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At least 127 records · Page 7Linked to original sources
PubMed2003

Identification and characterization of three new components of the mSin3A corepressor complex.

The mSin3A corepressor complex contains 7 to 10 tightly associated polypeptides and is utilized by many transcriptional repressors. Much of the corepressor function of mSin3A derives from associations with the histone deacetylases HDAC1 and HDAC2; however, the contributions of the other mSin3A-associated polypeptides remain largely unknown. We have purified an mSin3A complex from K562 erythroleukemia cells and identified three new mSin3A-associated proteins (SAP): SAP180, SAP130, and SAP45. SAP180 is 40% identical to a previously identified mSin3A-associated protein, RBP1. SAP45 is identical to mSDS3, the human ortholog of the SDS3p component of the Saccharomyces cerevisiae Sin3p-Rpd3p corepressor complex. SAP130 does not have detectable homology to other proteins. Coimmunoprecipitation and gel filtration data suggest that the new SAPs are, at the very least, components of the same mSin3A complex. Each new SAP repressed transcription when tethered to DNA. Furthermore, repression correlated with mSin3A binding, suggesting that the new SAPs are components of functional mSin3A corepressor complexes. SAP180 has two repression domains: a C-terminal domain, which interacts with the mSin3A-HDAC complex, and an N-terminal domain, which functions independently of mSin3A-HDAC. SAP130 has a repression domain at its C terminus that interacts with the mSin3A-HDAC complex and an N-terminal domain that probably mediates an interaction with a transcriptional activator. Together, our data suggest that these novel SAPs function in the assembly and/or enzymatic activity of the mSin3A complex or in mediating interactions between the mSin3A complex and other regulatory complexes. Finally, all three SAPs bind to the HDAC-interaction domain (HID) of mSin3A, suggesting that the HID functions as the assembly interface for the mSin3A corepressor complex.

Blotting, Northern↗
PubMed2001

Regulating the neoplastic phenotype using engineered transcriptional repressors.

We have applied engineered transcriptional repressors to specifically inhibit disease gene-activated pathways in oncogenesis. We have demonstrated that synthetic repressors combining PAX3 DNA binding domains with different repression domains, KRAB or SNAG, are able to specifically inhibit malignant growth and suppress tumorigenesis in alveolar rhabdomyosarcoma tumor cells transformed by the translocation-derived chimeric transcriptional activator, PAX3-FKHR. We discuss the potential applications of the engineered repressor strategy that relate to target gene analysis, mechanisms of repression, cell regulation, and possible anti-viral and cancer therapy.

Animals↗
PubMed2006

Runx2-mediated regulation of the zinc finger Osterix/Sp7 gene.

The zinc finger transcription factor Osterix (Osx) regulates bone formation and osteoblast differentiation in vitro and in vivo. We investigated the transcriptional mechanisms underlying the mouse Osx expression by isolating and characterizing its 5' upstream region. We performed 5' RACE on mRNA isolated from murine chondroprogenitor cells and determined a cap site of Osx approximately -99 nucleotides upstream of the initiation codon. Sequence analysis of this TATA-less promoter shows several putative response elements for Sox9, VDRE, Runx and Sp1. Transfection of the Osx promoter driving the luciferase reporter gene into C3H10T1/2 and ATDC5 cells shows a strong basal promoter activity between 565 bp and 2 kb. Deletion mutant analyses show that the most proximal 852 kb of the Osx promoter contains the highest activating domains, while strong repressive domains were identified between 1.8 and 2 kb. Over-expression experiments indicate that Runx2 significantly transactivates the Osx promoter by at least 2 fold indicating that Osx is downstream of Runx2 in mesenchymal cells. This up-regulation was abrogated when the Runx2 responsive element on the Osx promoter was mutated. Finally, we show that Runx2 specifically binds to this DNA element in the Osx promoter. Thus our results show for the first time Osx transcriptional regulation through the bone and cartilage related transcription factor Runx2.

Alternative Splicing↗
PubMed2000

Exogenous cdk4 overcomes reduced cdk4 RNA and inhibition of G1 progression in hematopoietic cells expressing a dominant-negative CBF - a model for overcoming inhibition of proliferation by CBF oncoproteins.

Core Binding Factor (CBF) is required for the development of definitive hematopoiesis, and the CBF oncoproteins AML1-ETO, TEL-AML1, and CBFbeta-SMMHC are commonly expressed in subsets of acute leukemia. CBFbeta-SMMHC slows the G1 to S cell cycle transition in hematopoietic cells, but the mechanism of this effect is uncertain. We have sought to determine whether inhibition of CBF-mediated trans-activation is sufficient to slow proliferation. We demonstrate that activation of KRAB-AML1-ER, a protein containing the AML1 DNA-binding domain, the KRAB repression domain, and the Estrogen receptor ligand binding domain, also slows G1, if its DNA-binding domain is intact. Also, exogenous AML1 overcame CBFbeta-SMMHC-induced inhibition of proliferation. Representational difference analysis (RDA) identified cdk4 RNA expression as an early target of KRAB-AML1 activation. Inhibition of CBF activities by KRAB-AML1-ER or CBFbeta-SMMHC rapidly reduced endogenous cdk4 mRNA levels, even in cells proliferating at or near control rates as a result of exogenous cdk4 expression. Over-expression of cdk4, especially a variant which cannot bind p16INK4a, overcame cell cycle inhibition resulting from activation of KRAB-AML1-ER, although cdk4 did not accelerate proliferation when expressed alone. These findings indicate that mutations which alter the expression of G1 regulatory proteins can overcome inhibition of proliferation by CBF oncoproteins. Oncogene (2000).

Animals↗
PubMed1997

MLL is fused to CBP, a histone acetyltransferase, in therapy-related acute myeloid leukemia with a t(11;16)(q23;p13.3).

The recurring translocation t(11;16)(q23;p13.3) has been documented only in cases of acute leukemia or myelodysplasia secondary to therapy with drugs targeting DNA topoisomerase II. We show that the MLL gene is fused to the gene that codes for CBP (CREB-binding protein), the protein that binds specifically to the DNA-binding protein CREB (cAMP response element-binding protein) in this translocation. MLL is fused in-frame to a different exon of CBP in two patients producing chimeric proteins containing the AT-hooks, methyltransferase homology domain, and transcriptional repression domain of MLL fused to the CREB binding domain or to the bromodomain of CBP. Both fusion products retain the histone acetyltransferase domain of CBP and may lead to leukemia by promoting histone acetylation of genomic regions targeted by the MLL AT-hooks, leading to transcriptional deregulation via aberrant chromatin organization. CBP is the first partner gene of MLL containing well defined structural and functional motifs that provide unique insights into the potential mechanisms by which these translocations contribute to leukemogenesis.

Acute Disease↗
PubMed2001

Regulatory mechanisms controlling expression of the DAN/TIR mannoprotein genes during anaerobic remodeling of the cell wall in Saccharomyces cerevisiae.

The DAN/TIR genes of Saccharomyces cerevisiae encode homologous mannoproteins, some of which are essential for anaerobic growth. Expression of these genes is induced during anaerobiosis and in some cases during cold shock. We show that several heme-responsive mechanisms combine to regulate DAN/TIR gene expression. The first mechanism employs two repression factors, Mox1 and Mox2, and an activation factor, Mox4 (for mannoprotein regulation by oxygen). The genes encoding these proteins were identified by selecting for recessive mutants with altered regulation of a dan1::ura3 fusion. MOX4 is identical to UPC2, encoding a binucleate zinc cluster protein controlling expression of an anaerobic sterol transport system. Mox4/Upc2 is required for expression of all the DAN/TIR genes. It appears to act through a consensus sequence termed the AR1 site, as does Mox2. The noninducible mox4Delta allele was epistatic to the constitutive mox1 and mox2 mutations, suggesting that Mox1 and Mox2 modulate activation by Mox4 in a heme-dependent fashion. Mutations in a putative repression domain in Mox4 caused constitutive expression of the DAN/TIR genes, indicating a role for this domain in heme repression. MOX4 expression is induced both in anaerobic and cold-shocked cells, so heme may also regulate DAN/TIR expression through inhibition of expression of MOX4. Indeed, ectopic expression of MOX4 in aerobic cells resulted in partially constitutive expression of DAN1. Heme also regulates expression of some of the DAN/TIR genes through the Rox7 repressor, which also controls expression of the hypoxic gene ANB1. In addition Rox1, another heme-responsive repressor, and the global repressors Tup1 and Ssn6 are also required for full aerobic repression of these genes.

Alleles↗
PubMed2005

RBP-Jkappa/SHARP recruits CtIP/CtBP corepressors to silence Notch target genes.

Notch is a transmembrane receptor that determines cell fates and pattern formation in all animal species. After ligand binding, proteolytic cleavage steps occur and the intracellular part of Notch translocates to the nucleus, where it targets the DNA-binding protein RBP-Jkappa/CBF1. In the absence of Notch, RBP-Jkappa represses Notch target genes through the recruitment of a corepressor complex. We and others have identified SHARP as a component of this complex. Here, we functionally demonstrate that the SHARP repression domain is necessary and sufficient to repress transcription and that the absence of this domain causes a dominant negative Notch-like phenotype. We identify the CtIP and CtBP corepressors as novel components of the human RBP-Jkappa/SHARP-corepressor complex and show that CtIP binds directly to the SHARP repression domain. Functionally, CtIP and CtBP augment SHARP-mediated repression. Transcriptional repression of the Notch target gene Hey1 is abolished in CtBP-deficient cells or after the functional knockout of CtBP. Furthermore, the endogenous Hey1 promoter is derepressed in CtBP-deficient cells. We propose that a corepressor complex containing CtIP/CtBP facilitates RBP-Jkappa/SHARP-mediated repression of Notch target genes.

Alcohol Oxidoreductases↗
PubMed2006

Regulation of Arabidopsis thaliana 4-coumarate:coenzyme-A ligase-1 expression by artificial zinc finger chimeras.

The use of artificial zinc finger chimeras to manipulate the expression of a gene of interest is a promising approach because zinc finger proteins can be engineered to bind any given DNA sequence in the genome. We have previously shown that a zinc finger chimera with a VP16 activation domain can activate a reporter gene in transgenic Arabidopsis thaliana (Sánchez, J.P., Ullman, C., Moore, M., Choo, Y. and Chua, N.H. (2002) Regulation of gene expression in Arabidopsis thaliana by artificial zinc finger chimeras. Plant Cell Physiol. 43, 1465-1472). Here, we report the use of artificial zinc finger chimeras to specifically regulate the 4-coumarate:coenzyme-A ligase-1 (At4CL1) gene in A. thaliana. At4CL1 is a key enzyme in lignin biosynthesis and the down-regulation of At4CL1 can lead to a decrease in lignin content, which has a significant commercial value for the paper industry. To this end, we designed zinc finger chimeras containing either an activation or a repression domain, which bind specifically to the At4CL1 promoter region. Transgenic lines expressing a zinc finger chimera with the VP16 activation domain showed an increase in At4CL1 expression and enzyme activity. In contrast, transgenic lines expressing a chimera with the KOX (KRAB) repression domain displayed repression of At4CL1 expression and enzyme activity. The activation of At4CL1 expression produced an increase in lignin content, and transgenic plant stems showed ectopic lignin distribution. Repression of the At4CL1 gene resulted in reduced lignin content, and lignin distribution in transgenic stems was severely diminished. Our results confirm and extend previous studies of gene regulation using various artificial zinc finger chimeras in animal and plant systems, and show that this system can be used to up- and down-regulate the expression of an endogenous plant gene such as At4CL1.

Arabidopsis↗
PubMed2002

Functional and physical interaction between the histone methyl transferase Suv39H1 and histone deacetylases.

The histone methyl transferase Suv39H1 is involved in silencing by pericentric heterochromatin. It specifically methylates K9 of histone H3, thereby creating a high affinity binding site for HP1 proteins. We and others have shown recently that it is also involved in transcriptional repression by the retinoblastoma protein Rb. Strikingly, both HP1 localisation and repression by Rb also require, at least in part, histone deacetylases. We found here that repression of a heterologous promoter by Suv39H1 is dependent on histone deacetylase activity. However, the enzymatic activity of Suv39H1 is not required, since the N-terminal part is by itself a transcriptional repression domain. Coimmunoprecipitation experiments indicated that Suv39H1 can physically interact with HDAC1, -2 and -3, therefore suggesting that transcriptional repression by Suv39H1 could be the consequence of histone deacetylases recruitment. Consistent with this interpretation, the N-terminal transcriptional repression domain of Suv39H1 bound the so-called 'core histone deacetylase complex', composed of HDAC1, HDAC2 and the Rb-associated proteins RbAp48 and RbAp46. Taken together, our results suggest that a complex containing both the Suv39H1 histone methyl transferase and histone deacetylases could be involved in heterochromatin silencing or transcriptional repression by Rb.

Binding Sites↗
PubMed2002

Functional domains and DNA-binding sequences of RFLAT-1/KLF13, a Krüppel-like transcription factor of activated T lymphocytes.

RFLAT-1/KLF13, a member of the Krüppel-like family of transcription factors, was identified as a transcription factor expressed 3-5 days after T lymphocyte activation. It binds to the promoter of the chemokine gene RANTES (regulated on activation normal T cell expressed and secreted) and regulates its "late" expression in activated T-cells. In this study, a series of experiments to define the functional domains of RFLAT-1/KLF13 were undertaken to further advance the understanding of the molecular mechanisms underlying transcriptional regulation by this factor. Using the GAL4 fusion system, distinct transcriptional activation and repression domains were identified. The RFLAT-1 minimum activation domain is localized to amino acids 1-35, whereas the repression domain resides in amino acids 67-168. Deletion analysis on the RFLAT-1 protein further supports these domain functions. The RFLAT-1 activation domain is similar to that of its closest family member, basic transcription element-binding protein 1. This domain is highly hydrophobic, and site-directed mutagenesis demonstrated that both negatively charged and hydrophobic residues are important for transactivation. The nuclear localization signal of RFLAT-1 was also identified using the RFLAT-1/green fluorescence protein fusion approach. RFLAT-1 contains two potent, independent nuclear localization signals; one is immediately upstream of the zinc finger DNA-binding domain, and the other is within the zinc fingers. Using mutational analysis, we also determined that the critical binding sequence of RFLAT-1 is CTCCC. The intact CTCCC box on the RANTES promoter is necessary for RFLAT-1-mediated RANTES transcription and is also required for the synergy between RFLAT-1 and NF-kappaB proteins.

3T3 Cells↗
PubMed2002

Repression and activation domains of RME1p structurally overlap, but differ in genetic requirements.

Rme1p, a repressor of meiosis in the yeast Saccharomyces cerevisiae, acts as both a transcriptional repressor and activator. Rme1p is a zinc-finger protein with no other homology to any protein of known function. The C-terminal DNA binding domain of Rme1p is essential for function. We find that mutations and progressive deletions in all three zinc fingers can be rescued by fusion of RME1 to the DNA binding domain of another protein. Thus, structural integrity of the zinc fingers is not required for the Rme1p-mediated effects on transcription. Using a series of mutant Rme1 proteins, we have characterized domains responsible for repression and activation. We find that the minimal transcriptional repression and activation domains completely overlap and lie in an 88-amino-acid N-terminal segment (aa 61-148). An additional transcriptional effector determinant lies in the first 31 amino acids of the protein. Notwithstanding the complete overlap between repression and activation domains of Rme1p, we demonstrated a functional difference between repression and activation: Rgr1p and Sin4p are absolutely required for repression but dispensable for activation.

Amino Acid Sequence↗
PubMed1996

Protein-protein interaction between the transcriptional repressor E4BP4 and the TBP-binding protein Dr1.

We have previously mapped a repression domain from the active transcriptional repressor E4BP4 to a 65 amino acid segment near the C-terminus of the polypeptide. Here we show that the E4BP4 repression domain interacts specifically with the TBP binding repressor protein Dr1. Mutants that affect the ability of E4BP4 to bring about transcriptional repression are also deficient in their binding of Dr1. The results are discussed in the light of evidence for squelching of a 'global' repressor by a DNA binding defective E4BP4 mutant.

Adenovirus E1A Proteins↗
PubMed1999

Evolutionary conserved mechanism of transcriptional repression by even-skipped.

Even-skipped (Eve) is a transcriptional repressor involved in segment formation in Drosophila melano-gaster. In order to gain further insights into the mechanism of action of Eve we tested whether it would function as a transcriptional repressor in mammalian cells. We found that Eve was indeed a potent repressor in two different mammalian cell types and at several promoters. In vitro transcription assays confirmed that Eve directly represses transcription initiation when specifically targeted to a promoter. We also found that, unlike the case with transcriptional activators, Eve does not repress transcription synergistically. Analysis of the effect of Eve on preinitiation complex assembly in a crude HeLa cell nuclear extract demonstrated that the Eve repression domain functions by preventing the assembly of TFIID with the promoter. Our data support the hypothesis that Eve contains an active repression domain that functions specifically to prevent preinitiation complex formation.

3T3 Cells↗
PubMed2001

A dynamic role for HDAC7 in MEF2-mediated muscle differentiation.

The overlapping expression profile of MEF2 and the class-II histone deacetylase, HDAC7, led us to investigate the functional interaction and relationship between these regulatory proteins. HDAC7 expression inhibits the activity of MEF2 (-A, -C, and -D), and in contrast MyoD and Myogenin activities are not affected. Glutathione S-transferase pulldown and immunoprecipitation demonstrate that the repression mechanism involves direct interactions between MEF2 proteins and HDAC7 and is associated with the ability of MEF2 to interact with the N-terminal 121 amino acids of HDAC7 that encode repression domain 1. The MADS domain of MEF2 mediates the direct interaction of MEF2 with HDAC7. MEF2 inhibition by HDAC7 is dependent on the N-terminal repression domain and surprisingly does not involve the C-terminal deacetylase domain. HDAC7 interacts with CtBP and other class-I and -II HDACs suggesting that silencing of MEF2 activity involves corepressor recruitment. Furthermore, we show that induction of muscle differentiation by serum withdrawal leads to the translocation of HDAC7 from the nucleus into the cytoplasm. This work demonstrates that HDAC7 regulates the function of MEF2 proteins and suggests that this class-II HDAC regulates this important transcriptional (and pathophysiological) target in heart and muscle tissue. The nucleocytoplasmic trafficking of HDAC7 and other class-II HDACs during myogenesis provides an ideal mechanism for the regulation of HDAC targets during mammalian development and differentiation.

Animals↗
PubMed1998

Cloning and characterization of mCtBP2, a co-repressor that associates with basic Krüppel-like factor and other mammalian transcriptional regulators.

Basic Krüppel-like factor (BKLF) is a zinc finger protein that recognizes CACCC elements in DNA. It is expressed highly in erythroid tissues, the brain and other selected cell types. We have studied the activity of BKLF and found that it is capable of repressing transcription, and have mapped its repression domain to the N-terminus. We carried out a two-hybrid screen against BKLF and isolated a novel clone encoding murine C-terminal-binding protein 2 (mCtBP2). mCtBP2 is related to human CtBP, a cellular protein which binds to a Pro-X-Asp-Leu-Ser motif in the C-terminus of the adenoviral oncoprotein, E1a. We show that mCtBP2 recognizes a related motif in the minimal repression domain of BKLF, and the integrity of this motif is required for repression activity. Moreover, when tethered to a promoter by a heterologous DNA-binding domain, mCtBP2 functions as a potent repressor. Finally, we demonstrate that mCtBP2 also interacts with the mammalian transcripition factors Evi-1, AREB6, ZEB and FOG. These results establish a new member of the CtBP family, mCtBP2, as a mammalian co-repressor targeting diverse transcriptional regulators.

Alcohol Oxidoreductases↗
PubMed1999

Nuclear DEAF-1-related (NUDR) protein contains a novel DNA binding domain and represses transcription of the heterogeneous nuclear ribonucleoprotein A2/B1 promoter.

Nuclear DEAF-1-related (NUDR) protein is a novel transcriptional regulator with sequence similarity to developmental and oncogenic proteins. NUDR protein deletions were used to localize the DNA binding domain between amino acids 167 and 368, and site-specific DNA photocross-linking indicated at least two sites of protein-DNA contact within this domain. The DNA binding domain contains a proline-rich region and a region with similarity to a Myc-type helix-loop-helix domain but does not include the zinc finger motif at the C terminus. Deoxyribonuclease I protection assays confirmed the presence of multiple NUDR binding motifs (TTC(C/G)G) in the heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) promoter and also in the 5'-untranslated region (UTR) of hNUDR cDNA. NUDR produced a 65-70% repression of the hnRNP A2/B1 promoter activity, and NUDR binding motifs in the 5'-UTR were found to mediate this repression. NUDR-dependent repression was also observed when the 5'-UTR of NUDR was placed onto a heterologous thymidine kinase promoter in an analogous 5'-UTR position but not when placed upstream of transcription initiation. These results suggest that NUDR may regulate the in vivo expression of hnRNP A2/B1 and NUDR genes and imply that inactivation of NUDR could contribute to the overexpression of hnRNP A2/B1 observed in some human cancers.

5' Untranslated Regions↗
PubMed2001

Biological activity of RE-1 silencing transcription factor (REST) towards distinct transcriptional activators.

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, RESTDeltaN and RESTDeltaC 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, RESTDeltaN or RESTDeltaC, 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.

Activating Transcription Factor 4↗
PubMed1997

Epstein-Barr virus immortalization: Notch2 interacts with CBF1 and blocks differentiation.

EBNA2 is essential for immortalization of B cells by Epstein-Barr virus. EBNA2 is tethered to responsive promoters through a cellular factor, CBF1. CBF1 also binds to the activated form of mammalian Notch1, providing a linkage between EBNA2 function and Notch signalling. However, Notch2 is the predominant form expressed in spleen. The degree to which these Notch homologs are functionally convergent is not known. We present evidence that Notch2 also signals through CBF1. As is the case for Notch1, Notch2 interacted with the minimal repression domain of CBF1 and was targeted to CBF1 through the intracellular, subtransmembrane domain. Additional characterization suggested that the interaction domain of Notch may be bipartite. The intracellular domain of Notch2 (Notch2IC) located to the nucleus. This activated form of Notch2 transactivated expression of a target gene containing upstream CBF1 binding sites. The use of CBF1 mutants carrying amino acid substitutions in the transcriptional repression domain revealed that activation of gene expression by Notch2 is also based on masking of CBF1-mediated repression. Targeting of Notch1 and targeting of Notch2 were found to be identical and distinguishable from targeting by EBNA2. Mutation of CBF1 at codons 249 to 251 abolished interaction with both Notch proteins but not with EBNA2. In a biological examination of Notch2 function in muscle cells, Notch2IC activated endogenous HES-1 gene expression and blocked muscle cell differentiation. Overall, the data imply that at least a subset of the intracellular events following signalling in cells expressing Notch2 are common to those in Notch1-expressing cells. The concept that EBNA2 functions by mimicking Notch signalling is therefore viable whether cells are expressing Notch1 or Notch2.

Animals↗