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PubMed2000

Tyrosine phosphorylation of HoxA10 decreases DNA binding and transcriptional repression during interferon gamma -induced differentiation of myeloid leukemia cell lines.

The DNA binding affinity of HoxA10 is increased by partnering with Pbx proteins. A consensus sequence for Pbx1-HoxA10 DNA binding has been derived, but genuine target genes have not been identified. We noted that the derived Pbx-HoxA10 DNA-binding consensus is similar to a repressor element in the CYBB promoter. The CYBB gene, which encodes the respiratory burst oxidase component gp91(phox), is expressed only in myeloid cells that have differentiated beyond the promyelocyte stage. In these studies, we demonstrate that interferon gamma (IFN-gamma)-induced differentiation of myeloid cell lines abolishes in vitro Pbx-HoxA10 binding to either the derived consensus or the similar CYBB sequence. We also demonstrate that HoxA10, overexpressed in myeloid cell lines, represses reporter gene expression from artificial promoter constructs with Pbx-HoxA10 binding sites. We determine that HoxA10 has endogenous repression domains that are not functionally altered by IFN-gamma treatment. However, IFN-gamma-induced differentiation of myeloid cell lines leads to HoxA10 tyrosine phosphorylation, which decreases in vitro DNA binding to Pbx-HoxA10 binding sites. Therefore, these investigations identify the CYBB gene as a potential target for HoxA10 and define repression of genes expressed in mature myeloid cells as a novel role for HoxA10 during myeloid differentiation.

Binding Sites↗
PubMed2006

NAB2 represses transcription by interacting with the CHD4 subunit of the nucleosome remodeling and deacetylase (NuRD) complex.

Early growth response (EGR) transactivators act as critical regulators of several physiological processes, including peripheral nerve myelination and progression of prostate cancer. The NAB1 and NAB2 (NGFI-A/EGR1-binding protein) transcriptional corepressors directly interact with three EGR family members (Egr1/NGFI-A/zif268, Egr2/Krox20, and Egr3) and repress activation of their target promoters. To understand the molecular mechanisms underlying NAB repression, we found that EGR activity is modulated by at least two repression domains within NAB2, one of which uniquely requires interaction with the CHD4 (chromodomain helicase DNA-binding protein 4) subunit of the NuRD (nucleosome remodeling and deacetylase) chromatin remodeling complex. Both NAB proteins can bind either CHD3 or CHD4, indicating that the interaction is conserved among these two protein families. Furthermore, we show that repression of the endogenous Rad gene by NAB2 involves interaction with CHD4 and demonstrate colocalization of NAB2 and CHD4 on the Rad promoter in myelinating Schwann cells. Finally, we show that interaction with CHD4 is regulated by alternative splicing of the NAB2 mRNA.

Alternative Splicing↗
PubMed1993

Protein-protein interactions in gene regulation: the cAMP-CRP complex sets the specificity of a second DNA-binding protein, the CytR repressor.

Maximal repression by the CytR protein depends on the formation of nucleoprotein complexes in which CytR interacts with DNA and with cAMP-cAMP receptor protein (CRP). Here we demonstrate that CytR regulates transcription from deoP2 promoters in which the entire CytR recognition sequence has been eliminated. Furthermore, CytR proteins deleted for the DNA-binding domain repress deoP2 in vivo and interact with deoP2 in vitro in a strictly cAMP-CRP-dependent fashion. These experiments show that the site of action of CytR can be specified by protein-protein interactions to cAMP-CRP, whereas CytR-DNA interactions may primarily serve to stabilize the nucleo-protein complex. This type of specificity mechanism may represent a general concept in the recruitment of DNA-binding proteins in combinatorial regulatory systems.

Amino Acid Sequence↗
PubMed2000

Transcriptional repression by the insulator protein CTCF involves histone deacetylases.

The highly conserved zinc-finger protein, CTCF, is a candidate tumor suppressor protein that binds to highly divergent DNA sequences. CTCF has been connected to multiple functions in chromatin organization and gene regulation including chromatin insulator activity and transcriptional enhancement and silencing. Here we show that CTCF harbors several autonomous repression domains. One of these domains, the zinc-finger cluster, silences transcription in all cell types tested and binds directly to the co-repressor SIN3A. Two distinct regions of SIN3A, the PAH3 domain and the extreme C-terminal region, bind independently to this zinc-finger cluster. Analysis of nuclear extract from HeLa cells revealed that CTCF is also capable of retaining functional histone deacetylase activity. Furthermore, the ability of regions of CTCF to retain deacetylase activity correlates with the ability to bind to SIN3A and to repress gene activity. We suggest that CTCF driven repression is mediated in part by the recruitment of histone deacetylase activity by SIN3A.

3T3 Cells↗
PubMed1999

UASrpg can function as a heterochromatin boundary element in yeast.

The HM loci in Saccharomyces cerevisiae constitute region-specific but gene-nonspecific repression domains, as a number of heterologous genes transcribed by RNA polymerase II or III are silenced when placed at these loci. The promoters of the Ashbya gossypii TEF gene and the S. cerevisiae TEF1 and TEF2 genes, however, are resistant to transcriptional silencing by the HM silencers in yeast. Moreover, when interposed between the HML alpha genes and the E silencer, certain segments of these promoters block the repression effect of the silencer on the alpha genes. All of these fragments contain UASrpg (upstream activation sequence of ribosome protein genes) composed of multiple binding sites for Rap1. In fact, a 149-bp segment consisting essentially of only three tandem Rap1-binding sites from the UASrpg of yeast TEF2 exhibits silencer-blocking activity. This element also exhibits insulating activity and orientation dependence characteristic of known chromatin boundary elements. Finally, the element blocks the physical spread of heterochromatin initiated at a silencer. This segment provides the first example of chromatin domain boundary or insulator elements in yeast.

Binding Sites↗
PubMed2006

Adenovirus type 5 early region 1B 156R protein promotes cell transformation independently of repression of p53-stimulated transcription.

Early region 1B (E1B) of adenovirus type 5 (Ad5) encodes at least five different polypeptides generated by alternative splicing of a common mRNA precursor. Two of these gene products, E1B-19K and E1B-55K, are individually capable of cooperating with the Ad5 E1A proteins to completely transform rodent cells in culture. Substantial evidence suggests that these two E1B proteins contribute to cell transformation by antagonizing growth arrest and apoptosis. Here, we performed genetic and biochemical analyses to assess the attributes of the remaining E1B proteins (E1B-156R, E1B-93R, and E1B-84R). Our results show that E1B-156R, which comprises the 79 amino-terminal and 77 carboxy-terminal amino acids of E1B-55K, also enhances focal transformation of primary rat cells in cooperation with E1A. Since E1B-156R seemed unable to relocalize p53 and inhibit its transactivating function, it must be assumed that it contributes to transformation independently of repression of p53-stimulated transcription. Furthermore, we discovered that E1B-156R contains a functional transcriptional repression domain and binds Ad5 E4orf6 and the cellular apoptosis regulator Daxx. While the ability to bind E4orf6 could indicate further biological functions of E1B-156R in viral infection, the interaction with Daxx might also be linked to its transforming potential. Taken together, these analyses introduce E1B-156R as a novel transformation-promoting E1B protein that acts without repressing p53 transactivation. Moreover, identification of the interaction partners E4orf6 and Daxx provides a first glance of E1B-156R's potential functions.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Role of direct interaction in BRCA1 inhibition of estrogen receptor activity.

The BRCA1 gene was previously found to inhibit the transcriptional activity of the estrogen receptor [ER-alpha] in human breast and prostate cancer cell lines. In this study, we found that breast cancer-associated mutations of BRCA1 abolish or reduce its ability to inhibit ER-alpha activity and that domains within the amino- and carboxyl-termini of the BRCA1 protein are required for the inhibition. BRCA1 inhibition of ER-alpha activity was demonstrated under conditions in which a BRCA1 transgene was transiently or stably over-expressed in cell lines with endogenous wild-type BRCA1 and in a breast cancer cell line that lacks endogenous functional BRCA1 (HCC1937). In addition, BRCA1 blocked the expression of two endogenous estrogen-regulated gene products in human breast cancer cells: pS2 and cathepsin D. The BRCA1 protein was found to associate with ER-alpha in vivo and to bind to ER-alpha in vitro, by an estrogen-independent interaction that mapped to the amino-terminal region of BRCA1 (ca. amino acid 1-300) and the conserved carboxyl-terminal activation function [AF-2] domain of ER-alpha. Furthermore, several truncated BRCA1 proteins containing the amino-terminal ER-alpha binding region blocked the ability of the full-length BRCA1 protein to inhibit ER-alpha activity. Our findings suggest that the amino-terminus of BRCA1 interacts with ER-alpha, while the carboxyl-terminus of BRCA1 may function as a transcriptional repression domain. Oncogene (2001) 20, 77 - 87.

BRCA1 Protein↗
PubMed2005

Sumoylation and acetylation play opposite roles in the transactivation of PLAG1 and PLAGL2.

PLAG1 (pleomorphic adenoma gene 1) and PLAGL2 (PLAG-like 2) are oncogenes involved in various malignancies. Thus the study of their regulatory mechanisms may lead to identification of novel therapeutic targets. In this study, we provide supporting evidence that sumoylation and acetylation regulate functions of PLAG1 and PLAGL2. A conserved transcriptional repression domain exists in both PLAG1 and PLAGL2, whose activity depends on the presence of three sumoylation motifs and an intact sumoylation pathway. In vivo sumoylation assays confirmed that lysines 244, 263, and 353 of PLAG1 and lysines 250, 269, and 356 of PLAGL2 are indeed sumoylation sites. Further study showed that sumoylation inhibits PLAG1-induced IGF-II expression in reporter assays. The repression mediated by sumoylation may be partially explained by its effect on the cellular localization of PLAG1 and PLAGL2, because sumoylation-deficient but not wild-type PLAG1 and PLAGL2 concentrate in the nucleolus. PLAG1 and PLAGL2 are also regulated by acetylation. They are acetylated and activated by p300 and deacetylated and repressed by HDAC7. Interestingly, the sumoylation-deficient mutant of PLAGL2 is acetylated at a lower level than its wild-type counterpart, suggesting that some of the lysine residues may be targets for both modifications. Finally, mutation of three lysine residues in sumoylation motifs significantly impairs the transformation ability of PLAG1 and PLAGL2, suggesting the essential roles of these sites in the oncogenic potential of PLAG proteins. Taken together, the activities of PLAG1 and PLAGL2 are tightly modulated by both sumoylation and acetylation, which have opposite effects on their transactivation. To our knowledge, this is the first demonstration that oncoproteins can be regulated by both sumoylation and acetylation.

Acetylation↗
PubMed2001

Transcriptional repression and developmental functions of the atypical vertebrate GATA protein TRPS1.

Known vertebrate GATA proteins contain two zinc fingers and are required in development, whereas invertebrates express a class of essential proteins containing one GATA-type zinc finger. We isolated the gene encoding TRPS1, a vertebrate protein with a single GATA-type zinc finger. TRPS1 is highly conserved between Xenopus and mammals, and the human gene is implicated in dominantly inherited tricho-rhino-phalangeal (TRP) syndromes. TRPS1 is a nuclear protein that binds GATA sequences but fails to transactivate a GATA-dependent reporter. Instead, TRPS1 potently and specifically represses transcriptional activation mediated by other GATA factors. Repression does not occur from competition for DNA binding and depends on a C-terminal region related to repressive domains found in Ikaros proteins. During mouse development, TRPS1 expression is prominent in sites showing pathology in TRP syndromes, which are thought to result from TRPS1 haploinsufficiency. We show instead that truncating mutations identified in patients encode dominant inhibitors of wild-type TRPS1 function, suggesting an alternative mechanism for the disease. TRPS1 is the first example of a GATA protein with intrinsic transcriptional repression activity and possibly a negative regulator of GATA-dependent processes in vertebrate development.

Algal Proteins↗
PubMed2005

SHP represses transcriptional activity via recruitment of histone deacetylases.

The orphan receptor short heterodimer partner (SHP) is a common partner for a great number of nuclear receptors, and it plays an important role in many diverse physiological events. In a previous study, we described SHP as a strong repressor of the androgen receptor (AR). Herein, we addressed the mechanism of action of its negative activity on transcription. We first investigated the intrinsic repressive potential of SHP and mapped two core repressive domains to the amino acids 170-210 and 210-240. From GST pull-down assays, we demonstrated a direct interaction between SHP and diverse histone deacetylases (HDACs) as well as a strong interaction between HDAC1 and SHP inhibitory domains. We further supported the evidence for an interaction between SHP and HDAC1 by showing their co-immunoprecipitation and provided evidence for the existence of a ternary complex comprising AR, SHP, and HDAC1. The use of trichostatin A (TSA), a specific inhibitor of HDAC activity, confirmed that HDACs significantly contribute to the intrinsic transrepressive activity of SHP. Finally, we showed that TSA reversed SHP-induced repression of AR, further emphasizing the relevance of the interaction between SHP and HDACs. This latter action affected in a very similar manner SHP-mediated repression of estrogen receptor alpha (ERalpha) transactivation. Altogether, our results indicate that SHP mediates most of its repressive effect through recruitment of HDACs and suggest that the physiological actions of SHP could be affected by HDAC inhibitors.

Animals↗
PubMed1998

Interaction of short-range repressors with Drosophila CtBP in the embryo.

Human CtBP attenuates transcriptional activation and tumorigenesis mediated by the adenovirus E1A protein. The E1A sequence motif that interacts with CtBP, Pro-X-Asp-Leu-Ser-X-Lys (P-DLS-K), is present in the repression domains of two unrelated short-range repressors in Drosophila, Knirps and Snail, and is essential for the interaction of these proteins with Drosophila CtBP (dCtBP). A P-element-induced mutation in dCtBP exhibits gene-dosage interactions with a null mutation in knirps, which is consistent with the occurrence of Knirps-dCtBP interactions in vivo. These observations suggest that CtBP and dCtBP are engaged in an evolutionarily conserved mechanism of transcriptional repression, which is used in both Drosophila and mammals.

Alcohol Oxidoreductases↗
PubMed1997

Design of polydactyl zinc-finger proteins for unique addressing within complex genomes.

Zinc-finger proteins of the Cys2-His2 type represent a class of malleable DNA-binding proteins that may be selected to bind diverse sequences. Typically, zinc-finger proteins containing three zinc-finger domains, like the murine transcription factor Zif268 and the human transcription factor Sp1, bind nine contiguous base pairs. To create a class of proteins that would be generally applicable to target unique sites within complex genomes, we have utilized structure-based modeling to design a polypeptide linker that fuses two three-finger proteins. Two six-fingered proteins were created and demonstrated to bind 18 contiguous bp of DNA in a sequence-specific fashion. Expression of these proteins as fusions to activation or repression domains allows transcription to be specifically up- or down-modulated within human cells. Polydactyl zinc-finger proteins should be broadly applicable as genome-specific transcriptional switches in gene therapy strategies and the development of novel transgenic plants and animals.

Amino Acid Sequence↗
PubMed2005

Regulation of co-repressive activity of and HDAC recruitment to RIP140 by site-specific phosphorylation.

Receptor interacting protein 140 (RIP140) is a versatile transcriptional co-repressor that contains several autonomous repressive domains (RDs). The N-terminal RD acts by recruiting histone deacetylases (HDACs). In a comprehensive proteomic analysis of RIP140 by MS, 11 phosphorylation sites of RIP140 are identified; among them five sites are located in the N-terminal RD including Ser104, Thr202, Thr207, Ser358, and Ser380. The role of phosphorylation of RIP140 in regulating its biological activity and the underlying mechanism are examined using a site-directed mutagenesis approach. Mutations mimicking constitutive phosphorylation or dephosphorylation are introduced. The N-terminal RD phosphorylation, mediated by the mitogen-activated protein kinase (MAPK), enhances its repressive activity through increased recruitment of HDAC. Mutations mimicking constitutive dephosphorylation at Thr202 or Thr207 significantly impair its repressive activity and HDAC recruitment, whereas mutation at Ser358 only slightly affects its HDAC recruitment and the repressive activity. Consistently, mutations mimicking constitutive phosphorylation at either Thr202 or Thr207 convert RIP140 into a more potent repressor, which is less responsive to a disturbance in the MAPK system. Furthermore, constitutive phosphorylation at both Thr202 and Thr207 residues renders RIP140 fully repressive and strongly interacting with HDAC. The activity of this mutant is resistant to the MAPK inhibitor, indicating an essential role for Thr202 and Thr207 in MAPK-mediated modulation of RIP140 function. The study provides insights into the modulation of RIP140 biological activity through a specific cellular signaling pathway that augments phosphorylation at specific residues of RIP140 molecule and alters its cofactor recruitment.

Adaptor Proteins, Signal Transducing↗
PubMedDate not supplied

A regulatory network for the efficient control of transgene expression.

BACKGROUND: Expression of heterologous genes in mammalian cells or organisms for therapeutic or experimental purposes often requires tight control of transgene expression. Specifically, the following criteria should be met: no background gene activity in the off-state, high gene expression in the on-state, regulated expression over an extended period, and multiple switching between on- and off-states. METHODS: Here, we describe a genetic switch system for controlled transgene transcription using chimeric repressor and activator proteins functioning in a novel regulatory network. In the off-state, the target transgene is actively silenced by a chimeric protein consisting of multimerized eukaryotic transcriptional repression domains fused to the DNA-binding tetracycline repressor. In the on-state, the inducer drug doxycycline affects both the derepression of the target gene promoter and activation by the GAL4-VP16 transactivator, which in turn is under the control of an autoregulatory feedback loop. RESULTS: The hallmark of this new system is the efficient transgene silencing in the off-state, as demonstrated by the tightly controlled expression of the highly cytotoxic diphtheria toxin A gene. Addition of the inducer drug allows robust activation of transgene expression. In stably transfected cells, this control is still observed after months of repeated cycling between the repressed and activated states of the target genes. CONCLUSIONS: This system permits tight long-term regulation when stably introduced into cell lines. The underlying principles of this network system should have general applications in biotechnology and gene therapy.

Animals↗
PubMed2001

The E2F6 transcription factor is a component of the mammalian Bmi1-containing polycomb complex.

The E2F transcription factors play a key role in the regulation of cellular proliferation and terminal differentiation. E2F6 is the most recently identified and the least well understood member of the E2F family. It is only distantly related to the other E2Fs and lacks the sequences responsible for both transactivation and binding to the retinoblastoma protein. Consistent with this finding, E2F6 can behave as a dominant negative inhibitor of the other E2F family members. In this study, we continue to investigate the possible role(s) of E2F6 in vivo. We report the isolation of RYBP, a recently identified member of the mammalian polycomb complex, as an E2F6-interacting protein. Mapping studies indicate that RYBP binds within the known "repression domain" of E2F6. Moreover, we demonstrate that endogenous E2F6 and polycomb group proteins, including RYBP, Ring1, MEL-18, mph1, and the oncoprotein Bmi1, associate with one another. These findings suggest that the biological properties of E2F6 are mediated through its ability to recruit the polycomb transcriptional repressor complex.

Animals↗
PubMed1995

Functional domains of the t(8;21) fusion protein, AML-1/ETO.

The AML-1/ETO fusion protein is created by the (8;21) translocation, the second most frequent chromosomal abnormality associated with acute myeloid leukemia. In the fusion protein the AML-1 runt homology domain, which is responsible for DNA binding and CBF beta interaction, is linked to ETO, a gene of unknown function. The primary sequences of the runt homology domain indicates no known DNA binding motifs, but is predicted to contain six beta-strands, two alpha-helices and a nucleotide binding motif. Mutagenesis of AML-1/ETO was performed to delimit the functional domains of the chimeric protein. Most mutations in the runt homology domain that resulted in reduced CBF beta binding also inhibited DNA binding, indicating that the DNA and CBF beta binding sequences are tightly linked. However, these activities were separated by a point mutation of residue 144, within the putative ATP binding motif, which nearly eliminated DNA binding, but did not affect CBF beta binding. Random mutagenesis identified the hydrophobic face of the amphipathic fifth beta-strand, adjacent to the putative ATP binding motif, as critical for both DNA and CBF beta binding. C-terminal deletion mutants of AML-1/ETO indicated that ETO sequences are essential for interference with AML-1B-mediated transcriptional activation, and that residue 540 defines the C-terminal boundary of a potential repression domain. Thus, these mutational analyses define the regions of AML-1/ETO which regulate its function and that may be important in promoting leukemia.

Adenosine Triphosphate↗
PubMed2006

The Tgif2 gene contains a retained intron within the coding sequence.

BACKGROUND: TGIF and TGIF2 are homeodomain proteins, which act as TGFbeta specific Smad transcriptional corepressors. TGIF recruits general repressors including mSin3 and CtBP. The related TGIF2 protein functions in a similar manner, but does not bind CtBP. In addition to repressing TGFbeta activated gene expression, TGIF and TGIF2 repress gene expression by binding directly to DNA. TGIF and TGIF2 share two major blocks of similarity, encompassing the homeodomain, and a conserved carboxyl terminal repression domain. Here we characterize two splice variants of the Tgif2 gene from mouse and demonstrate that the Tgif2 gene contains a retained intron. RESULTS: By PCR from mouse cDNA, we identified two alternate splice forms of the Tgif2 gene. One splice variant encodes the full length 237 amino acid Tgif2, whereas the shorter form results in the removal of 39 codons from the centre of the coding region. The generation of this alternate splice form occurs with the mouse RNA, but not the human, and both splice forms are present in all mouse tissues analyzed. Human and mouse Tgif2 coding sequences contain a retained intron, which in mouse Tgif2 is removed by splicing from around 25-50% of RNAs, as assessed by RT-PCR. This splicing event is dependent on sequences within the mouse Tgif2 coding sequence. Both splice forms of mouse Tgif2 encode proteins which are active transcriptional repressors, and can repress both TGFbeta dependent and independent transcription. In addition, we show that human and mouse Tgif2 interact with the transcriptional corepressor mSin3. CONCLUSION: These data demonstrate that the Tgif2 gene contains a retained intron, within the second coding exon. This retained intron is not removed from the human mRNA at a detectable level, but is spliced out in a significant proportion of mouse RNAs. This alternate splicing is dependent entirely on sequences within the mouse Tgif2 coding sequence, suggesting the presence of an exonic splicing enhancer. Both splice forms of mouse Tgif2 produce proteins which are functional transcriptional repressors.

Alternative Splicing↗
PubMed2004

Repression of nodal expression by maternal B1-type SOXs regulates germ layer formation in Xenopus and zebrafish.

B1-type SOXs (SOXs 1, 2, and 3) are the most evolutionarily conserved subgroup of the SOX transcription factor family. To study their maternal functions, we used the affinity-purified antibody antiSOX3c, which inhibits the binding of Xenopus SOX3 to target DNA sequences [Development. 130(2003)5609]. The antibody also cross-reacts with zebrafish embryos. When injected into fertilized Xenopus or zebrafish eggs, antiSOX3c caused a profound gastrulation defect; this defect could be rescued by the injection of RNA encoding SOX3DeltaC-EnR, a SOX3-engrailed repression domain chimera. In antiSOX3c-injected Xenopus embryos, normal animal-vegetal patterning of mesodermal and endodermal markers was disrupted, expression domains were shifted toward the animal pole, and the levels of the endodermal markers SOX17 and endodermin increased. In Xenopus, SOX3 acts as a negative regulator of Xnr5, which encodes a nodal-related TGFbeta-family protein. Two nodal-related proteins are expressed in the early zebrafish embryo, squint and cyclops; antiSOX3c-injection leads to an increase in the level of cyclops expression. In both Xenopus and zebrafish, the antiSOX3c phenotype was rescued by the injection of RNA encoding the nodal inhibitor Cerberus-short (CerS). In Xenopus, antiSOX3c's effects on endodermin expression were suppressed by injection of RNA encoding a dominant negative version of Mixer or a morpholino against SOX17alpha2, both of which act downstream of nodal signaling in the endoderm specification pathway. Based on these data, it appears that maternal B1-type SOX functions together with the VegT/beta-catenin system to regulate nodal expression and to establish the normal pattern of germ layer formation in Xenopus. A mechanistically conserved system appears to act in a similar manner in the zebrafish.

Amino Acid Sequence↗