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Transcriptional Repressor (transcriptional + repressor)
Selected AbstractsThe KRAB Domain of Zinc Finger Gene ZNF268: a Potential Transcriptional RepressorIUBMB LIFE, Issue 3 2003Yan Sun Abstract Nearly one-third of Krupple-type C2H2 zinc finger proteins have a krupple-associated box (KRAB) domain, which may act as a transcriptional repressor. ZNF268, which was novelty isolated from early human embryo, is a typical krupple-type C2H2 zinc finger protein with a conserved KRAB domain. In this report, the KRAB domain of ZNF268 is identified to localize in the nucleus and has transcriptional repressor activity. IUBMB Life, 55: 127-131, 2003 [source] Genetic basis of rett syndromeDEVELOPMENTAL DISABILITIES RESEARCH REVIEW, Issue 2 2002Ignatia B. Van den Veyver Abstract The origin of Rett syndrome has long been debated, but several observations have suggested an X-linked dominant inheritance pattern. We and others have pursued an exclusion-mapping strategy using DNA from a small number of familial Rett syndrome cases. This work resulted in the narrowing of the region likely to harbor the mutated gene to Xq27.3-Xqter. After systematic exclusion of several candidate genes, we discovered mutations in MECP2, the gene that encodes the transcriptional repressor, methyl-CpG-binding protein 2. Since then, nonsense, missense, or frameshift mutations have been found in at least 80% of girls affected with classic Rett syndrome. Sixty-four percent of mutations are recurrent C > T transitions at eight CpG dinucleotides mutation hotspots, while the C-terminal region of the gene is prone to recurrent multinucleotide deletions (11%). Most mutations are predicted to result in total or partial loss of function of MeCP2. There is no clear correlation between the type and position of the mutation and the phenotypic features of classic and variant Rett syndrome patients, and XCI appears to be a major determinant of phenotypic severity. Further research focuses on the pathogenic consequences of these mutations along the hypothesis of loss of transcriptional repression of a small number of genes that are essential for neuronal function in the maturing brain. MRDD Research Reviews 2002;8:82,86. © 2002 Wiley-Liss, Inc. [source] ghrelin is a novel target of Pax4 in endocrine progenitors of the pancreas and duodenumDEVELOPMENTAL DYNAMICS, Issue 1 2008Qian Wang Abstract Pax4 -deficient mice have a severe gastrointestinal endocrine deficiency: they lack most pancreatic cells that produce insulin or somatostatin and various duodenal endocrine cell types. Remarkably, Pax4 -deficient mice also have an overabundance of ghrelin-expressing cells in the pancreas and duodenum. Detailed analysis of the Pax4 nullizygous pancreas determined that the mutant islets are largely composed of a distinctive endocrine cell type that expresses ghrelin, glucagon, islet amyloid polypeptide (IAPP), and low levels of Pdx1. Lineage-tracing analysis revealed that most of these unique endocrine cells directly arose from Pax4 -deficient progenitors. Previous in vitro work reported that Pax4 is a transcriptional repressor of islet amyloid polypeptide (IAPP) and glucagon. In this study, we expanded those results by showing that Pax4 is also a repressor of gherlin. Together, our data further support the notion that Pax4 activity is necessary to establish appropriate patterns of gene expression in endocrine progenitors of the digestive tract. Developmental Dynamics 237:51,61, 2008. © 2007 Wiley-Liss, Inc. [source] Functional analysis of murine CBF1 during Drosophila developmentDEVELOPMENTAL DYNAMICS, Issue 4 2006Markus Kaspar Abstract Transcription factors of the CSL family are the main mediators of the Notch signalling pathway. The CSL factor in Drosophila is called Suppressor of Hairless (Su(H)) and it has been shown that it acts as a transcriptional repressor in the absence of a Notch signal and as a transcriptional activator in its presence in several developmental contexts. Furthermore, recent data suggest that Su(H) can also activate and maintain transcription of some target genes in a Notch-independent manner. However, although it has been shown that the mammalian CSL ortholog, CBF1, acts as a repressor of transcription in cell culture experiments, so far in vivo evidence for such a function has been lacking. Moreover, it is not known whether CBF1 can activate transcription in a Notch-independent manner, just like Su(H). Here we have investigated these questions by introducing murine CBF1 (mCBF1) and asked whether it can functionally replace Su(H) during Drosophila development. We found that this is indeed the case. We show that mCBF1 can act as a repressor of transcription and can activate and maintain the expression of some target genes in a Notch-independent manner. Our results, therefore, indicate that CBF1 can exert these functions also in its normal context, that is during mammalian development. Developmental Dynamics 235:918,927, 2006. © 2006 Wiley-Liss, Inc. [source] Xenopus aristaless-related homeobox (xARX) gene product functions as both a transcriptional activator and repressor in forebrain developmentDEVELOPMENTAL DYNAMICS, Issue 2 2005Daniel W. Seufert Abstract Mutations in the aristaless-related homeobox (ARX) gene have been found in patients with a variety of X-linked mental retardation syndromes with forebrain abnormalities, including lissencephaly. Arx is expressed in the developing mouse, Xenopus, and zebrafish forebrain. We have used whole-mount in situ hybridization, overexpression, and loss-of-function studies to investigate the involvement of xArx in Xenopus brain development. We verified that xArx is expressed in the prospective diencephalon, as the forebrain is patterned and specified during neural plate stages. Expression spreads into the ventral and medial telencephalon as development proceeds through neural tube and tadpole stages. Overexpression of xArx resulted in morphological abnormalities in forebrain development, including loss of rostral midline structures, syn- or anophthalmia, dorsal displacement of the nasal organ, and ventral neural tube hyperplasia. Additionally, there is a delay in expression of many molecular markers of brain and retinal development. However, expression of some markers, dlx5 and wnt8b, was enhanced in xArx -injected embryos. Loss-of-function experiments indicated that xArx was necessary for normal forebrain development. Expansion of wnt8b expression depended on xArx function as a transcriptional repressor, whereas ectopic expression of dlx5, accompanied by development of ectopic otic structures, depended on function of Arx as a transcriptional activator. These results suggest that Arx acts as a bifunctional transcriptional regulator in brain development. Developmental Dynamics 232:313,324, 2005. © 2004 Wiley-Liss, Inc. [source] The proto-oncogene BCL6 promotes survival of olfactory sensory neuronsDEVELOPMENTAL NEUROBIOLOGY, Issue 6 2010Joji M. Otaki Abstract For the mammalian olfactory epithelium to continually detect odorant, neuronal survival, apoptosis, and regeneration must be coordinated. Here, we showed that the proto-oncogene BCL6, which encodes a transcriptional repressor required for lymphocyte terminal differentiation, contributes to the survival of olfactory sensory neurons (OSNs). In the olfactory epithelia of the BCL6 null mutant mice, many OSNs were positive for both OMP and GAP43. The epithelium was relatively thinner, showing many apoptotic signals. These characters were phenotypically similar to those of the wild-type mice treated with nasal lectin irrigation, which acutely induces apoptosis of OSNs. Odorant receptors were expressed normally in the epithelia of the mutant mice, and their overall expression profile based on DNA microarray analyses was roughly similar to that of the apoptosis-induced olfactory epithelia of the wild-type mice. Experimental increase of BCL6 together with green fluorescent protein in OSNs using adenovirus-mediated gene transfer made the epifluorescence last longer than the control fluorescence without exogenous BCL6 after the nasal lectin irrigation, indicating that BCL6 made the infected neurons survive longer. We conclude that BCL6 plays an active role in the survival of OSNs as an anti-apoptotic factor and confers immature OSNs enough time to fully differentiate into mature ones. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 424-435, 2010 [source] Biological activity of RE-1 silencing transcription factor (REST) towards distinct transcriptional activatorsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2001Michael Lietz Abstract The zinc finger protein RE-1 silencing transcription factor (REST) is a transcriptional repressor that represses neuronal genes in non-neuronal tissues. We have analyzed the ability of REST and the REST mutants, REST,N and REST,C lacking either the N-terminal or C-terminal repression domains of REST, to inhibit transcription mediated by distinct transcriptional activator proteins. For this purpose we have designed an activator specific assay where transcription is activated as a result of only one distinct activation domain. In addition, binding sites for REST were inserted in the 5,-untranslated region or at a distant position downstream of the polyadenylation signal. The results show that REST or the REST mutants containing only one repression domain were able to block transcriptional activation mediated by the transcriptional activation domains derived from p53, AP2, Egr-1, and GAL4. Moreover, REST, as well as the REST mutants, blocked the activity of the phosphorylation-dependent activation domain of Elk1. However, the activity of the activation domain derived from cAMP response element binding protein 2 (CREB2), was not inhibited by REST, REST,N or REST,C, suggesting that REST is able to distinguish between distinct transcriptional activation domains. Additionally, the activator specific assay, together with a positive-dominant mutant of REST that activated instead of repressed transcription, was used in titration experiments to show that REST has transcriptional repression and no transcriptional activation properties when bound to the 5,-untranslated region of a gene. [source] The Ikaros family protein Eos associates with C-terminal-binding protein corepressorsFEBS JOURNAL, Issue 23 2002José Perdomo Eos is a zinc finger transcription factor of the Ikaros family. It binds typical GGGAA Ikaros recognition sites in DNA and functions as a transcriptional repressor. Here we show that Eos associates with the corepressor C-terminal-binding protein (CtBP). CtBP has previously been shown to bind Pro-X-Asp-Leu-Ser (PXDLS) motifs in several DNA-binding proteins. We note that Eos contains a related motif PEDLA, and we demonstrate that CtBP can bind this site weakly but that it also contacts additional regions of Eos. Consistent with this finding, mutation of the PEDLA motif does not negate CtBP binding or CtBP-mediated repression by Eos. CtBP has previously been shown to bind to a PXDLS-type motif in Ikaros, and we show that another Ikaros-related protein TRPS1 also contains a PXDLS CtBP contact motif within its repression domain. We conclude that several Ikaros family proteins utilize CtBP corepressors to inhibit gene expression. [source] Microbial interactions and differential protein expression in Staphylococcus aureus ,Candida albicans dual-species biofilmsFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 3 2010Brian M. Peters Abstract The fungal species Candida albicans and the bacterial species Staphylococcus aureus are responsible for a majority of hospital-acquired infections and often coinfect critically ill patients as complicating polymicrobial biofilms. To investigate biofilm structure during polymicrobial growth, dual-species biofilms were imaged with confocal scanning laser microscopy. Analyses revealed a unique biofilm architecture where S. aureus commonly associated with the hyphal elements of C. albicans. This physical interaction may provide staphylococci with an invasion strategy because candidal hyphae can penetrate through epithelial layers. To further understand the molecular mechanisms possibly responsible for previously demonstrated amplified virulence during coinfection, protein expression studies were undertaken. Differential in-gel electrophoresis identified a total of 27 proteins to be significantly differentially produced by these organisms during coculture biofilm growth. Among the upregulated staphylococcal proteins was l -lactate dehydrogenase 1, which confers resistance to host-derived oxidative stressors. Among the downregulated proteins was the global transcriptional repressor of virulence factors, CodY. These findings demonstrate that the hyphae-mediated enhanced pathogenesis of S. aureus may not only be due to physical interactions but can also be attributed to the differential regulation of specific virulence factors induced during polymicrobial growth. Further characterization of the intricate interaction between these pathogens at the molecular level is warranted, as it may aid in the design of novel therapeutic strategies aimed at combating fungal,bacterial polymicrobial infection. [source] Identification of a novel BTB-zinc finger transcriptional repressor, CIBZ, that interacts with CtBP corepressorGENES TO CELLS, Issue 9 2005Nobuhiro Sasai The transcriptional corepressor C-terminal binding protein (CtBP) is thought to be involved in development and oncogenesis, but the regulation of its corepressor activity is largely unknown. We show here that a novel BTB-zinc finger protein, CIBZ (CtBP-interacting BTB zinc finger protein; a mouse ortholog of rat ZENON that was recently identified as an e-box/dyad binding protein), redistributes CtBP to pericentromeric foci from a diffuse nuclear localization in interphase cells. CIBZ physically associates with CtBP via a conserved CtBP binding motif, PLDLR. When heterologously targeted to DNA, CIBZ represses transcription via two independent repression domains, an N-terminal BTB domain and a PLDLR motif-containing RD2 region, in a histone deacetylase-independent and -dependent manner, respectively. Mutation in the PLDLR motif abolishes the CIBZ-CtBP interaction and transcriptional repression activity of RD2, but does not affect the repression activity of the BTB domain. Furthermore, this PLDLR-mutated CIBZ cannot target CtBP to pericentromeric foci, although it is localized to the pericentromeric foci itself. These results suggest that at least one repression mechanism mediated by CIBZ is recruitment of the CtBP/HDAC complex to pericentromeric foci, and that CIBZ may regulate pericentromeric targeting of CtBP. [source] Upregulation of myosin Va by Snail is involved in cancer cell migration and metastasisINTERNATIONAL JOURNAL OF CANCER, Issue 1 2010Linxiang Lan Abstract Cell migration, which involves acto-myosin dynamics, cell adhesion, membrane trafficking and signal transduction, is a prerequisite for cancer cell metastasis. Here, we report that an actin-dependent molecular motor, unconventional myosin Va, is involved in this process and implicated in cancer metastasis. The mRNA expression of myosin Va is increased in a number of highly metastatic cancer cell lines and metastatic colorectal cancer tissues. Suppressing the expression of myosin Va by lentivirus-based RNA interference in highly metastatic cancer cells impeded their migration and metastasis capabilities both in vitro and in vivo. In addition, the levels of myosin Va in cancer cell lines are positively correlated with the expression of Snail, a transcriptional repressor that triggers epithelial,mesenchymal transition. Repression or overexpression of Snail in cancer cells caused reduced or elevated levels of myosin Va, respectively. Furthermore, Snail can bind to an E-box of the myosin Va promoter and induce its activity, which indicates that Snail might act as a transcriptional activator. These data demonstrate an essential role of myosin Va in cancer cell migration and metastasis, and suggest a novel target for Snail in its regulation of cancer progression. [source] TIP30 is associated with progression and metastasis of prostate cancerINTERNATIONAL JOURNAL OF CANCER, Issue 4 2008Hui Zhang Abstract Tat-interacting protein 30 (TIP30), a transcriptional repressor for ER,-mediated transcription, possesses several characteristics of a tumor suppressor in certain human and mouse cells. It is reported that deletion of TIP30 gene preferentially increases tumorigenesis in the female knockout mice. Here, we analyzed TIP30 gene expression in the databases of several DNA microarray studies of human prostate cancer and show that TIP30 is specifically overexpressed in metastatic prostate cancers. We demonstrate that TIP30 nuclear expression is associated with prostate cancer progression and metastasis by immunohistochemical analysis in primary and metastatic prostate cancers. Consistent with these data, we also show that knockdown of TIP30 expression, through use of a short hairpin RNA-expressing plasmid, suppresses the cellular growth of PC3 and LNCaP prostate cancer cells. Ectopic overexpression of TIP30 stimulates metastatic potential of prostate cancer cells in an in vitro invasion assay, whereas knockdown of TIP30 inhibits the prostate cancer cells invasion. Finally, we demonstrate that ectopic overexpression of TIP30 enhances androgen receptor mediated transcription, whereas knockdown of TIP30 results in a decreased transcription activity. These data provide evidence that TIP30 plays a role in prostate cancer progression and that TIP30 overexpression may promote prostate cancer cell growth and metastasis. © 2008 Wiley-Liss, Inc. [source] Id1 expression is transcriptionally regulated in radial growth phase melanomasINTERNATIONAL JOURNAL OF CANCER, Issue 8 2007Byungwoo Ryu Abstract Id genes have been demonstrated to be upregulated in a wide variety of human malignancies and their expression has been correlated with disease prognosis; however, little is known about the mechanisms of Id gene activation in tumors. We have previously shown that the helix-loop-helix transcription factor, Id1, is highly expressed in primary human melanomas during the radial growth phase and that Id1 is a transcriptional repressor of the familial melanoma gene CDKN2A. Here we use a series of melanoma cell lines that recapitulate the phenotypic characteristics of melanomas at varying stages of malignant progression to evaluate the expression levels of Id1 in this model system and determine the mechanism of Id1 dysregulation in these tumor cells. We find elevated protein levels of Id1 to be present consistently in radial growth phase tumor cells in accordance with our primary tumor data. Id1 transcript levels were also found to be elevated in these radial growth phase melanoma cells without any appreciable evidence of gene amplification and Id1 promoter activity was found to correlate with Id expression levels. We therefore conclude that Id1 expression is primarily regulated at the transcriptional level in radial growth phase melanomas and expect that therapies that target Id1 gene expression may be useful in the treatment of Id-associated malignancies. © 2007 Wiley-Liss, Inc. [source] The KRAB Domain of Zinc Finger Gene ZNF268: a Potential Transcriptional RepressorIUBMB LIFE, Issue 3 2003Yan Sun Abstract Nearly one-third of Krupple-type C2H2 zinc finger proteins have a krupple-associated box (KRAB) domain, which may act as a transcriptional repressor. ZNF268, which was novelty isolated from early human embryo, is a typical krupple-type C2H2 zinc finger protein with a conserved KRAB domain. In this report, the KRAB domain of ZNF268 is identified to localize in the nucleus and has transcriptional repressor activity. IUBMB Life, 55: 127-131, 2003 [source] RANKL Treatment Releases the Negative Regulation of the Poly(ADP-Ribose) Polymerase-1 on Tcirg1 Gene Expression During Osteoclastogenesis,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 11 2006Guillaume E Beranger Abstract The Tcirg1 gene encodes the osteoclast-specific a3 isoform of the V-ATPase a subunit. Using the mouse osteoclastic model RAW264.7 cells, we studied Tcirg1 gene expression, and we identified PARP-1 as a transcriptional repressor negatively regulated by RANKL during osteoclastogenesis. Introduction: The TCIRG1 gene encodes the a3 isoform of the V-ATPase a subunit, and mutations at this locus account for ,60% of infantile malignant osteopetrosis cases. Using RAW264.7 cells as an osteoclastic differentiation model, we undertook a transcriptional study of the mouse Tcirg1 gene focused on the 4-kb region upstream of the transcription starting point. Materials and Methods: The promoter activity of serial-deletion fragments of the Tcirg1 gene promoter was monitored throughout the RAW264.7 cell differentiation process. We next performed EMSA, UV cross-linking, affinity purification, mass spectrometry analysis, gel supershift, and siRNA transfection experiments to identify the factor(s) interacting with the promoter. Results: The ,3946/+113 region of the mouse Tcirg1 gene displayed a high basal promoter activity, which was enhanced by RANKL treatment of RAW264.7 cells. Constructs deleted up to ,1589 retained this response to RANKL. A deletion up to ,1402 induced a 3-fold enhancement of the basal activity, whereas RANKL response was not affected. EMSA experiments led us to identify within the ,1589/,1402 region, a 10-nucleotide sequence, which bound a nuclear protein present in nondifferentiated RAW264.7 cells. This interaction was lost using nuclear extracts derived from RANKL-treated cells. Affinity purification followed by mass spectrometry analysis and gel supershift assay allowed the identification of poly(ADP-ribose) polymerase-1 (PARP-1) as this transcriptional repressor, whereas Western blot experiments revealed the cleavage of the DNA-binding domain of PARP-1 on RANKL treatment. Finally, both PARP-1 depletion after siRNA transfection and RAW264.7 cell treatment by an inhibitor of PARP-1 activity induced an increase of a3 mRNA expression. Conclusions: We provide evidence that the basal transcription activity of the Tcirg1 gene is negatively regulated by the binding of PARP-1 protein to its promoter region in mouse pre-osteoclast. On RANKL treatment, PARP-1 protein is cleaved and loses its repression effect, allowing an increase of Tcirg1 gene expression that is critical for osteoclast function. [source] FIAT control of osteoblast activityJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2010René St-Arnaud Abstract The basic domain-leucine zipper transcription factor activating transcription factor 4 (ATF4) regulates most functions of the osteoblast. It is therefore not surprising that its activity should be regulated through several mechanisms. Factor inhibiting ATF4-mediated transcription (FIAT) is a leucine zipper nuclear molecule lacking a basic domain for DNA binding that interacts with ATF4 to repress its transcriptional activity. FIAT expression was monitored in parallel with ATF4 during osteoblastogenesis. The mechanism of ATF4 repression by FIAT was investigated through structure,function analysis. The physiological significance of FIAT expression in osteoblasts was studied through silencing FIAT in osteoblasts by RNA interference, as well as through characterization of two genetic mouse models: FIAT transgenic mice which overexpress FIAT in osteoblasts, and FIAT knockout mice. Studies to date show that FIAT and ATF4 are co-expressed in osteoblasts, and that FIAT inhibition of matrix mineralization requires dimerization with ATF4 through the second leucine zipper. Furthermore, transgenic mice overexpressing FIAT exhibit osteopenia. The phenotype of FIAT knockout mice is still under evaluation but the salient aspects are discussed here. Taken together, the results accumulated to date support the hypothesis that FIAT is a transcriptional repressor that modulates osteoblast function. J. Cell. Biochem. 109: 453,459, 2010. © 2009 Wiley-Liss, Inc. [source] The interaction of KCTD1 with transcription factor AP-2, inhibits its transactivation,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2009Xiaofeng Ding Abstract AP-2 is a transcription factor implicated in mammalian development, cell proliferation, apoptosis, and carcinogenesis. To identify potential AP-2,-interacting partners, a yeast two-hybrid screen was performed in human brain cDNA library. One of the identified clones encodes potassium channel tetramerization domain-containing 1 (KCTD1). We demonstrated the novel KCTD1,AP-2, interaction in vitro by GST pull-down assays and in vivo by co-immunoprecipitation assays and mapped the interaction domains to the N-termini of both proteins. In addition, we observed that the two proteins were completely co-localized in the nuclei of mammalian cells. Transient transfection assays using four promoters containing AP-2-binding sites confirmed that KCTD1 significantly repressed AP-2,-mediated transactivation through the BTB domain, whereas KCTD1 siRNA strongly relieved KCTD1-mediated repression of AP-2, transcriptional activity, and other BTB domain proteins such as PDIP1, KCTD10, and TNFAIP1 did not markedly inhibit the transcriptional activity of AP-2,, suggesting that KCTD1 specifically acts as a negative regulator of AP-2,. Finally, we found that KCTD1 interacted with three major members of the AP-2 family and inhibited their transcriptional activities. Taken together, our results indicate the novel function of KCTD1 as the transcriptional repressor for AP-2 family, especially for AP-2,. J. Cell. Biochem. 106: 285,295, 2009. © 2008 Wiley-Liss, Inc. [source] The Freud-1/CC2D1A family: Transcriptional regulators implicated in mental retardationJOURNAL OF NEUROSCIENCE RESEARCH, Issue 13 2007Anastasia Rogaeva Abstract The CC2D1A gene family consists of two homologous genes, Freud-1/CC2D1A and Freud-2/CC2D1B, that share conserved domains, including several DM14 domains that are specific to this protein family, a C-terminal helix-loop-helix domain, and a C2 calcium-dependent phospholipid binding domain. Although the function of Freud-2 is unknown, Freud-1 has been shown to function as a transcriptional repressor of the serotonin-1A receptor gene that binds to a novel DNA element (FRE, 5,-repressor element). The DNA binding and repressor activities of Freud-1 are inhibited by calcium-calmodulin-dependent protein kinase. Recently, a deletion in the CC2D1A gene has been linked to nonsyndromic mental retardation. This deletion results in the truncation of the helix-loop-helix DNA binding and the C2 domains, crucial for Freud-1 repressor activity, and hence is predicted to generate an inactive or weakly dominant negative protein. The possible mechanisms by which inactivation of Freud-1 could lead to abnormal cortical development and cognitive impairment and the potential roles of Freud-1 gene targets are discussed. © 2007 Wiley-Liss, Inc. [source] Functional analysis of NsrR, a nitric oxide-sensing Rrf2 repressor in Neisseria gonorrhoeaeMOLECULAR MICROBIOLOGY, Issue 1 2009Vincent M. Isabella Summary Nitric oxide (NO) has been shown to be an important component of the human immune response, and as such, it is important to understand how pathogenic organisms respond to its presence. In Neisseria gonorrhoeae, recent work has revealed that NsrR, an Rrf2-type transcriptional repressor, can sense NO and control the expression of genes responsible for NO metabolism. A highly pure extract of epitope-tagged NsrR was isolated and mass spectroscopic analysis suggested that the protein contained a [2Fe,2S] cluster. NsrR/DNA interactions were thoroughly analysed in vitro. Using EMSA analysis, NsrR::FLAG was shown to interact with predicted operators in the norB, aniA and nsrR upstream regions with a Kd of 7, 19 and 35 nM respectively. DNase I footprint analysis was performed on the upstream regions of norB and nsrR, where NsrR was shown to protect the predicted 29 bp binding sites. The presence of exogenously added NO inhibited DNA binding by NsrR. Alanine substitution of C90, C97 or C103 in NsrR abrogated repression of norB::lacZ and inhibited DNA binding, consistent with their presumed role in co-ordination of a NO-sensitive Fe,S centre required for DNA binding. [source] Membrane localization itself but not binding to IICBGlc is directly responsible for the inactivation of the global repressor Mlc in Escherichia coliMOLECULAR MICROBIOLOGY, Issue 3 2004Yuya Tanaka Summary Mlc is a global transcriptional repressor involved in the regulation of genes linked to glucose metabolism. The activity of Mlc is modulated through the interaction with a major glucose transporter, IICBGlc, in response to external glucose. To understand how IICBGlc,Mlc interaction controls the repressor activity of Mlc, we attempted to isolate Mlc mutants that retain the ability to repress target genes even in the presence of glucose. The Mlc mutants were tested for their ability to interact with IICBGlc. Mutants in which a single amino acid substitution occurs in the N-terminal portion were no longer able to bind to IICBGlc, suggesting that the N-terminal region of Mlc is primarily responsible for the interaction with IICBGlc. To examine whether the Mlc,IICBGlc interaction and/or the membrane localization of Mlc per se are essential for the inactivation of Mlc, the properties of several hybrid proteins in which either IIBGlc or Mlc is fused to membrane proteins were analysed. The cytoplasmic IIBGlc domain failed to inhibit the Mlc action although it retains the ability to bind Mlc in cells. However, it gained the ability to inhibit the Mlc activity when it was fused to a membrane protein LacY. In addition, we showed that Mlc is inactivated when fused to membrane proteins but not when fused to cytoplasmic proteins. We conclude that the IICBGlc,Mlc interaction is dispensable for the inactivation of Mlc, and that membrane localization is directly responsible for the inactivation of Mlc. [source] TBX3 and its splice variant TBX3 + exon 2a are functionally similarPIGMENT CELL & MELANOMA RESEARCH, Issue 3 2008Willem M.H. Hoogaars Summary Tbx3, a member of the conserved family of T-box developmental transcription factors, is a transcriptional repressor required during cardiogenesis for the formation and specification of the sinoatrial node, the pacemaker of the heart. Both the TBX3 and the highly related TBX2 genes are also associated with several cancers, most likely as a consequence of their powerful anti-senescence properties mediated via suppression p14Arf and p21CIP expression. In melanoma, the TBX2 gene is frequently amplified and inhibition of Tbx2 function leads to senescence and up-regulation of p21CIP, a Tbx2 target gene. Tbx3 + 2a is a splice variant containing an extra 20 amino acids encoded by exon 2a inserted into the highly conserved T-box DNA-binding domain. We find here that Tbx3 + 2a is evolutionary conserved and that similar insertions are largely absent from the T-box domains of other T-box factors. Tbx3 + 2a has been reported to lack DNA-binding ability and act as a functional antagonist of Tbx3. By contrast, we now demonstrate that both Tbx3 and Tbx3 + 2a bind the consensus T-element, the p21CIP1 promoter, and the Nppa cardiac target gene. Both isoforms also function as repressors of p21CIP1 and Nppa promoter activity and interact with homeobox factor Nkx2-5. When ectopically expressed in the embryonic heart of mice, Tbx3 and Tbx3 + 2a both suppressed chamber formation and repressed expression of cardiac chamber markers Nppa and Cx40. The results suggest that in the assays used, Tbx3 and Tbx3 + 2a are functionally equivalent and that like Tbx2, Tbx3 may also function as an anti-senescence factor in melanoma. [source] Transcriptional profiling of hexaploid wheat (Triticum aestivum L.) roots identifies novel, dehydration-responsive genesPLANT CELL & ENVIRONMENT, Issue 5 2007MOHSEN MOHAMMADI ABSTRACT We used a long-oligonucleotide microarray to identify transcripts that increased or decreased in abundance in roots of dehydration-tolerant hexaploid bread wheat, in response to withholding of water. We observed that the major classes of dehydration-responsive genes (e.g. osmoprotectants, compatible solutes, proteases, glycosyltransferases/hydrolases, signal transducers components, ion transporters) were generally similar to those observed previously in other species and osmotic stresses. More specifically, we highlighted increases in transcript expression for specific genes including those putatively related to the synthesis of asparagine, trehalose, oligopeptide transporters, metal-binding proteins, the gamma-aminobutyric acid (GABA) shunt and transcription factors. Conversely, we noted a decrease in transcript abundance for diverse classes of glutathione and sulphur-related enzymes, specific amino acids, as well as MATE-efflux carrier proteins. From these data, we identified a novel, dehydration-induced putative AP2/ERF transcription factor, which we predict to function as a transcriptional repressor. We also identified a dehydration-induced ,little protein' (LitP; predicted mass: 8 kDa) that is highly conserved across spermatophytes. Using qRT-PCR, we compared the expression patterns of selected genes between two related wheat genotypes that differed in their susceptibility to dehydration, and confirmed that these novel genes were highly inducible by water limitation in both genotypes, although the magnitude of induction differed. [source] Crystal structure of an enhancer of rudimentary homolog (ERH) at 2.1 Å resolutionPROTEIN SCIENCE, Issue 7 2005Ryoichi Arai Abstract The enhancer of rudimentary gene, e(r), of Drosophila melanogaster encodes an enhancer of rudimentary (ER) protein with functions implicated in pyrimidine biosynthesis and the cell cycle. The ER homolog (ERH) is highly conserved among vertebrates, invertebrates, and plants. Xenopus laevis ERH was reported to be a transcriptional repressor. Here we report the 2.1 Å crystal structure of murine ERH (Protein Data Bank ID 1WZ7), determined by the multiwavelength anomalous dispersion (MAD) method. The monomeric structure of ERH comprises a single domain consisting of three ,-helices and four ,-strands, which is a novel fold. In the crystal structure, ERH assumes a dimeric structure, through interactions between the ,-sheet regions. The formation of an ERH dimer is consistent with the results of analytical ultracentrifugation. The residues at the core region and at the dimer interface are highly conserved, suggesting the conservation of the dimer formation as well as the monomer fold. The long flexible loop (44,53) is also significantly conserved, suggesting that this loop region may be important for the functions of ERH. In addition, the putative phosphorylation sites are located at the start of the ,2-strand (Thr18) and at the start of the ,1-helix (Ser24), implying that the phosphorylation might cause some structural changes. [source] Facile chemical synthesis and equilibrium unfolding properties of CopGPROTEIN SCIENCE, Issue 7 2004Thomas E. Wales Abstract The 45-amino acid polypeptide chain of the homodimeric transcriptional repressor, CopG, was chemically synthesized by stepwise solid phase peptide synthesis (SPPS) using a protocol based on Boc-chemistry. The product obtained from the synthesis was readily purified by reversed-phase HPLC to give a good overall yield (21% by weight). Moreover, the synthetic CopG constructs prepared in this work folded into three-dimensional structures similar to the wild-type protein prepared using conventional recombinant methods as judged by far UV-CD spectroscopy. A fluorescent CopG analog, (Y39W)CopG, was also designed and chemically synthesized to facilitate biophysical studies of CopG's protein folding and assembly reaction. The guanidinium chloride-induced equilibrium unfolding properties of the wild-type CopG and (Y39W)CopG constructs in this work were characterized and used to develop a model for CopG's equilibrium unfolding reaction. Our results indicate that CopG's folding and assembly reaction is well modeled by a two-state process involving folded dimer and unfolded monomer. Using this model, ,Gf and m -values of ,13.42 ± 0.04 kcal/mole dimer and 1.92 ± 0.01 kcal/(mole M) were calculated for CopG. [source] Effects of Th2 pulmonary inflammation in mice with bleomycin-induced pulmonary fibrosisRESPIROLOGY, Issue 6 2008Hirokuni HIRATA Background and objective: Leucocytes, especially lymphocytes and neutrophils, as well as alveolar macrophages, that infiltrate into the lung are involved in the development of pulmonary fibrosis. However, the role of T helper (Th)2-type inflammation, mediated by Th2 cells and eosinophils, in fibrosis remains unknown. Transgenic mice deficient in the transcriptional repressor, Bcl6, display an attenuation of Th2 cytokine production. We studied the effects of Th2-type pulmonary inflammation on bleomycin-induced pulmonary fibrosis using Bcl6 transgenic mice. Methods: Bleomycin was administered to ovalbumin (OVA)-sensitized Bcl6 transgenic and wild-type mice by intratracheal instillation during sequential OVA antigen challenge. Concentrations of transforming growth factor-,1 in the BAL fluid were measured 2 weeks after bleomycin administration. At the same time lung tissue was examined histopathologically, and homogenized to assess collagen levels and Th1/Th2 cytokine mRNA expression. Results: Although OVA-sensitized, bleomycin-treated Bcl6 transgenic mice had markedly lower numbers of eosinophils in both BAL and lung tissue compared with OVA-sensitized, bleomycin-treated wild-type mice, the development of pulmonary fibrosis in response to bleomycin was similar in Bcl6 transgenic mice and wild-type mice. Conclusion: These results suggest that Th2-dominant inflammation in the lung is not essential for the development of bleomycin-induced pulmonary fibrosis. [source] Ternary complex formation between HvMYBS3 and other factors involved in transcriptional control in barley seedsTHE PLANT JOURNAL, Issue 2 2006I. Rubio-Somoza Summary The SHAQKYF R1MYB transcription factor (TF) HvMYBS3 from barley is an activator of gene expression both during endosperm development and in aleurone cells upon seed germination. Its mRNA was detected as early as 10 days after flowering in developing barley endosperm, with a peak at 18 days, and in aleurone cells at 8 h after water imbibition, as shown by Northern blot and in situ hybridization analyses. The HvMYBS3 protein expressed in bacteria binds to oligonucleotides containing a GATA core derived from the promoters of: (i) the developing endosperm gene Itr1 (5,- GATAAGATA -3,) encoding trypsin inhibitor BTI-CMe, and (ii) the post-germinating aleurone gene Amy6.4 (5,-TATCCAC-3,/5,-GTGGATA -3,) encoding a high-pI , -amylase. Transient expression experiments in co-bombarded developing endosperms and in barley aleurone layers demonstrated that HvMYBS3 trans -activated transcription both from Itr1 and Amy6.4 promoters, in contrast with a previously reported seed-expressed R1MYB, HvMCB1, which was an activator of Itr1 and a transcriptional repressor of the Amy6.4 gene. In the yeast three-hybrid system, the HvMYBS3 protein formed a ternary complex with BPBF and BLZ2, two important seed TFs. However, no binary interactions could be detected between HvMYBS3 and BLZ2 or between HvMYBS3 and BPBF. [source] HvMCB1, a R1MYB transcription factor from barley with antagonistic regulatory functions during seed development and germinationTHE PLANT JOURNAL, Issue 1 2006Ignacio Rubio-Somoza Summary The functional analysis of hydrolase gene promoters induced by gibberellin (GA) in barley aleurone cells upon germination has identified a tripartite GA-response complex (GARC) containing a 5,- TATCCAC-3, box as well as the GA-responsive element (GARE) recognized by GAMYB and the pyrimidine box interacting with the DOF transcription factors BPBF and SAD. We show here that the MCB1 gene encoding a R1MYB protein binds to the 5,- TATCCAC-3, (GATA core) box in vitro and is a transcriptional repressor of a GA-induced amylase (Amy6.4) promoter in bombarded aleurone layers. Northern blot and mRNA in situ hybridization analyses showed that the MCB1 transcripts accumulate in the aleurone cells upon germination, as well as in endosperm tissues during seed development. The HvMCB1 protein expressed in bacteria binds in a specific manner to a 27-mer oligonucleotide containing the 5,-TATCCAC-3, sequence, derived from the promoter region of the Amy6.4 gene. Accumulation of the MCB1 transcript diminished in response to external GA incubation in aleurone cells, and in transient expression experiments HvMCB1 repressed transcription of the Amy6.4 promoter in GA-treated aleurone layers and reversed the GAMYB-mediated activation of this amylase promoter. In contrast, during endosperm maturation HvMCB1 acted as a transcription activator of the seed-specific Itr1 gene promoter through binding to a 5,-GATAAGATA-3, box. [source] Expression, purification, crystallization and preliminary crystallographic studies of the Enterococcus faecalis cytolysin repressor CylR2ACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2004Adelia Razeto The expression of an exotoxin called cytolysin contributes to the virulence of Enterococcus faecalis, one of the organisms responsible for antibiotic resistant infections acquired in hospitals. The DNA-binding protein CylR2 is a transcriptional repressor of cytolysin. At a specific cell density, cytolysin triggers signaling events, which result in the dissociation of CylR2 from its DNA-binding site. CylR2 was overexpressed in Escherichia coli and purified and crystals diffracting to 1.9,Å were obtained in two different crystal forms. One crystal form belongs to space group P41, with unit-cell parameters a = 63.7, b = 63.7, c = 41.2,Å, , = , = , = 90°, and the other belongs to space group P1, with unit-cell parameters a = 36.9, b = 45.0, c = 47.7,Å, , = 67, , = 90, , = 66°. [source] CtBP family proteins: More than transcriptional corepressorsBIOESSAYS, Issue 1 2003G. Chinnadurai CtBP family proteins predominantly function as transcriptional corepressors. Studies with mutant mouse suggest that the two mouse genes, Ctbp1 and Ctbp2, play unique and redundant gene regulatory roles during development.1Ctbp1 -deficient mice are viable, but are small and die early, while Ctbp2 deficiency leads to embryonic lethality. Ctbp2 -null mutation causes defects in axial patterning, heart morphogenesis and neural development. The Ctbp2 mutant phenotype is more severe in the absence of Ctbp1. The studies with Ctbp2 mutant embryos suggest that CtBP can also activate transcription. A plant CtBP homolog, Angustifolia (AN), has recently been identified.2,3AN controls polar elongation of leaf cells via the microtubule cytoskeleton. Microarray analysis suggests that AN also functions as a transcriptional repressor. Thus, the CtBP family proteins control cellular processes by serving as transcriptional activators and regulators of the cytoskeleton as well as transcriptional corepressors. BioEssays 25:9,12, 2003. © 2002 Wiley Periodicals, Inc. [source] Taming the fierce roller: an "enhanced" understanding of cellular differentiation in VolvoxBIOESSAYS, Issue 1 2002Stephen M. Miller Few organisms offer a better opportunity to explore the mechanisms of cellular differentiation, and their origins, than Volvox. Volvox consists of just two cell types, germ and soma, and is the most complex member of a family of green algae that includes unicellular and multicellular relatives. At the heart of the cell-fate determination program of Volvox carteri is the regA gene, which encodes a putative transcriptional repressor that prevents somatic cells from expressing reproductive functions. Stark et al.(1) have dissected the regA gene to determine how its expression is restricted to somatic cells. Their results suggest that regA expression is controlled by multiple enhancers, the most important of which prevents transcription in reproductive cells. While these findings shed light on Volvox development, they also raise a new set of questions about the mechanisms that control the germ,soma dichotomy in this organism. BioEssays 24:3,7, 2002. © 2002 John Wiley & Sons, Inc. [source] | |||||||||||||||||||||||||||||||