exposure or diet. A key regulator of BAT is the gene encoding PR domain containing 16 (Prdm16), whose expression can drive differentiation of myogenic and white fat precursors to brown adipocytes. Here we show that after cold exposure, the muscle-enriched miRNA-133 is markedly downregulated in BAT and subcutaneous white adipose tissue (SAT) as a result of decreased expression of its transcriptional regulator Mef2. miR-133 directly targets and negatively regulates PRDM16, and inhibition of miR-133 or Mef2 promotes differentiation of precursors from BAT and SAT to mature brown adipocytes, thereby leading to increased mitochondrial activity. Forced expression of miR-133 in brown adipogenic conditions prevents the differentiation to brown adipocytes in both BAT and SAT precursors. Our results point to Mef2 and miR-133 as central upstream regulators of Prdm16 and hence of brown adipogenesis in response to cold exposure in BAT and SAT.

Major histocompatibility complex (MHC) class II deficiency, or bare lymphocyte syndrome (BLS), is a disease of gene regulation. Patients with BLS have been classified into at least three complementation groups (A, B, and C) believed to correspond to three distinct MHC class II regulatory genes. The elucidation of the molecular basis for this disease will thus clarify the mechanisms controlling the complex regulation of MHC class II genes. Complementation groups B and C are characterized by a lack of binding of RFX, a nuclear protein that normally binds specifically to the X box cis-acting element present in the promoters of all MHC class II genes. We have now purified RFX to near homogeneity by affinity chromatography. Using an in vitro transcription system based on the HLA-DRA promoter, we show here that extracts from RFX-deficient cells from patients with BLS (BLS cells) in groups B and C, which are transcriptionally inactive in this assay, can be complemented to full transcriptional activity by the purified RFX. As expected, purified RFX also restores a completely normal pattern of X box-binding complexes in these mutant extracts. This provides the first direct functional evidence that RFX is an activator of MHC class II gene transcription and that its absence is indeed responsible for the regulatory defect in MHC class II gene expression in patients with BLS.

The Hedgehog (Hh)-Gli signaling pathway is an essential pathway involved in development and cancer. It controls the Gli code-the sum of all activator and repressor functions of the Gli transcription factors. Through the Gli code, and Gli1 in particular, it modulates the fate and behavior of stem and cancer stem cells, as well as tumor growth and survival in many human cancer types. It also affects recurrence and metastasis and is enhanced in advanced tumors, where it promotes an embryonic stem (ES) cell-like gene expression signature. A central component of this signature, Nanog, is critical for glioblastoma and cancer stem cell survival and expansion. Gli1 activity is also enhanced by several oncogenic proteins, including Ras, Myc, and Akt, and by loss of tumor suppressors, such as p53 and PTEN. The oncogenic load boosts Gli1 levels, which supports tumor progression, and promotes a critical threshold of Gli1 activity that allows cells to enter the metastatic transition. In colon cancers, this transition is defined by enhanced Hh-Gli and, surprisingly, by repressed Wnt-Tcf signaling. Together our data support a model in which the Gli code, and Gli1 in particular, acts as a key sensor that responds to both Hh signals and the oncogenic load. We hypothesize that, in turn, the Gli-regulated ES-like factors induce a reprogramming event in cancer stem cells that promotes high invasion, growth and/or metastasis. Targeting the Gli code, the autoregulatory Gli1-Nanog module and interacting partners and pathways thus offers new therapeutic possibilities.

Resveratrol, a polyphenol compound, is known for its effects on energy homeostasis. With properties of energy sensors mediating effects of calorie restriction, sirtuins are targets of resveratrol. The mammalian sirtuin homolog SIRT1 is a protein deacetylase playing a role in glucose metabolism and islet function. Here, we investigated the effects of resveratrol and possible link with SIRT1 in β-cells. Insulinoma INS-1E cells and human islets were cultured with resveratrol before analyzing their physiological responses. Treatment of INS-1E cells for 24 h with 25 μM resveratrol resulted in marked potentiation of glucose-stimulated insulin secretion. This effect was associated with elevated glycolytic flux, resulting in increased glucose oxidation, ATP generation, and mitochondrial oxygen consumption. Such changes correlated with up-regulation of key genes for β-cell function, i.e. Glut2, glucokinase, Pdx-1, Hnf-1α, and Tfam. In human islets, chronic resveratrol treatment similarly increased both the glucose secretory response and expression of the same set of genes, eventually restoring the glucose response in islets obtained from one type 2 diabetic donor. Overexpression of Sirt1 in INS-1E cells potentiated resveratrol effects on insulin secretion. Conversely, inhibition of SIRT1 achieved either by expression of an inactive mutant or by using the EX-527 inhibitor, both abolished resveratrol effects on glucose responses. Treatment of INS-1E cells with EX-527 also prevented resveratrol-induced up-regulation of Glut2, glucokinase, Pdx-1, and Tfam. Resveratrol markedly enhanced the glucose response of INS-1E cells and human islets, even after removal of the compound from the medium. These effects were mediated by and fully dependent on active SIRT1, defining a new role for SIRT1 in the regulation of insulin secretion.

In a subset of poorly differentiated and highly aggressive carcinoma, a chromosomal translocation, t(15;19)(q13;p13), results in an in-frame fusion of the double bromodomain protein, BRD4, with a testis-specific protein of unknown function, NUT (nuclear protein in testis). In this study, we show that, after binding to acetylated chromatin through BRD4 bromodomains, the NUT moiety of the fusion protein strongly interacts with and recruits p300, stimulates its catalytic activity, initiating cycles of BRD4-NUT/p300 recruitment and creating transcriptionally inactive hyperacetylated chromatin domains. Using a patient-derived cell line, we show that p300 sequestration into the BRD4-NUT foci is the principal oncogenic mechanism leading to p53 inactivation. Knockdown of BRD4-NUT released p300 and restored p53-dependent regulatory mechanisms leading to cell differentiation and apoptosis. This study demonstrates how the off-context activity of a testis-specific factor could markedly alter vital cellular functions and significantly contribute to malignant cell transformation.

Cellular functions including transcription regulation, DNA repair, and DNA replication need to be tightly regulated. DNA sequence can contribute to the regulation of these mechanisms. This is exemplified by the consensus sequences that allow the binding of specific transcription factors, thus regulating transcription rates. Another layer of regulation resides in modifications that do not affect the DNA sequence itself but still results in the modification of chromatin structure and properties, thus affecting the readout of the underlying DNA sequence. These modifications are dubbed as "epigenetic modifications" and include, among others, histone modifications, DNA methylation, and small RNAs. While these events can independently regulate cellular mechanisms, recent studies indicate that joint activities of different epigenetic modifications could result in a common outcome. In this chapter, I will attempt to recapitulate the best known examples of collaborative activities between epigenetic modifications. I will emphasize mostly on the effect of crosstalks between epigenetic modifications on transcription regulation, simply because it is the most exposed and studied aspect of epigenetic interactions. I will also summarize the effect of epigenetic interactions on DNA damage response and DNA repair. The involvement of epigenetic crosstalks in cancer formation, progression, and treatment will be emphasized throughout the manuscript. Due to space restrictions, additional aspects involving histone replacements [Park, Y. J., and Luger, K. (2008). Histone chaperones in nucleosome eviction and histone exchange. Curr. Opin. Struct. Biol.18, 282-289.], histone variants [Boulard, M., Bouvet, P., Kundu, T. K., and Dimitrov, S. (2007). Histone variant nucleosomes: Structure, function and implication in disease. Subcell. Biochem. 41, 71-89; Talbert, P. B., and Henikoff, S. (2010). Histone variants-Ancient wrap artists of the epigenome. Nat. Rev. Mol. Cell Biol.11, 264-275.], and histone modification readers [de la Cruz, X., Lois, S., Sanchez-Molina, S., and Martinez-Balbas, M. A. (2005). Do protein motifs read the histone code? Bioessays27, 164-175; Grewal, S. I., and Jia, S. (2007). Heterochromatin revisited. Nat. Rev. Genet.8, 35-46.] will not be addressed in depth in this chapter, and the reader is referred to the reviews cited here.

Hypoxia-inducible factor 1 (HIF-1), a transcriptional activator that mediates cellular response to hypoxia and a promising target of anticancer therapy, is essential for adaptation to low oxygen conditions, embryogenesis and tumor progression. HIF-1 is a heterodimer of HIF-1alpha, expression of which is controlled by oxygen levels as well as by various oxygen-independent mechanisms, and HIF-1beta (or ARNT), which is constitutively expressed. In this work, we investigate the phosphorylation of the N-terminal heterodimerization (PAS) domain of HIF-1alpha and identify Ser247 as a major site of in vitro modification by casein kinase 1delta (CK1delta). Mutation of this site to alanine, surprisingly, enhanced the transcriptional activity of HIF-1alpha, a result phenocopied by inhibition or small interfering RNA (siRNA)-mediated silencing of CK1delta under hypoxic conditions. Conversely, overexpression of CK1delta or phosphomimetic mutation of Ser247 to aspartate inhibited HIF-1alpha activity without affecting its stability or nuclear accumulation. Immunoprecipitation and in vitro binding experiments suggest that CK1-dependent phosphorylation of HIF-1alpha at Ser247 impairs its association with ARNT, a notion also supported by modeling the structure of the complex between HIF-1alpha and ARNT PAS-B domains. We suggest that modification of HIF-1alpha by CK1 represents a novel mechanism that controls the activity of HIF-1 during hypoxia by regulating the interaction between its two subunits. J Cell Sci

The X box of major histocompatibility complex class II promoters is essential for proper expression of class II genes. Here we show that two distinct protein-DNA complexes (A and B), which exhibit similar binding characteristics and identical contact points on the X box, can be formed. This suggests the existence of a family of related X box-binding factors. Complex B (and not complex A) is specifically affected in primary combined immunodeficiency, a congenital defect in class II gene regulation. RFX1, the first X box-binding protein cloned, encodes a functionally relevant factor present in complex A and not in complex B as originally suspected. This report also illustrates the need for caution in correlating specific cloned proteins with nuclear factors identified by DNA-binding assays, particularly when dealing with families of related proteins.

Mutations in the transcription factor Pdx1 cause maturity-onset diabetes of the young 4 (MODY4). Islet transduction with dominant-negative Pdx1 (RIPDN79PDX1) impairs mitochondrial metabolism and glucose-stimulated insulin secretion (GSIS). Transcript profiling revealed suppression of nuclear-encoded mitochondrial factor A (TFAM). Herein, we show that Pdx1 suppression in adult mice reduces islet TFAM expression coinciding with hyperglycemia. We define TFAM as a direct target of Pdx1 both in rat INS1 cells and human islets. Adenoviral overexpression of TFAM along with RIPDN79PDX1 in isolated rat islets rescued mitochondrial DNA (mtDNA) copy number and restored respiratory chain activity as well as glucose-induced ATP synthesis and insulin secretion. CGP37157, which blocks the mitochondrial Na(+)/Ca(2+) exchanger, restored ATP generation and GSIS in RIPDN79PDX1 islets, thereby bypassing the transcriptional defect. Thus, the genetic control by the beta cell-specific factor Pdx1 of the ubiquitous gene TFAM maintains beta cell mtDNA vital for ATP production and normal GSIS.

OBJECTIVES: Transplantation of in vitro generated islets or insulin-producing cells represents an attractive option to overcome organ shortage. The aim of this study was to isolate, expand, and characterize cells from human exocrine pancreas and analyze their potential to differentiate into beta cells. METHODS: Fibroblast-like cells growing out of human exocrine tissue were characterized by flow cytometry and by their capacity to differentiate into mesenchymal cell lineages. During cell expansion and after differentiation toward beta cells, expression of transcription factors of endocrine pancreatic progenitors was analyzed by reverse transcription polymerase chain reaction. RESULTS: Cells emerged from 14/18 human pancreatic exocrine fractions and were expanded up to 40 population doublings. These cells displayed surface antigens similar to mesenchymal stem cells from bone marrow. A culture of these cells in adipogenic and chondrogenic differentiation media allowed differentiation into adipocyte- and chondrocyte-like cells. During expansion, cells expressed transcription factors implicated in islet development such as Isl1, Nkx2.2, Nkx6.1, nestin, Ngn3, Pdx1, and NeuroD. Activin A and hepatocyte growth factor induced an expression of insulin, glucagon, and glucokinase. CONCLUSIONS: Proliferating cells with characteristics of mesenchymal stem cells and endocrine progenitors were isolated from exocrine tissue. Under specific conditions, these cells expressed little insulin. Human pancreatic exocrine tissue might thus be a source of endocrine cell progenitors.

In certain cell types, apoptosis in response to extracellular stimuli like Fas depends on a mitochondrial amplificatory loop: the apical caspase-8 cleaves and activates the BH3-only member of the Bcl-2 family BID. In turn, BID induces the release of cytochrome c from mitochondria to the cytoplasm, where it is required to fully activate effector caspases. In this issue of The Journal of Cell Biology, Gonzalvez et al. (see p. 681) show that when caspase-8 activation and production of functional BID is required, it is performed on mitochondrial platforms provided by the mitochondrion-specific lipid cardiolipin. Cardiolipin anchors caspase-8 at contact sites between inner and outer mitochondrial membranes, facilitating its self activation. These findings suggests that like other second messengers such as Ca2+ and cAMP, production of apoptotic messengers can be compartmentalized in close proximity to their intracellular target.

International audience; Transactivation-transformation domain-associated protein (TRRAP) is a component of several multiprotein histone acetyltransferase (HAT) complexes implicated in transcriptional regulation. TRRAP was shown to be required for the mitotic checkpoint and normal cell cycle progression. MRE11, RAD50, and NBS1 (product of the Nijmegan breakage syndrome gene) form the MRN complex that is involved in the detection, signaling, and repair of DNA double-strand breaks (DSBs). By using double immunopurification, mass spectrometry, and gel filtration, we describe the stable association of TRRAP with the MRN complex. The TRRAP-MRN complex is not associated with any detectable HAT activity, while the isolated other TRRAP complexes, containing either GCN5 or TIP60, are. TRRAP-depleted extracts show a reduced nonhomologous DNA end-joining activity in vitro. Importantly, small interfering RNA knockdown of TRRAP in HeLa cells or TRRAP knockout in mouse embryonic stem cells inhibit the DSB end-joining efficiency and the precise nonhomologous end-joining process, further suggesting a functional involvement of TRRAP in the DSB repair processes. Thus, TRRAP may function as a molecular link between DSB signaling, repair, and chromatin remodeling.

Prostate cancer is the most common solid tumor in men, and it shares with all cancers the hallmark of elevated, nonhomeostatic cell proliferation. Here we have tested the hypothesis that the SONIC HEDGEHOG (SHH)–GLI signaling pathway is implicated in prostate cancer. We report expression of SHH–GLI pathway components in adult human prostate cancer, often with enhanced levels in tumors versus normal prostatic epithelia. Blocking the pathway with cyclopamine or anti-SHH antibodies inhibits the proliferation of GLI1 + / PSA + primary prostate tumor cultures. Inversely, SHH can potentiate tumor cell proliferation, suggesting that autocrine signaling may often sustain tumor growth. In addition, pathway blockade in three metastatic prostate cancer cell lines with cyclopamine or through GLI1 RNA interference leads to inhibition of cell proliferation, suggesting cell-autonomous pathway activation at different levels and showing an essential role for GLI1 in human cells. Our data demonstrate the dependence of prostate cancer on SHH–GLI function and suggest a novel therapeutic approach.

The Williams-Beuren syndrome (WBS) is a contiguous gene syndrome caused by chromosomal rearrangements at chromosome band 7q11.23. Several endocrine phenotypes, in particular impaired glucose tolerance and silent diabetes, have been described for this clinically complex disorder. The WBSCR14 gene, one of the genes mapping to the WBS critical region, encodes a member of the basic-helix-loop-helix leucine zipper family of transcription factors, which dimerizes with the Max-like protein, Mlx. This heterodimeric complex binds and activates, in a glucose-dependent manner, carbohydrate response element (ChoRE) motifs in the promoter of lipogenic enzymes. We identified five novel WBSCR14-interacting proteins, four 14-3-3 isotypes and NIF3L1, which form a single polypeptide complex in mammalian cells. Phosphatase treatment abrogates the association between WBSCR14 and 14-3-3, as shown previously for multiple 14-3-3 interactors. WBSCR14 is exported actively from the nucleus through a CRM1-dependent mechanism. This translocation is contingent upon the ability to bind 14-3-3. Through this mechanism the 14-3-3 isotypes directly affect the WBSCR14:Mlx complexes, which activate the transcription of lipogenic genes.

Two hereditary human leukemia syndromes are severe congenital neutropenia (SCN), caused by mutations in the gene ELA2, encoding the protease neutrophil elastase, and familial platelet disorder with acute myelogenous leukemia (AML), caused by mutations in the gene AML1, encoding the transcription factor core-binding factor alpha (CBFalpha). In mice, CBFalpha regulates the expression of ELA2, suggesting a common link for both diseases. However, gene-targeted mouse models have failed to reproduce either human disease, thus prohibiting further in vivo studies in mice. Here we investigate CBFalpha regulation of the human ELA2 promoter, taking advantage of bone marrow obtained from patients with either illness. In particular, we have identified novel ELA2 promoter substitutions (-199 C to A) within a potential motif for lymphoid enhancer factor-1 (LEF-1), a transcriptional mediator of Wnt/beta-catenin signaling, in SCN patients. The LEF-1 motif lies adjacent to a potential CBFalpha binding site that is in a different position in human compared with mouse ELA2. We find that LEF-1 and CBFalpha co-activate ELA2 expression. In vitro, the high mobility group domain of LEF-1 interacts with the runt DNA binding and proline-, serine-, threonine-rich activation domains of CBFalpha. ELA2 transcript levels are up-regulated in bone marrow of an SCN patient with the -199 C to A substitution. Conversely, a mutation of the CBFalpha activation domain, found in a patient with familial platelet disorder with AML, fails to stimulate the ELA2 promoter in vitro, and bone marrow correspondingly demonstrates reduced ELA2 transcript. Observations in these complementary patients indicate that LEF-1 cooperates with CBFalpha to activate ELA2 in vivo and also suggest the possibility that up-regulating promoter mutations can contribute to SCN. Two hereditary AML predisposition syndromes may therefore intersect via LEF-1, potentially linking them to more generalized cancer mechanisms.

Diminished Sonic Hedgehog (Shh) signaling is associated with the most common forebrain defect in humans, holoprosencephaly (HPE), which includes cyclopia, a phenotype also seen in mice and other vertebrates with defective Shh signaling. The secreted protein Shh acts as a crucial factor that patterns the ventral forebrain and is required for the division of the primordial eye field and brain into two discrete halves. Gli2 is one of three vertebrate transcription factors implicated as obligatory mediators of Shh signal transduction. Here, we show that loss-of-function mutations in the human GLI2 gene are associated with a distinctive phenotype (within the HPE spectrum) whose primary features include defective anterior pituitary formation and pan-hypopituitarism, with or without overt forebrain cleavage abnormalities, and HPE-like midfacial hypoplasia. We also demonstrate that these mutations lack GLI2 activity. We report on a functional association between GLI2 and human disease and highlight the role of GLI2 in human head development.

The Sonic hedgehog (Shh) pathway plays an important role in the development of many tissues and organs. The secreted ligand Shh has been shown to act as a mitogen, morphogen and survival factor in different contexts whereas the three Gli transcription factors act as Shh mediators in a context-dependent combinatorial fashion. The common wisdom has been that Gli protein function is subject to Shh signaling. One can ask how Gli proteins act and what the nature of Shh signaling during CNS dorsal-ventral patterning is. Is it possible that Hedgehog signals are only one of several ways to regulate Gli activity? Moreover, in light of the partial rescue of the neural tube phenotype of Shh or Smoothened mutant embryos in Shh(-/-);Gli3(-/-), Smoothened(-/-);Gli3(-/-), and Shh(-/-);Rab23(-/-) double null embryos, one can consider the roles that the Shh-Gli pathway may have taken to orchestrate congruent prepattern and growth, and the importance of creating the correct number of precursors in patterning mechanisms.

Gliomas are highly resistant to conventional treatment. Improved knowledge of the molecular defects of glioma cells offers new avenues for the development of gene therapy strategies. Transfer of the p53 gene has proven effective in suppressing proliferation in human glioma cell lines. However, several human glioma cell lines are resistant to p53-induced cell death. The E2F family of transcription factors are pivotal for the regulation of cell-cycle and cell-death related genes in gliomas. In the present study, we sought a more effective strategy for glioma treatment by examining the therapeutic potential of the simultaneous transfer of p53 and E2F-2 to gliomas. Trypan blue cell viability assays and flow cytometric cell-cycle analysis demonstrated that the transfer of both p53 and E2F-2 induced cell death in D-54 MG, a p53-resistant glioma cell line. In addition, transfer of E2F-2 did not interfere with the apoptotic properties of exogenous wild-type p53 in U-251 MG cells. Finally, the expression of E2F-2 in D-54 MG cells suppressed the expression of the apoptotic molecule mdm-2 induced by exogenous p53 in these cells. These results show that co-expression of E2F-2 and p53 enhances the anti-cancer effect of p53 in gliomas. Int J Oncol

Major histocompatibility complex class II (MHCII) molecules drive the development, activation and homeostasis of CD4* T-helper cells. They play a central role in key processes of the adaptive immune system, such as the generation of T-cell-mediated immune responses, the regulation of antibody production and the development and maintenance of tol erance. It is thus not surprising that the absence of MHCII expression results in a severe primary immunodeficiency disease (the bare lymphocyte syndrome (BLS)). The genetic defects responsible for BLS do not lie within the MHCII locus, but in genes encoding transcription factors required for MHCII expression. A great deal of our current knowledge about the mechanisms regulating expression of MHCII genes has been derived from the study of BLS. Four different MHCII regulatory genes have been identified. These genes encode RFXANK, RFXS, RFXAP and CIITA. The first three are subunits of RFX, a ubiquitously expressed factor that binds to the promoters of all MHCII genes. RFX binds co-operatively with other factors to form a highly stable multiprotein complex referred to as the MHCII enhanceosome. This enhanceosome serves as a landing pad for the co-activator CIITA, which is recruited via protein-protein interactions CIITA is the master control factor for MHCII expression. The highly regulated expression pattern of CIITA ultimately dictates the cell type specificity, induction and level of MHCII expression.

Mutations in the hepatocyte nuclear factor-1alpha (HNF-1alpha) have been linked to subtype 3 of maturity-onset diabetes of the young (MODY3), which is characterized by a primary defect in insulin secretion. The role of HNF-1alpha in the regulation of pancreatic beta-cell function was investigated. Gene manipulation allowed graded overexpression of HNF-1alpha and controlled dominant-negative suppression of HNF-1alpha function in insulinoma INS-1 cells. We show that HNF-1alpha is essential for insulin gene transcription, as demonstrated by a pronounced decrease in insulin mRNA expression and in insulin promoter activity under dominant-negative conditions. The expression of genes involved in glucose transport and metabolism including glucose transporter-2 and L-type pyruvate kinase is also regulated by HNF-1alpha. Loss of HNF-1alpha function leads to severe defects in insulin secretory responses to glucose and leucine, resulting from impaired glucose utilization and mitochondrial oxidation. The nutrient-evoked ATP production and subsequent changes in plasma membrane potential and intracellular Ca2+ were diminished by suppression of HNF-1alpha function. These results suggest that HNF-1alpha function is essential for maintaining insulin storage and nutrient-evoked release. The defective mitochondrial oxidation of metabolic substrates causes impaired insulin secretion, indicating a molecular basis for the diabetic phenotype of MODY3 patients.

Sporadic basal cell carcinoma (BCC) is the most common type of malignant cancer in fair-skinned adults. Familial BCCs and a fraction of sporadic BCCs have lost the function of Patched (Ptc), a Sonic hedgehog (Shh) receptor that acts negatively on this signalling pathway. Overexpression of Shh can induce BCCs in mice. Here we show that ectopic expression of the zinc-finger transcription factor Gli1 in the embryonic frog epidermis results in the development of tumours that express endogenous Gli1. We also show that Shh and the Gli genes are normally expressed in hair follicles, and that human sporadic BCCs consistently express Gli1 but not Shh or Gli3. Because Gli1, but not Gli3, acts as a target and mediator of Shh signalling, our results suggest that expression of Gli1 in basal cells induces BCC formation. Moreover, loss of Ptc or overexpression of Shh cannot be the sole causes of Gli1 induction and sporadic BCC formation, as they do not occur consistently. Thus any mutations leading to the expression of Gli1 in basal cells are predicted to induce BCC formation.

Hepatitis B virus gene expression is to a large extent under the control of enhancer I (EnhI). The activity of EnhI is strictly dependent on the enhancer factor C (EF-C) site, an inverted repeat that is bound by a ubiquitous nuclear protein known as EF-C. Here we report the unexpected finding that EF-C is in fact identical to RFX1, a novel transcription factor previously cloned by virtue of its affinity for the HLA class II X-box promoter element. This finding has allowed us to provide direct evidence that RFX1 (EF-C) is crucial for EnhI function in HepG2 hepatoma cells; RFX1-specific antisense oligonucleotides appear to inhibit EnhI-driven expression of the hepatitis B virus major surface antigen gene, and in transfection assays, RFX1 behaves as a potent transactivator of EnhI. Interestingly, transactivation of EnhI by RFX1 (EF-C) is not observed in cell lines that are not of liver origin, suggesting that the ubiquitous RFX1 protein cooperates with liver-specific factors.

To study the molecular mechanisms of circadian gene expression, we have sought to identify genes whose expression in mouse liver is regulated by the transcription factor DBP (albumin D-site-binding protein). This PAR basic leucine zipper protein accumulates according to a robust circadian rhythm in nuclei of hepatocytes and other cell types. Here, we report that the Cyp2a4 gene, encoding the cytochrome P450 steroid 15alpha-hydroxylase, is a novel circadian expression gene. This enzyme catalyzes one of the hydroxylation reactions leading to further metabolism of the sex hormones testosterone and estradiol in the liver. Accumulation of CYP2A4 mRNA in mouse liver displays circadian kinetics indistinguishable from those of the highly related CYP2A5 gene. Proteins encoded by both the Cyp2a4 and Cyp2a5 genes also display daily variation in accumulation, though this is more dramatic for CYP2A4 than for CYP2A5. Biochemical evidence, including in vitro DNase I footprinting on the Cyp2a4 and Cyp2a5 promoters and cotransfection experiments with the human hepatoma cell line HepG2, suggests that the Cyp2a4 and Cyp2a5 genes are indeed regulated by DBP. These conclusions are corroborated by genetic studies, in which the circadian amplitude of CYP2A4 and CYP2A5 mRNAs and protein expression in the liver was significantly impaired in a mutant mouse strain homozygous for a dbp null allele. These experiments strongly suggest that DBP is a major factor controlling circadian expression of the Cyp2a4 and Cyp2a5 genes in the mouse liver.