Response Elements

The murine 2′-5′ oligoadenylate synthetase 1a (Oas1a) and Oas1b genes are type 1 IFN responsive genes. Oas1a is an active synthetase with broad antiviral activity mediated through RNase L. Oas1b is inactive but can inhibit Oas1a synthetase activity and mediate a flavivirus-specific antiviral activity through an unknown RNase L-independent mechanism. Analysis of promoter elements regulating gene transcription confirmed that an IFN-stimulated response element (ISRE) is required for IFN beta-activation but neither the overlapping IRF binding site present in both promoters nor the adjacent Oas1b NF-kappa B site is required. Mutation of the overlapping STAT site negatively affected IFN beta-induction of Oas1a but not of Oas1b. Also, IFN beta induction of Oas1a was STAT1-and STAT2-dependent, while induction of Oas1b was STAT1-independent but STAT2-dependent. The two promoters differ at a single nucleotide in the STAT site. The data indicate that these two duplicated genes can be differentially regulated by IFN beta.

The yeast Saccharomyces cerevisiae responds to environmental stress by rapidly altering the expression of large sets of genes. We report evidence that the transcriptional repressors Nrg1 and Nrg2 (Nrg1/Nrg2), which were previously implicated in glucose repression, regulate a set of stress-responsive genes. Genome-wide expression analysis identified 150 genes that were upregulated in nrg1Δ nrg2Δ double mutant cells, relative to wild-type cells, during growth in glucose. We found that many of these genes are regulated by glucose repression. Stress response elements (STREs) and STRE-like elements are overrepresented in the promoters of these genes, and a search of available expression data sets showed that many are regulated in response to a variety of environmental stress signals. In accord with these findings, mutation of NRG1 and NRG2 enhanced the resistance of cells to salt and oxidative stress and decreased tolerance to freezing. We present evidence that Nrg1/Nrg2 not only contrib...

A central mechanism in cellular defence against oxidative or electrophilic stress is mediated by transcriptional induction of genes via the ARE (antioxidant-response element), a cis-acting sequence present in the regulatory regions of genes involved in the detoxification and elimination of reactive oxidants and electrophiles. The ARE binds different bZIP (basic-region leucine zipper) transcription factors, most notably Nrf2 (nuclear factor-erythroid 2-related factor 2) that functions as a transcriptional activator via heterodimerization with small Maf proteins. Although ARE activation by Nrf2 is relatively well understood, the mechanisms by which ARE-mediated signalling is down-regulated are poorly known. Transcription factor BACH1 [BTB (broad-complex, tramtrack and bric-a-brac) and CNC (cap'n'collar protein) homology 1] binds to ARE-like sequences, functioning as a transcriptional repressor in a subset of ARE-regulated genes, thus antagonizing the activator function of Nrf2...

The transforming growth factor (TGF)-␤/Smad3 signaling pathway is considered a central mediator of pathological organ fibrosis; however, contribution of Smad2/3-independent TGF-␤ signaling has not been fully explored. The present study utilized previously a described model of scleroderma (SSc) fibrosis based on forced expression of the TGF-␤RI (ALK5) (Pannu, J., Gardner, H., Shearstone, J. R., Smith, E., and Trojanowska, M. (2006) Arthritis Rheum. 54, 3011-3021). This study was aimed at determining the molecular mechanisms underlying the profibrotic program in this model. We demonstrate that the TGF-␤RI-dependent up-regulation of collagen and CCN2 (CTGF) does not involve Smad2/3 activation but is mediated by ALK1/Smad1 and ERK1/2 pathways. The following findings support this conclusion: (i) Smad2 and -3 were not phosphorylated in response to TGF-␤RI, (ii) a TGF-␤RI mutant defective in Smad2/3 activation, ALK5(3A), potently stimulated collagen production, (iii) elevation of TGF-␤RI triggered sustained association of ALK5 with ALK1 and high levels of Smad1 phosphorylation, (iv) blockade of Smad1 via small interfering RNA abrogated collagen and CCN2 up-regulation in this model, (v) elevated TGF-␤RI led to a prolonged activation of ERK1/2, (vi) the pharmacologic inhibitor of ERK1/2 inhibited Smad1 phosphorylation and abrogated profibrotic effects of elevated TGF␤-RI. Additional experiments demonstrated that a GC-rich response element located ؊6 to ؊16 (upstream of the transcription start site) in the CCN2 promoter mediated Smad1-dependent increased promoter activity in this model. This element was shown previously to mediate up-regulation of the CCN2 promoter in SSc fibroblasts. In conclusion, this study defines a novel ALK1/Smad1-and ERK1/2-dependent, Smad3-independent mode of TGF-␤ signaling that may operate during chronic stages of fibrosis in SSc. Transforming growth factor-␤ (TGF-␤) 2 is a pleiotropic cytokine with diverse functions in many cell types (1). The Smad pathway is a central mediator of TGF-␤ signaling (2). The canonical Smad pathway is activated upon binding of TGF-␤ to the heteromeric serine-threonine kinase receptors, TGF-␤RII and -RI (ALK5), which leads to phosphorylation of Smad2 and -3, oligomerization with Smad4, nuclear translocation, and activation of various transcription programs (1). Furthermore, a more complex TGF-␤ signaling has recently been described in endothelial cells, in which TGF-␤ signals through two distinct type I receptors, ALK5 and ALK1, and their respective signal transducers: Smad2 and Ϫ3 for ALK5 and Smad1 and Ϫ5 for ALK1 (3). There is also evidence for various modifications of basic TGF-␤/Smad signaling, especially in pathological conditions such as cancer or fibrosis (2). Whereas the canonical TGF-␤/Smad pathway has been studied in detail, other modes of TGF-␤ signaling are still poorly understood. Because TGF-␤ is a potent inducer of extracellular matrix (ECM) synthesis, it has long been considered a primary mediator of organ fibrosis (4). Numerous studies have demonstrated that blockade of TGF-␤ signaling ameliorated fibrotic response in various experimental models of fibrosis (5-9). Moreover, a specific role of Smad3 in fibrosis was demonstrated in mice lacking Smad3 (10). Smad3-null mice showed reduction of fibrosis in different organs, including lung, skin, liver, and kidney (10). Surprisingly, however, production of ECM was unaffected during cutaneous wound healing in Smad3-null mice, suggesting the existence of Smad3-independent mechanisms involved in physiologic tissue repair by dermal fibroblasts (11). Animal models, at best, represent only certain aspects of human fibrotic diseases, which are chronic and progressive and may take many years to develop. Despite numerous studies, the specific contribution of TGF-␤ signaling to the human fibrosis has not been fully elucidated and, in particular, the basis for the chronic nature of TGF-␤ signaling in fibrosis remains unknown. One of the characteristics of fibrotic lesions is the aberrant expression of TGF-␤ receptors. For example, an increased ratio of TGF-␤RI to RII expression levels have been

Cellular retinol-binding protein (CRBP) type III (CRBP-III) belongs to the family of intracellular lipid-binding proteins, which includes the adipocyte-binding protein aP2. In the cytosol, CRBP-III binds retinol, the precursor of retinyl ester and the active metabolite retinoic acid. The goal of the present work is to understand the regulation of CRBP-III expression and its role in lipid metabolism. Using EMSAs, luciferase reporter assays, and chromatin immunoprecipitation assays, we found that CRBP-III is a direct target of peroxisome proliferator-activated receptor-γ (PPARγ). Moreover, CRBP-III expression was induced in adipose tissue of mice after treatment with the PPARγ agonist rosiglitazone. To examine a potential role of CRBP-III in regulating lipid metabolism in vivo, CRBP-III-deficient (C-III-KO) mice were maintained on a high-fat diet (HFD). Hepatic steatosis was decreased in HFD-fed C-III-KO compared with HFD-fed wild-type mice. These differences were partly explained by ...

lular retinol-binding protein (CRBP) type III (CRBP-III) belongs to the family of intracellular lipid-binding proteins, which includes the adipocyte-binding protein aP2. In the cytosol, CRBP-III binds retinol, the precursor of retinyl ester and the active metabolite retinoic acid. The goal of the present work is to understand the regulation of CRBP-III expression and its role in lipid metabolism. Using EMSAs, luciferase reporter assays, and chromatin immunoprecipitation assays, we found that CRBP-III is a direct target of peroxisome proliferatoractivated receptor-␥ (PPAR␥). Moreover, CRBP-III expression was induced in adipose tissue of mice after treatment with the PPAR␥ agonist rosiglitazone. To examine a potential role of CRBP-III in regulating lipid metabolism in vivo, CRBP-III-deficient (C-III-KO) mice were maintained on a high-fat diet (HFD). Hepatic steatosis was decreased in HFD-fed C-III-KO compared with HFD-fed wild-type mice. These differences were partly explained by decreased serum free fatty acid levels and decreased free fatty acid efflux from adipose tissue of C-III-KO mice. In addition, the lack of CRBP-III was associated with reduced food intake, increased respiratory energy ratio, and altered body composition, with decreased adiposity and increased lean body mass. Furthermore, expression of genes involved in mitochondrial fatty acid oxidation in brown adipose tissue was increased in C-III-KO mice, and C-III-KO mice were more cold tolerant than wild-type mice fed an HFD. In summary, we demonstrate that CRBP-III is a PPAR␥ target gene and plays a role in lipid and whole body energy metabolism.

Overexpression of sterol regulatory element-binding protein-1c (SREBP-1c) in ␤ cells causes impaired insulin secretion and ␤ cell dysfunction associated with diminished pancreatic duodenal homeodomain transcription factor-1 (PDX-1) expression in vitro and in vivo. To identify the molecular mechanism responsible for this effect, the mouse Pdx-1 gene promoter (2.7 kb) was analyzed in ␤ cell and non-␤ cell lines. Despite no apparent sterol regulatory element-binding protein-binding sites, the Pdx-1 promoter was suppressed by SREBP-1c in ␤ cells in a dose-dependent manner. PDX-1 activated its own promoter. The E-box (؊104/؊99 bp) in the proximal region, occupied by ubiquitously expressed upstream stimulatory factors (USFs), was crucial for the PDX-1-positive autoregulatory loop through direct PDX-1⅐USF binding. This positive feedback activation was a prerequisite for SREBP-1c suppression of the promoter in non-␤ cells. SREBP-1c and PDX-1 directly interact through basic helix-loop-helix and homeobox domains, respectively. This robust SREBP-1c⅐PDX-1 complex interferes with PDX-1⅐USF formation and inhibits the recruitment of PDX-1 coactivators. SREBP-1c also inhibits PDX-1 binding to the previously described PDX-1-binding site (؊2721/؊2646 bp) in the distal enhancer region of the Pdx-1 promoter. Endogenous up-regulation of SREBP-1c in INS-1 cells through the activation of liver X receptor and retinoid X receptor by 9-cis-retinoic acid and 22-hydroxycholesterol inhibited PDX-1 mRNA and protein expression. Conversely, SREBP-1c RNAi restored Pdx-1 mRNA and protein levels. Through these multiple mechanisms, SREBP-1c, when induced in a lipotoxic state, repressed PDX-1 expression contributing to the inhibition of insulin expression and ␤ cell dysfunction.

The physiological androgens testosterone and 5␣-dihydrotestosterone regulate the development and maintenance of primary and secondary male sexual characteristics through binding to the androgen receptor (AR), a ligand-dependent transcription factor. In addition, a number of nonreproductive tissues of both genders are subject to androgen regulation. AR is also a central target in the treatment of prostate cancer. A large number of studies over the last decade have characterized many regulatory aspects of the AR pathway, such as androgendependent transcription programs, AR cistromes, and coregulatory proteins, mostly in cultured cells of prostate cancer origin. Moreover, recent work has revealed the presence of pioneer/licensing factors and chromatin modifications that are important to guide receptor recruitment onto appropriate chromatin loci in cell lines and in tissues under physiological conditions. Despite these advances, current knowledge related to the mechanisms responsible for receptor-and tissue-specific actions of androgens is still relatively limited. Here, we review topics that pertain to these specificity issues at different levels, both in cultured cells and tissues in vivo, with a particular emphasis on the nature of the steroid, the response element sequence, the AR cistromes, pioneer/licensing factors, and coregulatory proteins. We conclude that liganded AR and its DNA-response elements are required but are not sufficient for establishment of tissue-specific transcription programs in vivo, and that AR-selective actions over other steroid receptors rely on relaxed rather than increased stringency of cis-elements on chromatin. (Endocrine Reviews 36: 357-384, 2015) I. Introduction II. Steroid-Receptor Interaction A. Physiological steroids vs cognate receptors B. Androgens and selective androgen receptor modulators C. Modified androgens as progestins D. Antiandrogens III. Receptor-Chromatin Interaction A. Overview of steroid receptor cistromes B. Response elements: shared and selective for AR C. The role of ligand in receptor-chromatin interaction D. GR as regulator of the AR pathway E. Competition between receptors for chromatin binding IV. Pioneer Factors in Androgen Signaling A. General outline of pioneering/licensing proteins in steroid signaling B. Receptor specificity of pioneer factors C. Tissue specificity of pioneer factors D. Chromatin modifications and pioneer factors E. Pioneer factors in hormone-dependent cancers V. Coregulatory Proteins in Androgen Signaling A. Overview of coregulatory proteins and their functions B. The role of ligand in coregulator recruitment C. Receptor-and tissue-specificity of coregulatory proteins VI. Concluding Remarks and Future Directions

Although it is well known that RET gene is strongly activated by retinoic acid (RA) in neuroblastoma cells, the mechanisms underlying such activation are still poorly understood. Here we show that a complex series of molecular events, that include modifications of both chromatin and DNA methylation state, accompany RA-mediated RET activation. Our results indicate that the primary epigenetic determinants of RA-induced RET activation differ between enhancer and promoter regions. At promoter region, the main mark of RET activation was the increase of H3K4me3 levels while no significant changes of the methylation state of H3K27 and H3K9 were observed. At RET enhancer region a bipartite chromatin domain was detected in unstimulated cells and a prompt demethylation of H3K27me3 marked RET gene activation upon RA exposure. Moreover, ChIP experiments demonstrated that EZH2 and MeCP2 repressor complexes were associated to the heavily methylated enhancer region in the absence of RA while both complexes were displaced during RA stimulation. Finally, our data show that a demethylation of a specific CpG site at the enhancer region could favor the displacement of MeCP2 from the heavily methylated RET enhancer region providing a novel potential mechanism for transcriptional regulation of methylated RA-regulated loci.

Polychlorinated biphenyls (PCBs) are environmental compounds that disrupt the endocrine system, and exposure to low doses causes abnormalities, particularly in the developing central nervous system. Because they are structurally similar to thyroid hormone (TH), PCBs might affect systems involving this hormone. We previously found, using reporter assays, that hydroxylated-PCB at doses as low as 10–10m suppress TH-induced transcriptional activation of TH receptor (TR). To understand the molecular mechanisms of this process, we examined whether PCBs alter coactivator or corepressor interaction with TR. Polychlorinated biphenyls suppressed steroid receptor coactivator-1 enhanced TR-mediated transcription, but did not reduce TR/steroid receptor coactivator-1 interaction in mammalian two-hybrid and glutathione S-transferase pull-down studies. Thus, the suppression was probably not caused by coactivator dissociation. Nuclear receptor co-repressor was not recruited to TR by PCBs either in v...

In Gram-positive bacteria, the catabolite control protein A (CcpA) functions as the master transcriptional regulator of carbon catabolite repression/regulation (CCR). To effect CCR, CcpA binds a phosphoprotein, either HPr-Ser 46 -P or Crh-Ser 46 -P. Although Crh and histidine-containing protein (HPr) are structurally homologous, CcpA binds Crh-Ser 46 -P more weakly than HPr-Ser 46 -P. Moreover, Crh can form domain-swapped dimers, which have been hypothesized to be functionally relevant in CCR. To understand the molecular mechanism of Crh-Ser 46 -P regulation of CCR, we determined the structure of a CcpA-(Crh-Ser 46 -P)-DNA complex. The structure reveals that Crh-Ser 46 -P does not bind CcpA as a dimer but rather interacts with CcpA as a monomer in a manner similar to that of HPr-Ser 46 -P. The reduced affinity of Crh-Ser 46 -P for CcpA as compared with that of HPr-Ser 46 P is explained by weaker Crh-Ser 46 -P interactions in its contact region I to CcpA, which causes this region to shift away from CcpA. Nonetheless, the interface between CcpA and helix ␣2 of the second contact region (contact region II) of Crh-Ser 46 -P is maintained. This latter finding demonstrates that this contact region is necessary and sufficient to throw the allosteric switch to activate cre binding by CcpA.

Infection with human T-cell leukemia virus type 1 (HTLV-1) is characterized by long latency periods, indicating that viral gene expression is under tight control. There is presently little information available regarding the nature of extracellular stimuli that can transactivate the regulatory elements of HTLV-1 (i.e., long terminal repeat [LTR]). To gain insight into the biological importance of externally induced activation pathways in virus gene expression, primary and established T cells were transfected with HTLV-1-based reporter gene vectors and then were treated with agents that cross-linked the T-cell receptor (TCR) or the costimulatory CD28 molecule with prostaglandin E2 (PGE2). We demonstrated that a potent induction of HTLV-1 LTR-driven reporter gene activity was seen only when the three agents were used in combination. Interestingly, similar observations were made when using C91/PL, a cell line that carries integrated HTLV-1 proviral DNA. This TCR-CD28-PGE2-mediated incr...

Estrogen receptors (ERs ␣ and ␤) enhance transcription in response to estrogens by binding to estrogen response elements (EREs) within target genes and utilizing transactivation functions (AF-1 and AF-2) to recruit p160 coactivator proteins. The ERs also enhance transcription in response to estrogens and antiestrogens by modulating the activity of the AP-1 protein complex. Here, we examine the role of AF-1 and AF-2 in ER action at AP-1 sites. Estrogen responses at AP-1 sites require the integrity of the ER␣ AF-1 and AF-2 activation surfaces and the complementary surfaces on the p160 coactivator GRIP1 (glucocorticoid receptor interacting protein 1), the NID/AF-1 region, and NR boxes. Thus, estrogen-liganded ER␣ utilizes the same protein-protein contacts to transactivate at EREs and AP-1 sites. In contrast, antiestrogen responses are strongly inhibited by ER␣ AF-1 and weakly inhibited by AF-2. Indeed, ER␣ truncations that lack AF-1 enhance AP-1 activity in the presence of antiestrogens, but not estrogens. This phenotype resembles ER␤, which naturally lacks constitutive AF-1 activity. We conclude that the ERs enhance AP-1 responsive transcription by distinct mechanisms with different requirements for ER transactivation functions. We suggest that estrogen-liganded ER enhances AP-1 activity via interactions with p160s and speculate that antiestrogen-liganded ER enhances AP-1 activity via interactions with corepressors. (Molecular Endocrinology 13: 1672-1685, 1999)

Estrogen receptor (ER) binds to estrogen response elements in target genes and recruits a coactivator complex of CBP-pl60 that mediates stimulation of transcription. ER also activates transcription at AP-1 sites that bind the Jun/Fos transcription factors, but not ER. We review the evidence regarding mechanisms whereby ER increases the activity of Jun/Fos and propose two pathways of ER action depending on the ER (a or b) and on the ligand. We propose that estrogen-ERa complexes use their activation functions (AF-1 and AF-2) to bind to the p 160 component of the coactivator complex recruited by Jun/Fos and trigger the coactivator to a higher state of activity. We propose that selective estrogen receptor modulator (SERM) complexes with ERb and with truncated ERa derivatives use their DNA binding domain to titrate histone deacetylase (HDAC)-repressor complexes away from the Jun/Fos coactivator complex, thereby allowing unfettered activity of the coactivators. Finally, we consider the possible physiological significance of ER action at AP-1 sites.

To test the role of CACCC box on g-globin gene activation, the CACCC box was deleted or mutated and g-gene expression was monitored in transgenic mice. Disruption of the CACCC box had no effect on g-gene expression in the cells of embryonic erythropoiesis but it strikingly reduced g-gene expression in fetal erythropoiesis, and abolished g-gene expression in adult erythroid cells. The CACCC mutation diminished HS formation, as well as TBP and polII recruitment at the g-gene promoter; however, it only resulted in slight or no effects on histone H3 and H4 acetylation in adult erythropoiesis. Our findings indicate that each basic cis element of the proximal g-gene promoter, i.e. CACCC, CCAAT or TATA box, can be disrupted without affecting the activation of g gene in embryonic erythroid cells. We propose that the trans factors recruited by the three boxes interact with each other to form a 'promoter complex'. In embryonic erythropoiesis the locus control region enhancer is able to interact with the complex even when components normally binding to one of the motifs are missing, but it can only activate an intact 'promoter complex' in adult erythroid cells.

Objective-MEIS1 is a Hox cofactor known for its role in development and strongly linked to normal and leukemic hematopoiesis. Although previous studies have focused on identifying protein partners of MEIS1 and its transcriptionaly regulated targets, little is known about the upstream transcriptional regulators of this tightly regulated gene. Understanding the regulation of MEIS1 is important toward the understanding of normal hematopoiesis and leukemogenesis. Materials and Methods-Here we describe our studies focusing on the evolutionary conserved putative MEIS1 promoter region. Phylogenetic sequence analysis followed and reporter assays in MEIS1 expressing (K562) and non-expressing (HL60) leukemic cell line models were used to identify key regulatory regions and potential transcription factor binding sites within the candidate promoter region followed by functional and expression studies of one identified regulator in both cell lines and primary human cord blood and leukemia samples. Truncation and mutation studies coupled with reporter assays revealed that a conserved ETS family member binding site located 289bp upstream of the annotated human MEIS1 transcription start site (AHTSS) is required for promoter activity. Of the three ETS family members tested, only ELF1 was enriched on the MEIS1 promoter as assessed by both electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) experiments in K562. This finding was confirmed in MEIS1 expressing primary human samples. Moreover, siRNA -mediated knockdown of ELF1 in K562 cells was associated with a decreased MEIS1 expression. Conclusions-We conclude that the ETS transcription factor ELF1 is an important positive regulator of MEIS1 expression.

The inhibitory effect of stress on reproductive function is potentially mediated by high concentrations of circulating glucocorticoids (GCs) acting via the GC receptor (GR). Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic neuropeptide that inhibits gonadotropin secretion. GnIH may mediate stress-induced reproductive dysfunction. However, it is not yet known whether GC-bound GR is directly involved in GnIH transcription. Here, we demonstrated the localization of GR mRNA in GnIH neurons in the paraventricular nucleus of quail, suggesting that GC can directly regulate GnIH transcription. We next showed that 24 hours of treatment with corticosterone (CORT) increase GnIH mRNA expression in the quail diencephalon. We further investigated the mechanism of activation of GnIH transcription by CORT using a GnIH-expressing neuronal cell line, rHypoE-23, derived from rat hypothalamus. We found the expression of GR mRNA in rHypoE-23 cells and increased GnIH mRNA expression by 24 hours o...

Hypoxia induces apoptosis but also triggers adaptive mechanisms to ensure cell survival. Here we show that the prosurvival effects of hypoxia-inducible factor 1 (HIF-1) in endothelial cells are mediated by neuron-derived orphan receptor 1 (NOR-1). The overexpression of NOR-1 decreased the rate of endothelial cells undergoing apoptosis in cultures exposed to hypoxia, while the inhibition of NOR-1 increased cell apoptosis. Hypoxia upregulated NOR-1 mRNA levels in a time-and dose-dependent manner. Blocking antibodies against VEGF or SU5614 (a VEGF receptor 2 inhibitor) did not prevent hypoxia-induced NOR-1 expression, suggesting that NOR-1 is not induced by the autocrine secretion of VEGF in response to hypoxia. The reduction of HIF-1␣ protein levels by small interfering RNAs, or by inhibitors of the phosphatidylinositol-3 kinase (PI3K)/Akt pathway or mTOR, significantly counteracted hypoxia-induced NOR-1 upregulation. Intracellular Ca 2؉ was involved in hypoxia-induced PI3K/Akt activation and in the downstream NOR-1 upregulation. A hypoxia response element mediated the transcriptional activation of NOR-1 induced by hypoxia as we show by transient transfection and chromatin immunoprecipitation assays. Finally, the attenuation of NOR-1 expression reduced both basal and hypoxia-induced cIAP2 (cellular inhibitor of apoptosis protein 2) mRNA levels, while NOR-1 overexpression upregulated cIAP2. Therefore, NOR-1 is a downstream effector of HIF-1 signaling involved in the survival response of endothelial cells to hypoxia.

Accumulated findings have indicated that hepatitis B virus (HBV) DNA integrates into the cellular DNA of HBV-infected chronic hepatitis tissues. The integrated sequence (IS) of HBV DNA at the virus-cell junction is conserved in a 25-bp region which is adjacent to direct repeat 1. A cellular protein which we purified from the nuclear extract of HepG2 cells binds to the IS and was designated IS binding protein 3 (ISBP3). The amino acid sequence of ISBP3 was determined and found to be identical to that of transcription initiation factor Yin and Yang 1 (YY1). An antibody against C-terminal amino acids of YY1 recognized ISBP3 in a Western blot analysis and an electrophoretic mobility shift assay. Furthermore, ISBP3 also interacted with Y3, which corresponds to the YY1 binding sequence, to enhance intramolecular recombination of polyomavirus DNA. Although YY1 is known as a transcription factor, the IS did not exhibit any effect on the transcription of precore and pregenome RNAs. The possible involvement of YY1 in the intramolecular recombination of linear replicative HBV DNA has been examined (Y. Hayashi et al., unpublished data). Data suggest that YY1 is involved in the joining reaction between HBV DNA and cellular DNA to form the virus-cell junction.

Accumulated findings have indicated that hepatitis B virus (HBV) DNA integrates into the cellular DNA of HBV-infected chronic hepatitis tissues. The integrated sequence (IS) of HBV DNA at the virus-cell junction is conserved in a 25-bp region which is adjacent to direct repeat 1. A cellular protein which we purified from the nuclear extract of HepG2 cells binds to the IS and was designated IS binding protein 3 (ISBP3). The amino acid sequence of ISBP3 was determined and found to be identical to that of transcription initiation factor Yin and Yang 1 (YY1). An antibody against C-terminal amino acids of YY1 recognized ISBP3 in a Western blot analysis and an electrophoretic mobility shift assay. Furthermore, ISBP3 also interacted with Y3, which corresponds to the YY1 binding sequence, to enhance intramolecular recombination of polyomavirus DNA. Although YY1 is known as a transcription factor, the IS did not exhibit any effect on the transcription of precore and pregenome RNAs. The possi...

The cholesterol-sensing nuclear receptor liver X receptor (LXR) and the glucose-sensing transcription factor carbohydrate responsive element-binding protein (ChREBP) are central players in regulating glucose and lipid metabolism in liver. We have previously shown that LXR regulates ChREBP transcription and activity; however, the underlying mechanisms are unclear. In the current study, we demonstrate that LXRα and ChREBPα interact physically, and show a high co-occupancy at regulatory regions in the mouse genome. LXRα co-activates ChREBPα, and regulates ChREBP-specific target genes in vitro and in vivo. This co-activation is dependent on functional recognition elements for ChREBP, but not for LXR, indicating that ChREBPα recruits LXRα to chromatin in trans. The two factors interact via their key activation domains; ChREBPα's low glucose inhibitory domain (LID) and the ligand-binding domain (LBD) of LXRα. While unliganded LXRα co-activates ChREBPα, ligand-bound LXRα surprisingly represses ChREBPα activity on ChREBP-specific target genes. Mechanistically, this is due to a destabilized LXRα:ChREBPα interaction, leading to reduced ChREBP-binding to chromatin and restricted activation of glycolytic and lipogenic target genes. This liganddriven molecular switch highlights an unappreciated role of LXRα that was overlooked due to LXR lipogenesis-promoting function.

The cholesterol-sensing nuclear receptor liver X receptor (LXR) and the glucose-sensing transcription factor carbohydrate responsive element-binding protein (ChREBP) are central players in regulating glucose and lipid metabolism in the liver. More knowledge of their mechanistic interplay is needed to understand their role in pathological conditions like fatty liver disease and insulin resistance. In the current study, LXR and ChREBP co-occupancy was examined by analyzing ChIP-seq datasets from mice livers. LXR and ChREBP interaction was determined by Co-immunoprecipitation (CoIP) and their transactivity was assessed by real-time quantitative polymerase chain reaction (qPCR) of target genes and gene reporter assays. Chromatin binding capacity was determined by ChIP-qPCR assays. Our data show that LXRα and ChREBPα interact physically and show a high co-occupancy at regulatory regions in the mouse genome. LXRα co-activates ChREBPα and regulates ChREBP-specific target genes in vitro and...

ABSTRACTThe cholesterol-sensing nuclear receptor liver X receptor (LXR) and the glucose-sensing transcription factor carbohydrate responsive element-binding protein (ChREBP) are central players in regulating glucose and lipid metabolism in liver. We have previously shown that LXR regulates ChREBP transcription and activity; however, the underlying mechanisms are unclear. In the current study, we demonstrate that LXRα and ChREBPα interact physically, and show a high co-occupancy at regulatory regions in the mouse genome. LXRα co-activates ChREBPα, and regulates ChREBP-specific target genesin vitroandin vivo. This co-activation is dependent on functional recognition elements for ChREBP, but not for LXR, indicating that ChREBPα recruits LXRα to chromatin intrans. The two factors interact via their key activation domains; ChREBPα’s low glucose inhibitory domain (LID) and the ligand-binding domain (LBD) of LXRα. While unliganded LXRα co-activates ChREBPα, ligand-bound LXRα surprisingly r...

Despite internal complexity, algae and bacteria have coexisted since the early stages of evolution. This coevolution follows relatively simple laws that can be clearly expressed using mathematical models. This paper performs a quantitative analysis, motivated from the perspective of control theory, of a classical model from the literature. The model has been developed using data from an in vivo experimental two-species system where the bacterium Mesorhizobium loti supplies the vitamin B12 required for growth to the freshwater green alga Lobomonas rostrata and where the action of the B12 riboswitch is known to be a determinant of system behaviour. Analysis of the model both before and after the add-back of nutrients is carried out. A focus is exploring the robustness of the system. The paper first describes a simple model of algal-bacterial growth and analysis is undertaken. The effect of system parameters and control mechanisms is quantified. Motivated by the inherent switching action within the biology, a sliding mode interpretation of the control mechanisms is hypothesized based on knowledge of the maximum carrying capacities for each growth. The results of a range of experiments reported in the literature are used to validate the assertions.

Despite internal complexity, algae and bacteria have coexisted since the early stages of evolution. This coevolution follows relatively simple laws that can be clearly expressed using mathematical models. This paper performs a quantitative analysis, motivated from the perspective of control theory, of a classical model from the literature. The model has been developed using data from an in vivo experimental two-species system where the bacterium Mesorhizobium loti supplies the vitamin B12 required for growth to the freshwater green alga Lobomonas rostrata and where the action of the B12 riboswitch is known to be a determinant of system behaviour. Analysis of the model both before and after the add-back of nutrients is carried out. A focus is exploring the robustness of the system. The paper first describes a simple model of algal-bacterial growth and analysis is undertaken. The effect of system parameters and control mechanisms is quantified. Motivated by the inherent switching action within the biology, a sliding mode interpretation of the control mechanisms is hypothesized based on knowledge of the maximum carrying capacities for each growth. The results of a range of experiments reported in the literature are used to validate the assertions.

Despite internal complexity, algae and bacteria have coexisted since the early stages of evolution. This co- evolution follows relatively simple laws that can be clearly expressed using mathematical models. This paper performs a quantitative analysis, motivated from the perspective of control theory, of a classical model from the literature. The model has been developed using data from an in vivo experimental two-species system where the bacterium Mesorhizobium loti supplies the vitamin $B_{12}$ required for growth to the freshwater green alga Lobomonas rostrata and where the action of the $B_{12}$ riboswitch is known to be a determinant of system behaviour. Analysis of the model both before and after the add-back of nutrients is carried out. A focus is exploring the robustness of the system. The paper first describes a simple model of algal-bacterial growth and analysis is undertaken. The effect of system parameters and control mechanisms is quantified. Motivated by the inherent sw...

Tamoxifen-resistant (TAMR) estrogen receptor-positive (ER؉) breast cancer is characterized by elevated Erb-B2 receptor tyrosine kinase 2 (ERBB2) expression. However, the underlying mechanisms responsible for the increased ERBB2 expression in the TAMR cells remain poorly understood. Herein, we reported that the ERBB2 expression is regulated at the posttranscriptional level by miR26a/b and the RNA-binding protein human antigen R (HuR), both of which associate with the 3-UTR of the ERBB2 transcripts. We demonstrated that miR26a/b inhibits the translation of ERBB2 mRNA, whereas HuR enhances the stability of the ERBB2 mRNA. In TAMR ER؉ breast cancer cells with elevated ERBB2 expression, we observed a decrease in the level of miR26a/b and an increase in the level of HuR. The forced expression of miR26a/b or the depletion of HuR decreased ERBB2 expression in the TAMR cells, resulting in the reversal of tamoxifen resistance. In contrast, the inactivation of miR26a/b or forced expression of HuR decreased tamoxifen responsiveness of the parental ER؉ breast cancer cells. We further showed that the increase in HuR expression in the TAMR ER؉ breast cancer cells is attributable to an increase in the HuR mRNA isoform with shortened 3-UTR, which exhibits increased translational activity. This shortening of the HuR mRNA 3-UTR via alternative polyadenylation (APA) was observed to be dependent on cleavage stimulation factor subunit 2 (CSTF2/CstF-64), which is up-regulated in the TAMR breast cancer cells. Taken together, we have characterized a model in which the interplay between miR26a/b and HuR posttranscriptionally up-regulates ERBB2 expression in TAMR ER؉ breast cancer cells.

The important physiological actions of the thyroid hormones are mediated by binding to nuclear thyroid hormone receptors (TRs), encoded by two genes TRalpha and TRbeta. These receptors act as hormone-dependent transcription factors by binding to DNA motifs located in the regulatory regions of target genes and recruiting coregulators (coactivators and corepresors), which alter chromatin structure. Novel thyromimetics have been developed that bind preferentially TRbeta could be used for treatment of hyperlipidemia and obesity. TRbeta gene mutations cause resistance to thyroid hormones (RTH), characterized by inappropriately high thyroid-stimulating hormone (TSH) levels due to lack of feedback inhibition of thyroid hormones on the hypothalamus and pituitary gland, and to reduced sensitivity of other TRbeta target tissues to thyroid hormones. Very recently, patients heterozygous for TRalpha mutations have been identified. These patients exhibit clinical symptoms of hypothyroidism in TRalpha target tissues such as intestine or hearth and near normal circulating TSH and thyroid hormone levels.

To investigate the effect of sulforaphane (SFN) on regulation of NF-E2-related factor-2 (Nrf2)-antioxidant response element (ARE) pathway in liver injury induced by intestinal ischemia/reperfusion (I/R). Rats were divided randomly into four experimental groups: control, SFN control, intestinal I/R and SFN pretreatment groups (n = 8 in each group). The intestinal I/R model was established by clamping the superior mesenteric artery for 1 h and 2 h reperfusion. In the SFN pretreatment group, surgery was performed as in the intestinal I/R group, with intraperitoneal administration of 3 mg/kg SFN 1 h before the operation. Intestine and liver histology was investigated. Serum levels of aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were measured. Liver tissue superoxide dismutase (SOD), myeloperoxidase (MPO), glutathione (GSH) and glutathione peroxidase (GSH-Px) activity were assayed. The liver transcription factor Nrf2 and heme oxygenase-1 (HO-1) were determined by immunohistochemical analysis and Western blotting analysis. Intestinal I/R induced intestinal and liver injury, characterized by histological changes as well as a significant increase in serum AST and ALT levels (AST:

The white rot fungus Phanerochaete chrysosporium metabolizes a range of xenobiotics via P450 mono-oxygenation, particularly under peroxidase-suppressing culture conditions. Here we report the cloning and analysis of the gene from this fungus for the cytochrome P450 oxidoreductase (CPR) and its dierentially terminated cDNAs. Using a PCR-based approach with degenerate primers, a 285-bp genomic fragment was isolated from the two widely studied strains BKM-F 1767 and ME 446, and was identi®ed as a CPR gene segment based on sequence comparison with the database. A clone containing the full-length CPR gene was isolated from a BKM-F 1767 genomic library using the PCR-generated segment as a probe, and the 3937-bp insert was sequenced by gene walking. Based on the detection of conserved CPR motifs, a coding region of 2381 bp was identi®ed with a 991-bp segment 5¢ to the putative ATG start codon. Two cDNAs with dierentially terminated transcripts were isolated and sequenced. Comparison of the gene and the cDNA sequences con-®rmed the presence of three introns (62 bp, 50 bp, and 58 bp). Sequence identity and a phylogenetic comparison of the deduced protein (736 aa) with other CPRs in the database suggested that P. chrysosporium CPR is the largest CPR known and is more closely related to animal (36±38%) and yeast (37±38%) CPRs than to plant CPRs (33±35%). The availability of this gene will facilitate further studies on understanding the potent xenobiotic mono-oxygenation systems in this model white rot fungus.

SENP1 (SUMO-specific protease 1) has been shown to be essential for the stability and activity of hypoxia-inducible factor 1 (HIF-1α) under hypoxia conditions. However, it is unknown how SENP1 activation and hypoxia signaling are coordinated in the cellular response to hypoxia. Here, we report the essential role of SENP1 in endothelial cells as a positive regulator of hypoxia-driven VEGF production and angiogenesis. SENP1 expression is increased in endothelial cells following exposure to hypoxia. Silencing of HIF-1α blocks SENP1 expression in cell response to hypoxia. Mutation of the hypoxia response element (HRE) on the Senp1 promoter abolishes its transactivation in response to hypoxia. Moreover, silencing of SENP1 expression decreases VEGF production and abrogates the angiogenic functions of endothelial cell. We also find that the elongated endothelial cells in embryonic brain section and vascular endothelial cells in embryonic renal glomeruli in Senp1−/− mice are markedly reduce...

SARA, an early endosomal protein, plays a key role in TGFb signalling, as it presents SMAD2 and SMAD3 for phosphorylation by the activated TGFb receptors. Here, we show that ERBIN is a new SARA-interacting protein that can be recruited by SARA to early endosomes. ERBIN was recently shown to bind and segregate phosphorylated SMAD2 and SMAD3 (SMAD2/3) in the cytoplasm, thereby inhibiting SMAD2/3-dependent transcription. SARA binds to ERBIN using a new domain, which we have called the ERBID (ERBIN-binding domain), whereas ERBIN binds to SARA using a domain (amino acids 1208-1265) that also interacts with SMAD2 and SMAD3, which we have called the SSID (SARA-and SMAD-interacting domain). We additionally show that SARA competes with SMAD2/3 for binding to ERBIN. In agreement, overexpression of SARA or the ERBID peptide reverses the inhibitory effect of ERBIN on SMAD2/3-dependent transcription. Taken together, these data suggest that the response of cells to TGFb and activin A can be influenced by the relative concentrations of SARA, ERBIN and SMAD2/3.

A mutant screen was conducted in Arabidopsis that was based on deregulated expression of auxin-responsive transgenes. Two different tightly regulated (i.e., very low expression in the absence of auxin treatment and very high expression after exogenous auxin treatment) auxin-responsive promoters were used to drive the expression of both a ␤ -glucuronidase ( GUS ) reporter gene and a hygromycin phosphotransferase ( HPH )-selectable marker gene. This screen yielded several mutants, and five of the mutations ( axe1-1 to axe1-5 ) mapped to the same locus on chromosome 5. A map-based cloning approach was used to locate the axe1 mutations in an Arabidopsis RPD3 -like histone deacetylase gene, referred to as HDA6 . The axe1 mutant plants displayed increased expression of the GUS and HPH transgenes in the absence of auxin treatment and increased auxin-inducible expression of the transgenes compared with nonmutant control plants. None of a variety of endogenous, natural auxin-inducible genes in the mutant plants were upregulated like the transgenes, however. Results of treatment with the DNA methylation inhibitor 5-aza-2 -deoxycytidine suggest that the axe1 mutations affect transgene silencing; however, histone deacetylase inhibitors had no affect on transgene silencing in mutant or control plants. The specific effect of AtHDA6 mutations on the auxin-responsive transgenes implicates this RPD3-like histone deacetylase as playing a role in transgene silencing. Furthermore, the effect of AtHDA6 on transgene silencing may be independent of its histone deacetylase activity.

A mutant screen was conducted in Arabidopsis that was based on deregulated expression of auxin-responsive transgenes. Two different tightly regulated (i.e., very low expression in the absence of auxin treatment and very high expression after exogenous auxin treatment) auxin-responsive promoters were used to drive the expression of both a ␤ -glucuronidase ( GUS ) reporter gene and a hygromycin phosphotransferase ( HPH )-selectable marker gene. This screen yielded several mutants, and five of the mutations ( axe1-1 to axe1-5 ) mapped to the same locus on chromosome 5. A map-based cloning approach was used to locate the axe1 mutations in an Arabidopsis RPD3 -like histone deacetylase gene, referred to as HDA6 . The axe1 mutant plants displayed increased expression of the GUS and HPH transgenes in the absence of auxin treatment and increased auxin-inducible expression of the transgenes compared with nonmutant control plants. None of a variety of endogenous, natural auxin-inducible genes in the mutant plants were upregulated like the transgenes, however. Results of treatment with the DNA methylation inhibitor 5-aza-2 -deoxycytidine suggest that the axe1 mutations affect transgene silencing; however, histone deacetylase inhibitors had no affect on transgene silencing in mutant or control plants. The specific effect of AtHDA6 mutations on the auxin-responsive transgenes implicates this RPD3-like histone deacetylase as playing a role in transgene silencing. Furthermore, the effect of AtHDA6 on transgene silencing may be independent of its histone deacetylase activity.

Hepatic glucose production is critical for basal brain function and survival when dietary glucose is unavailable. Glucose-6-phosphatase (G6Pase) is an essential, rate-limiting enzyme that serves as a terminal gatekeeper for hepatic glucose release into the plasma. Mutations in G6Pase result in Von Gierke's disease (glycogen storage disease-1a), a potentially fatal genetic disorder. We have identified the transcriptional coactivator SRC-2 as a regulator of fasting hepatic glucose release, a function that SRC-2 performs by controlling the expression of hepatic G6Pase. SRC-2 modulates G6Pase expression directly by acting as a coactivator with the orphan nuclear receptor RORα. In addition, SRC-2 ablation, in both a whole-body and liver-specific manner, resulted in a Von Gierke's disease phenotype in mice. Our results position SRC-2 as a critical regulator of mammalian glucose production. In mammals, when exogenous glucose is not available, between meals and during fasting, the liver produces glucose from substrates such as glycogen, lactate, pyruvate, glycerol, and amino acids (1). During fasting, the liver contributes about 85 to 90% of the glucose load, and the remainder is contributed by the kidney (1,2). This function of the liver is vital for basal brain function, and thus for the survival of the fasting organism (1,3). Glucose-6-phosphatase (G6Pase) is an essential, rate-limiting enzyme that catalyzes the terminal reaction that produces glucose during fasting (2,. This reaction is common to glycogenolysis and gluconeogenesis, the two processes that provide the body with glucose when the organism is subjected to a fast. This metabolic location, at the crossroad between glycogenolysis and gluconeogenesis, allows G6Pase to control both the short-term and longer- †To whom correspondence should be addressed.

Hepatic glucose production is critical for basal brain function and survival when dietary glucose is unavailable. Glucose-6-phosphatase (G6Pase) is an essential, rate-limiting enzyme that serves as a terminal gatekeeper for hepatic glucose release into the plasma. Mutations in G6Pase result in Von Gierke's disease (glycogen storage disease-1a), a potentially fatal genetic disorder. We have identified the transcriptional coactivator SRC-2 as a regulator of fasting hepatic glucose release, a function that SRC-2 performs by controlling the expression of hepatic G6Pase. SRC-2 modulates G6Pase expression directly by acting as a coactivator with the orphan nuclear receptor RORα. In addition, SRC-2 ablation, in both a whole-body and liver-specific manner, resulted in a Von Gierke's disease phenotype in mice. Our results position SRC-2 as a critical regulator of mammalian glucose production. In mammals, when exogenous glucose is not available, between meals and during fasting, the liver produces glucose from substrates such as glycogen, lactate, pyruvate, glycerol, and amino acids (1). During fasting, the liver contributes about 85 to 90% of the glucose load, and the remainder is contributed by the kidney (1,2). This function of the liver is vital for basal brain function, and thus for the survival of the fasting organism (1,3). Glucose-6-phosphatase (G6Pase) is an essential, rate-limiting enzyme that catalyzes the terminal reaction that produces glucose during fasting (2,. This reaction is common to glycogenolysis and gluconeogenesis, the two processes that provide the body with glucose when the organism is subjected to a fast. This metabolic location, at the crossroad between glycogenolysis and gluconeogenesis, allows G6Pase to control both the short-term and longer- †To whom correspondence should be addressed.

Glutathione peroxidase 3 (GPx3), an antioxidant enzyme, acts as a modulator of redox signaling, has immunomodulatory function, and catalyzes the detoxification of reactive oxygen species (ROS). GPx3 has been identified as a tumor suppressor in many cancers. Although hyper-methylation of the GPx3 promoter has been shown to down-regulate its expression, other mechanisms by which GPx3 expression is regulated have not been reported. The aim of this study was to further elucidate the mechanisms of GPx3 regulation. GPx3 gene analysis predicted the presence of ten glucocorticoid response elements (GREs) on the GPx3 gene. This result prompted us to investigate whether GPx3 expression is regulated by the glucocorticoid receptor (GR), which is implicated in tumor response to chemotherapy. The corticosteroid dexamethasone (Dex) was used to examine the possible relationship between GR and GPx3 expression. Dex significantly induced GPx3 expression in H1299, H1650, and H1975 cell lines, which exhibit low levels of GPx3 expression under normal conditions. The results of EMSA and ChIP-PCR suggest that GR binds directly to GRE 6 and 7, both of which are located near the GPx3 promoter. Assessment of GPx3 transcription efficiency using a luciferase reporter system showed that blocking formation of the GR-GRE complexes reduced luciferase activity by 7-8-fold. Suppression of GR expression by siRNA transfection also induced down-regulation of GPx3. These data indicate that GPx3 expression can be regulated independently via epigenetic or GR-mediated mechanisms in lung cancer cells, and suggest that GPx3 could potentiate glucocorticoid (GC)mediated anti-infla-mmatory signaling in lung cancer cells. 1

)-dependent histone/protein deacetylase, has been extensively studied recently for its critical role in the regulation of physiology, calorie restriction and aging. Studies on laboratory mice showed that expression of SIRT1 can be induced by starvation in a p53-dependent manner and requires the p53-binding sites present in the Sirt1 promoter. However, it remains to be determined whether these findings based on rodents apply to human beings. In this paper, we characterized a putative p53-binding element in the human SIRT1 promoter that might be required for the up-regulation of SIRT1 in response to nutritional stress. The p53-binding site in the promoter of human SIRT1 is more deviant from the consensus sequence than the corresponding sequence in the mouse Sirt1. There is a C to A change at the second half site in human SIRT1, thus disrupting the core-binding element CWWG in the canonical RRRCWWGYYY. To test whether such sequence change would affect its binding with p53 and the SIRT1 expression under stress, we studied various human cell lines with different p53 status and cells with ectopic expression of functionally distinct p53. We found that serum withdrawal also up-regulates human SIRT1 gene expression in a p53-dependent manner and that the p53binding element in SIRT1 is required for the up-regulation. Thus, the mechanism responsible for the regulation of SIRT1 expression by p53 is conserved between mice and human beings.

The interaction between the vascular endothelium and hemodynamic forces (and more specifically, fluid shear stress), induced by the flow of blood, plays a major role in vascular remodeling and in new blood vessels formation via a process termed arteriogenesis. Tie1 is an orphan tyrosine kinase receptor expressed almost exclusively in endothelial cells and is required for normal vascular development and maintenance. The present study demonstrates that Tie1 expression is rapidly down-regulated in endothelial cells exposed to shear stress, and more so to shear stress changes. This down-regulation is accompanied by a rapid cleavage of Tie1 and binding of the cleaved Tie1 45 kDa endodomain to Tie2. The rapid cleavage of Tie1 is followed by a transcriptional down-regulation in response to shear stress. The activity of the Tie1 promoter is suppressed by shear stress and by tumor necrosis factor α. Shear stress-induced transcriptional suppression of Tie1 is mediated by a negative shear stress response element, localized in a region of 250 bp within the promoter. The rapid down-regulation of Tie1 by shear stress changes and its rapid binding to Tie2 may be required for destabilization of endothelial cells in order to initiate the process of vascular restructuring. Key words: vascular endothelium • biomechanical forces • vascular restructuring • shear stress response element s blood flows through the arteries, it imparts physical forces to the vascular wall that regulate several important physiological responses in blood vessels and that are also implicated in the development of arterial wall pathologies. It is now well established that the primary role of hemodynamic forces (mainly shear stress-a frictional force acting at the interface between the flowing blood and the vessel wall), acting through the endothelium, is to cause chronic restructuring of blood vessels (1-6) and to initiate blood vessel formation through a process termed arteriogenesis (7, 8).

Chromosome organization inside the nucleus is not random but rather is determined by a variety of factors, including interactions between chromosomes and nuclear components such as the nuclear envelope or nuclear matrix. Such interactions may be critical for proper nuclear organization, chromosome partitioning during cell division, and gene regulation. An important, but poorly documented subset, includes interactions between specific chromosomal regions. Interactions of this type are thought to be involved in long-range promoter regulation by distant enhancers or locus control regions and may underlie phenomena such as transvection. Here, we used an in vivo microscopy assay based on Lac Repressor/operator recognition to show that Mcp, a polycomb response element from the Drosophila bithorax complex, is able to mediate physical interaction between remote chromosomal regions. These interactions are tissue specific, can take place between multiple Mcp elements, and seem to be stable on...

Nodal and Smad2/3 signals play pivotal roles in mesendoderm induction and axis determination during late blastulation and early gastrulation in vertebrate embryos. However, Smad2/3 direct target genes during those critical developmental stages have not been systematically identified. Here, through ChIPchip assay, we show that the promoter/enhancer regions of 679 genes are bound by Smad2 in the zebrafish early gastrulas. Expression analyses confirm that a significant proportion of Smad2 targets are indeed subjected to Nodal/Smad2 regulation at the onset of gastrulation. The co-existence of DNA-binding sites of other transcription factors in the Smad2-bound regions allows the identification of well known Smad2-binding partners, such as FoxH1 and Lef1/␤-catenin, as well as many previously unknown Smad2 partners, including Oct1 and Gata6, during embryogenesis. We demonstrate that Oct1 physically associates with and enhances the transcription and mesendodermal induction activity of Smad2, whereas Gata6 exerts an inhibitory role in Smad2 signaling and mesendodermal induction. Thus, our study systemically uncovers a large number of Smad2 targets in early gastrulas and suggests cooperative roles of Smad2 and other transcription factors in controlling target gene transcription, which will be valuable for studying regulatory cascades during germ layer formation and patterning of vertebrate embryos.