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At least 307 records · Page 17Linked to original sources
PubMed2000

Histone deacetylase-independent transcriptional repression by methyl-CpG-binding protein 2.

Methyl-CpG-binding protein 2 (MeCP2) contains a transcriptional repression domain (TRD), which can act by recruitment of a large transcriptional co-repressor complex containing histone deacetylases HDAC1 and 2. We demonstrate here that transient transcription from the SV40 enhancer/promoter or the SV40 promoter is strongly repressed in a histone deacetylase-independent manner, since repression is not alleviated by Trichostatin A (TSA). In a mutational analysis, repression depends on a conserved 30 residue sequence containing two clusters of basic amino acids. Mutation of the first of these clusters inhibits in vitro interaction between TRD and mSin3A. Furthermore, a subdomain of the TRD containing the conserved 30-residue sequence and 16 flanking amino acids was sufficient to compromise VP16-activated transcription. In summary, our results indicate an alternative, histone deacetylase-independent pathway of transcriptional repression by MeCP2.

3T3 Cells↗
PubMed2001

Brinker requires two corepressors for maximal and versatile repression in Dpp signalling.

decapentaplegic (dpp) encodes a Drosophila transforming growth factor-beta homologue that functions as a morphogen in the developing embryo and in adult appendage formation. In the wing imaginal disc, a Dpp gradient governs patterning along the anteroposterior axis by inducing regional expression of diverse genes in a concentration-dependent manner. Recent studies show that responses to graded Dpp activity also require an input from a complementary and opposing gradient of Brinker (Brk), a transcriptional repressor protein encoded by a Dpp target gene. Here we show that Brk harbours a functional and transferable repression domain, through which it recruits the corepressors Groucho and CtBP. By analysing transcriptional outcomes arising from the genetic removal of these corepressors, and by ectopically expressing Brk variants in the embryo, we demonstrate that these corepressors are alternatively used by Brk for repressing some Dpp-responsive genes, whereas for repressing other distinct target genes they are not required. Our results show that Brk utilizes multiple means to repress its endogenous target genes, allowing repression of a multitude of complex Dpp target promoters.

Alcohol Oxidoreductases↗
PubMed2005

ZNF649, a novel Kruppel type zinc-finger protein, functions as a transcriptional suppressor.

Cardiac differentiation involves a cascade of coordinated gene expression that regulates cell proliferation and matrix protein formation in a defined temporo-spatial manner. Many of the KRAB-ZFPs are involved in cardiac development or cardiovascular diseases. Here we report the identification and characterization of a novel human zinc-finger gene named ZNF649. The cDNA of ZNF649 is 3176 bp, encoding a protein of 505 amino acids in the nuclei. Northern blot analysis indicates that ZNF649 is expressed in most of the examined human adult and embryonic tissues. ZNF649 is a transcription suppressor when fused to GAL-4 DNA-binding domain and cotransfected with VP-16. Overexpression of ZNF649 in COS-7 cells inhibits the transcriptional activities of SRE and AP-1. Deletion analysis with a series of truncated fusion proteins indicates that the KRAB motif is a basal repression domain when the truncated fusion proteins were assayed for the transcriptional activities of SRE and AP-1. These results suggest that ZNF649 protein may act as a transcriptional repressor in mitogen-activated protein kinase signaling pathway to mediate cellular functions.

Amino Acid Sequence↗
PubMed2005

ZNF328, a novel human zinc-finger protein, suppresses transcriptional activities of SRE and AP-1.

The zinc finger proteins containing the Kruppel-associated box domain (KRAB-ZFPs) are the single largest class of transcription factors in human genome. Many of the KRAB-ZFPs are involved in cardiac development or cardiovascular diseases. Here, we have identified a novel human KRAB zinc finger gene, named ZNF328, from the human fetal heart cDNA library. The complete sequence of ZNF328 cDNA contains a 2376-bp open reading frame (ORF) and encodes a 792 amino acid protein with an N-terminal KRAB domain and classical zinc finger C2H2 motifs in the C-terminus. Northern blot analysis indicates that the protein is expressed in most of the examined human adult and embryonic tissues. ZNF328 is a transcription suppressor when fused to Gal-4 DNA-binding domain and cotransfected with VP-16. Overexpression of ZNF328 in COS-7 cells inhibits the transcriptional activities of SRE and AP-1. Deletion analysis with a series of truncated fusion proteins indicates that the KRAB motif is a basal repression domain when cotransfected with VP-16. Similar results were obtained when the truncated fusion proteins were assayed for the transcriptional activities of SRE and AP-1. These results suggest that ZNF328 protein may act as a transcriptional repressor in mitogen-activated protein kinase (MAPK) signaling pathway to mediate cellular functions.

Amino Acid Sequence↗
PubMed1997

Repression by Ume6 involves recruitment of a complex containing Sin3 corepressor and Rpd3 histone deacetylase to target promoters.

Sin3 and Rpd3 negatively regulate a diverse set of yeast genes. A mouse Sin3-related protein is a transcriptional corepressor, and a human Rpd3 homolog is a histone deacetylase. Here, we show that Sin3 and Rpd3 are specifically required for transcriptional repression by Ume6, a DNA-binding protein that regulates genes involved in meiosis. A short region of Ume6 is sufficient to repress transcription, and this repression domain mediates a two-hybrid and physical interaction with Sin3. Coimmunoprecipitation and two-hybrid experiments indicate that Sin3 and Rpd3 are associated in a complex distinct from TFIID and Pol II holoenzyme. Rpd3 is specifically required for repression by Sin3, and artificial recruitment of Rpd3 results in repression. These results suggest that repression by Ume6 involves recruitment of a Sin3-Rpd3 complex and targeted histone deacetylation.

DNA-Binding Proteins↗
PubMed1998

Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex.

Cytosine residues in the sequence 5'CpG (cytosine-guanine) are often postsynthetically methylated in animal genomes. CpG methylation is involved in long-term silencing of certain genes during mammalian development and in repression of viral genomes. The methyl-CpG-binding proteins MeCP1 and MeCP2 interact specifically with methylated DNA and mediate transcriptional repression. Here we study the mechanism of repression by MeCP2, an abundant nuclear protein that is essential for mouse embryogenesis. MeCP2 binds tightly to chromosomes in a methylation-dependent manner. It contains a transcriptional-repression domain (TRD) that can function at a distance in vitro and in vivo. We show that a region of MeCP2 that localizes with the TRD associates with a corepressor complex containing the transcriptional repressor mSin3A and histone deacetylases. Transcriptional repression in vivo is relieved by the deacetylase inhibitor trichostatin A, indicating that deacetylation of histones (and/or of other proteins) is an essential component of this repression mechanism. The data suggest that two global mechanisms of gene regulation, DNA methylation and histone deacetylation, can be linked by MeCP2.

Acetylation↗
PubMed2001

A new functional domain of Bcl6 family that recruits histone deacetylases.

The proto-oncogene Bcl6 and its family gene, BAZF, encode a sequence-specific transcriptional repressor which contains the BTB/POZ domain in NH(2)-terminal region and zinc finger motifs in COOH-terminal region. The BTB/POZ domain and the middle portion of Bcl6 and BAZF are known to display transrepressor activity. Since we have identified the identical 17-amino acid (aa) sequence in the middle portion of Bcl6 and BAZF, the 17aa region may be another repressive domain of the middle portion. The reporter gene assay indicates that the 27aa sequence including the 17aa region recruits histone deacetylases to express transrepressor activity. Furthermore, overexpression of Bcl6 or Bcl6(POZ-) (Bcl6 deleted with the BTB/POZ domain) induced apoptosis in NIH3T3 cells, and the apoptosis was inhibited by the addition of histone deacetylase inhibitor in the culture. However, apoptosis was not induced in NIH3T3 cells by overexpression of Bcl6(POZ-) deleted with the 17aa region. These results indicate that the 17aa region in the middle portion of Bcl6 is a functional domain of transrepressor activity and is responsible for inducibility of apoptosis in NIH3T3 cells.

3T3 Cells↗
PubMed1996

Functional analysis of histones H2A and H2B in transcriptional repression in Saccharomyces cerevisiae.

The presence of H2A-H2B dimers in nucleosomes can inhibit the binding of transcription factors to chromatin templates. To study the roles of histones H2A and H2B in transcriptional repression in vivo, mutant forms of these histones were analyzed in two different assay systems. Two repression domains were identified in H2A. One domain includes residues that fall in the beginning of the H2A-H2B dimerization region, and the second is in the H2A N terminus, a region of potential interactions with nonhistone proteins. The function of H2A and H2B in one repression assay was found to be dependent on three SPT (suppressor of Ty) genes whose products are important for chromatin-mediated repression. These results suggest that repressive chromatin structure may be established through the interactions of the Spt proteins with these histones. In contrast, other proteins, the products of the HIR (histone regulation) genes, may function to direct H2A and H2B to specific promoters.

Base Sequence↗
PubMed1997

Overexpression, purification, characterization, and crystallization of the BTB/POZ domain from the PLZF oncoprotein.

The BTB/POZ domain defines a conserved region of about 120 residues and has been found in over 40 proteins to date. It is located predominantly at the N terminus of Zn-finger DNA-binding proteins, where it may function as a repression domain, and less frequently in actin-binding and poxvirus-encoded proteins, where it may function as a protein-protein interaction interface. A prototypic human BTB/POZ protein, PLZF (promyelocytic leukemia zinc finger) is fused to RARalpha (retinoic acid receptor alpha) in a subset of acute promyelocytic leukemias (APLs), where it acts as a potent oncogene. The exact role of the BTB/POZ domain in protein-protein interactions and/or transcriptional regulation is unknown. We have overexpressed, purified, characterized, and crystallized the BTB/POZ domain from PLZF (PLZF-BTB/POZ). Gel filtration, dynamic light scattering, and equilibrium sedimentation experiments show that PLZF-BTB/POZ forms a homodimer with a Kd below 200 nM. Differential scanning calorimetry and equilibrium denaturation experiments are consistent with the PLZF-BTB/POZ dimer undergoing a two-state unfolding transition with a Tm of 70.4 degrees C, and a DeltaG of 12.8 +/- 0.4 kcal/mol. Circular dichroism shows that the PLZF-BTB/POZ dimer has significant secondary structure including about 45% helix and 20% beta-sheet. We have prepared crystals of the PLZF-BTB/POZ that are suitable for a high resolution structure determination using x-ray crystallography. The crystals form in the space group I222 or I212121 with a = 38.8, b = 77.7, and c = 85.3 A and contain 1 protein subunit per asymmetric unit with approximately 40% solvent. Our data support the hypothesis that the BTB/POZ domain mediates a functionally relevant dimerization function in vivo. The crystal structure of the PLZF-BTB/POZ domain will provide a paradigm for understanding the structural basis underlying BTB/POZ domain function.

Amino Acid Sequence↗
PubMed1999

cDNA cloning and expression of rat homeobox gene, Hex, and functional characterization of the protein.

We isolated two cDNA clones of rat Hex, a homeobox protein, studied its expression in rat liver and various cells, and characterized the protein. The levels of Hex mRNA were only slightly increased in liver of rats refed with a high-carbohydrate diet or after partial hepatectomy. Whereas the expression of Hex mRNA was detected in hepatocytes isolated from adult rat liver and also in highly differentiated hepatoma cells, no Hex mRNA was detected in poorly differentiated hepatoma cells. Hex mRNA was also detected in liver from embryo aged 15 days. Expression of Hex was increased in F9 cells during differentiation into visceral endoderm cells by treatment with retinoic acid. This stimulation occurred prior to an increase in the level of alpha-fetoprotein mRNA. When fusion-protein expression vectors of GAL4 DNA-binding domain and Hex were co-transfected with luciferase reporter plasmid, with or without five copies of the GAL4-binding site, into HepG2 cells, the luciferase activities were decreased in concentration- and GAL4-binding site-dependent manners. This repression did not require the presence of the homeodomain, which is located between the amino acid residues 137 and 196. Its repression domain was mapped between the residues 45 and 136 in the proline-rich N-terminal region. In addition, the homeodomain was responsible for DNA-binding of Hex. These results indicate that Hex functions as a transcriptional repressor and may be involved in the differentiation and/or maintenance of the differentiated state in hepatocytes.

Amino Acid Sequence↗
PubMed2004

MdmX represses E2F1 transactivation.

Based on knockout mouse studies, Mdm2 and MdmX have been identified as critical regulators of the p53 tumor suppressor protein, at least during early development. While many of the functions attributed to Mdm2 and MdmX involve p53 and overexpression of each gene appears to have oncogenic activities, a number of studies have suggested that each protein also possesses p53-independent functions. While examining the effect of Mdm2 overexpression on E2F1 transactivation we uncovered a novel MdmX function, the ability to inhibit E2F1 transactivation in a p53 and Mdm2 independent manner. Using a series of MdmX deletion mutants the central region of MdmX, amino acids 128-444 appears to possess the repressive domain. While an in vivo association of MdmX with either E2F1 or DP1 was not observed, a slight reduction in DP1 and an increased cytoplasmic localization of E2F1 were seen in cells overexpressing MdmX. These results suggest that elevated MdmX expression may repress E2F1-regulated genes like p14ARF and thus represent another regulatory mechanism in the Rb-p53 signaling pathway.

Blotting, Western↗
PubMed2003

Scanning the human genome with combinatorial transcription factor libraries.

Despite the critical importance of transcription factors in mediating gene regulation, there exists no general, genome-wide tool that uses transcription factors to induce or silence a target gene or select for a particular phenotype. In the strategy described here, we prepared large combinatorial libraries of artificial transcription factors comprising three or six zinc-finger domains, and selected transcription factor-DNA interactions able to upregulate several genes in human cells. Selected transcription factors either induced the expression of an endothelial-specific differentiation marker, VE-cadherin, in non-endothelial cell lines or, when combined with a repression domain, knocked down expression. Potential binding sites for a number of these transcription factors were mapped along the promoter of CDH5, the gene encoding VE-cadherin. Transcription factor libraries represent a useful approach for studying and modulating gene function in cells and potentially in whole organisms.

Cell Line↗
PubMed2006

Differential transcriptional regulation by mouse single-minded 2s.

Single-minded 1 and 2 are unique members of the basic helix-loop-helix Per-Arnt-Sim family as they are transcriptional repressors. Here we report the identification and transcriptional characterization of mouse Sim2s, a splice variant of Sim2, which is missing the carboxyl Pro/Ala-rich repressive domain. Sim2s is expressed at high levels in kidney and skeletal muscle; however, the ratio of Sim2 to Sim2s mRNA differs between these tissues. Similar to full-length Sim2, Sim2s interacts with Arnt and to a lesser extent, Arnt2. The effects of Sim2s on transcriptional regulation through hypoxia, dioxin, and central midline response elements are different than that of full-length Sim2. Specifically, Sim2s exerts a less repressive effect on hypoxia-induced gene expression than full-length Sim2, but is just as effective as Sim2 at repressing TCDD-induced gene expression from a dioxin response element. Interestingly, Sim2s bind to and activates expression from a central midline response element-controlled reporter through an Arnt transactivation domain-dependent mechanism. The differences in expression pattern, protein interactions, and transcriptional activities between Sim2 and Sim2s may reflect differential roles each isoform plays during development or in tissue-specific effects on other protein-mediated pathways.

Alternative Splicing↗
PubMed2006

A L225A substitution in the human tumour suppressor HIC1 abolishes its interaction with the corepressor CtBP.

HIC1 (hypermethylated in cancer) is a tumour suppressor gene located in 17p13.3, a region frequently hypermethylated or deleted in many types of prevalent human tumour. HIC1 is also a candidate for a contiguous-gene syndrome, the Miller-Dieker syndrome, a severe form of lissencephaly accompanied by developmental anomalies. HIC1 encodes a BTB/POZ-zinc finger transcriptional repressor. HIC1 represses transcription via two autonomous repression domains, an N-terminal BTB/POZ and a central region, by trichostatin A-insensitive and trichostatin A-sensitive mechanisms, respectively. The HIC1 central region recruits the corepressor CtBP (C-terminal binding protein) through a conserved GLDLSKK motif, a variant of the consensus C-terminal binding protein interaction domain PxDLSxK/R. Here, we show that HIC1 interacts with both CtBP1 and CtBP2 and that this interaction is stimulated by agents increasing NADH levels. Furthermore, point mutation of two CtBP2 residues forming part of the structure of the recognition cleft for a PxDLS motif also ablates the interaction with a GxDLS motif. Conversely, in perfect agreement with the structural data and the universal conservation of this residue in all C-terminal binding protein-interacting motifs, mutation of the central leucine residue (leucine 225 in HIC1) abolishes the interaction between HIC1 and CtBP1 or CtBP2. As expected from the corepressor activity of CtBP, this mutation also impairs the HIC1-mediated transcriptional repression. These results thus demonstrate a strong conservation in the binding of C-terminal binding protein-interacting domains despite great variability in their amino acid sequences. Finally, this L225A point mutation could also provide useful knock-in animal models to study the role of the HIC1-CtBP interaction in tumorigenesis and in development.

Alcohol Oxidoreductases↗
PubMed2000

The interaction of the carboxyl terminus-binding protein with the Smad corepressor TGIF is disrupted by a holoprosencephaly mutation in TGIF.

The homeodomain protein TGIF represses transcription in part by recruiting histone deacetylases. TGIF binds directly to DNA to repress transcription or interacts with TGF-beta-activated Smads, thereby repressing genes normally activated by TGF-beta. Loss of function mutations in TGIF result in holoprosencephaly (HPE) in humans. One HPE mutation in TGIF results in a single amino acid substitution in a conserved PLDLS motif within the amino-terminal repression domain. We demonstrate that TGIF interacts with the corepressor carboxyl terminus-binding protein (CtBP) via this motif. CtBP, which was first identified by its ability to bind the adenovirus E1A protein, interacts both with gene-specific transcriptional repressors and with a subset of polycomb proteins. Efficient repression of TGF-beta-activated gene responses by TGIF is dependent on interaction with CtBP, and we show that TGIF is able to recruit CtBP to a TGF-beta-activated Smad complex. Disruption of the PLDLS motif in TGIF abolishes the interaction of CtBP with TGIF and compromises the ability of TGIF to repress transcription. Thus, at least one HPE mutation in TGIF appears to prevent CtBP-dependent transcriptional repression by TGIF, suggesting an important developmental role for the recruitment of CtBP by TGIF.

Alcohol Oxidoreductases↗
PubMed1997

Characterization of mechanisms of interleukin-6 gene repression by estrogen receptor.

Estrogens are the most effective agents available for preventing osteoporosis, and their principal role in bone metabolism is the inhibition of interleukin-6 (IL-6) production in osteoblasts and bone marrow stromal cells. We examined the mechanism of inhibitory effect of estrogens on the 190 bp proximal promoter of the IL-6 gene. Promoter activity induced by transfection of both NF-kappaB p65 subunit and NF-IL6 was decreased by 45% by estradiol (E2)-estrogen receptor (ER) complexes. The inhibitory effect of E2 was also observed on a mutant IL-6 promoter in which the NF-IL6 binding site was disrupted. E2 repressed the wild-type promoter activity induced by NF-kappaB p65 subunit alone, but had no effect on that induced by NF-IL6 alone. These findings suggested that estrogens inhibit IL-6 production by interfering with the function of NF-kappaB rather than that of NF-IL6. The ER mutant, HE19, which does not contain the A/B domain, repressed the induction by NF-kappaB to the same extent as wild-type ER HE0, whereas the effect of C-terminal deletion mutant, HE21, was only marginal. The antiestrogen, 4-hydroxytamoxifen (OHT), had no effect on IL-6 promoter activity, suggesting that E2-induced conformational change of the hormone binding domain plays an important role in protein-protein interaction between ER and NF-kappaB. E2 had no effect on the nuclear translocation of NF-kappaB, and electrophoretic mobility shift assay showed that the presence of E2-ER complexes did not affect the ability of NF-kappaB to bind to specific DNA sequences.

Animals↗
PubMed2000

Mechanism of transcriptional regulation by methyl-CpG binding protein MBD1.

MBD1 is a mammalian protein that binds symmetrically methylated CpG sequences and regulates gene expression in association with DNA methylation. This protein possesses a conserved sequence, named methyl-CpG binding domain (MBD), among a family of methyl-CpG binding proteins that mediate the biological consequences of the methylation. In addition, MBD1 has at least five isoforms due to alternative splicing events, resulting in the presence of CXXC1, CXXC2, and CXXC3 in MBD1 isoforms v1 (MBD1v1) and MBD1v2, and CXXC1 and CXXC2 in MBD1v3 and -v4. In the present study, we have investigated the significance of MBD, CXXC, and the C-terminal transcriptional repression domain (TRD) in MBD1. A bacterially expressed MBD binds efficiently to densely methylated rather than to sparsely methylated DNAs. In both methylation-deficient Drosophila melanogaster SL2 cells and mammalian CHO-K1 cells, MBD1v1 represses transcription preferentially from both unmethylated and sparsely methylated promoters, while MBD1v3 inhibits densely methylated but not unmethylated promoter activities. The CXXC3 sequence in MBD1v1 is responsible for the ability to bind unmethylated promoter. Furthermore, we have constructed mutant-type MBD1s in which the functionally important residues Arg22, Arg30, Asp32, Tyr34, Arg44, Ser45, and Tyr52 are changed to alanine to investigate the correlation between the structure and function of the MBD in MBD1. Excepting those for Ser45 and Tyr52, none of the recombinant MBD mutants bound to the densely methylated or unmethylated DNAs, and green fluorescent protein-fused MBD1 mutants did not localize properly in the nucleus. All the MBD1v1 and -v3 mutants lost the activity of methylation-dependent gene repression. Based on these findings we have concluded that MBD1 acts as a transcriptional regulator depending on the density of methyl-CpG pairs through the cooperation of MBD, CXXC, and TRD sequences.

Amino Acid Motifs↗
PubMed1996

Truncated mammalian Notch1 activates CBF1/RBPJk-repressed genes by a mechanism resembling that of Epstein-Barr virus EBNA2.

The Notch/Lin-12/Glp-1 receptor family participates in cell-cell signaling events that influence cell fate decisions. Although several Notch homologs and receptor ligands have been identified, the nuclear events involved in this pathway remain incompletely understood. A truncated form of Notch, consisting only of the intracellular domain (NotchIC), localizes to the nucleus and functions as an activated receptor. Using both an in vitro binding assay and a cotransfection assay based on the two-hybrid principle, we show that mammalian NotchIC interacts with the transcriptional repressor CBF1, which is the human homolog of Drosophila Suppressor of Hairless. Cotransfection assays using segments of mouse NotchIC and CBF1 demonstrated that the N-terminal 114-amino-acid region of mouse NotchIC contains the CBF1 interactive domain and that the cdc10/ankyrin repeats are not essential for this interaction. This result was confirmed in immunoprecipation assays in which the N-terminal 114-amino-acid segment of NotchIC, but not the ankyrin repeat region, coprecipitated with CBF1. Mouse NotchIC itself is targeted to the transcriptional repression domain (aa179 to 361) of CBF1. Furthermore, transfection assays in which mouse NotchIC was targeted through Gal4-CBF1 or through endogenous cellular CBF1 indicated that NotchIC transactivates gene expression via CBF1 tethering to DNA. Transactivation by NotchIC occurs partially through abolition of CBF1-mediated repession. This same mechanism is used by Epstein-Barr virus EBNA2. Thus, mimicry of Notch signal transduction is involved in Epstein-Barr virus-driven immortalization.

Amino Acid Sequence↗