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At least 1,063 records · Page 59Linked to original sources
PubMed1995

Functional reconstruction of trans regulation of the Ultrabithorax promoter by the products of two antagonistic genes, trithorax and Polycomb.

Maintenance of the "on-off" state of Drosophila homeotic genes in Antennapedia and bithorax complexes requires activities of the trithorax and Polycomb groups of genes. To identify cis-acting sequences for functional reconstruction of regulation by both trithorax and Polycomb, we examined the expression patterns of several Ubx-lacZ transgenes that carry upstream fragments corresponding to a region of approximately 50 kb. A 14.5-kb fragment from the postbithorax/bithoraxoid region of Ultrabithorax exhibited proper regulation by both trithorax and Polycomb in the embryonic central nervous system. Using a Drosophila haploid cell line for transient expression, we found that trithorax or Polycomb can function independently through this upstream fragment to activate or repress the Ultrabithorax promoter, respectively. Studies of deletion mutants of trithorax and Polycomb demonstrated that trithorax-dependent activation requires the central zinc-binding domain, while Polycomb-dependent repression requires the intact chromodomain. In addition, trithorax-dependent activity can be abrogated by increasing the amount of Polycomb, suggesting a competitive interaction between the products of trithorax and Polycomb. Deletion analysis of this fragment demonstrated that a 440-bp fragment contains response elements for both trithorax and Polycomb. Furthermore, we showed that the integrity of the proximal promoter region is essential for trithorax-dependent activation, implicating a long-range interaction for promoter activation.

Animals↗
PubMed2005

Identification of growth factor independent-1 (GFI1) as a repressor of 25-hydroxyvitamin D 1-alpha hydroxylase (CYP27B1) gene expression in human prostate cancer cells.

The hormone 1,25-dihydroxyvitamin D (1,25D) may play a protective role in prostate cancer. 25-hydroxyvitamin D 1-alpha hydroxylase (CYP27B1) is the enzyme responsible for the regulation of cellular 1,25D levels. CYP27B1 is substantially repressed in prostate cancer cells. We have investigated the molecular basis for this inhibition. First, we identify a repressive region between -997 and -1200 in the human CYP27B1 promoter following transient transfection analysis in the prostate cancer cell lines DU145, PC3 and LNCaP. Next, we demonstrate a role for the transcription factor growth factor independent-1 (GFI1) in the repression of CYP27B1. Electrophoretic mobility assays with nuclear extracts from prostate cancer cell lines established binding of GFI1 to the sequence 5'-TGGTACAATCATAACTCACTGCAG-3' present at -997 to -1200 in the repressive region. Site directed mutagenesis of the core GFI1 binding sequence (5'-AATC-3') substantially increased while forced expression of GFI1 decreased the expression of the CYP27B1 reporter construct. Importantly, GFI1 repression is dependent on an intact GFI1 binding site in the -997 to -1200 region. GFI1 is an oncoprotein known to form a large protein complex with co-repressors that recruit histone deacetylases. We propose that the formation of such a repressive complex on the inhibitory domain of the CYP27B1 gene in prostate cancer cells could lead to silencing of either the nearby enhancer or proximal promoter domains and lead to cancer progression by reducing local production of 1,25D. These studies provide the basis for a more detailed understanding of CYP27B1 repression in prostate cancer cells and could provide a novel insight in future diagnosis and treatment.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMed2003

Identification of a C-terminal poly(A)-binding protein (PABP)-PABP interaction domain: role in cooperative binding to poly (A) and efficient cap distal translational repression.

The poly(A)-binding protein (PABP), bound to the 3' poly(A) tail of eukaryotic mRNAs, plays critical roles in mRNA translation and stability. PABP autoregulates its synthesis by binding to a conserved A-rich sequence present in the 5'-untranslated region of PABP mRNA and repressing its translation. PABP is composed of two parts: the highly conserved N terminus, containing 4 RNA recognition motifs (RRMs) responsible for poly(A) and eIF4G binding; and the more variable C terminus, which includes the recently described PABC domain, and promotes intermolecular interaction between PABP molecules as well as cooperative binding to poly(A). Here we show that, in vitro, GST-PABP represses the translation of reporter mRNAs containing 20 or more A residues in their 5'-untranslated regions and remains effective as a repressor when an A61 tract is placed at different distances from the cap, up to 126 nucleotides. Deletion of the PABP C terminus, but not the PABC domain alone, significantly reduces its ability to inhibit translation when bound to sequences distal to the cap, but not to proximal ones. Moreover, cooperative binding by multiple PABP molecules to poly(A) requires the C terminus, but not the PABC domain. Further analysis using pull-down assays shows that the interaction between PABP molecules, mediated by the C terminus, does not require the PABC domain and is enhanced by the presence of RRM 4. In vivo, fusion proteins containing parts of the PABP C terminus fused to the viral coat protein MS2 have an enhanced ability to prevent the expression of chloramphenicol acetyltransferase reporter mRNAs containing the MS2 binding site at distal distances from the cap. Altogether, our results identify a proline- and glutamine-rich linker located between the RRMs and the PABC domain as being strictly required for PABP/PABP interaction, cooperative binding to poly(A) and enhanced translational repression of reporter mRNAs in vitro and in vivo.

Amino Acid Sequence↗
PubMed1999

Cloning and characterization of the EAP30 subunit of the ELL complex that confers derepression of transcription by RNA polymerase II.

The product of the human oncogene ELL encodes an RNA polymerase II transcription factor that undergoes frequent translocation in acute myeloid leukemia (AML). In addition to its elongation activity, ELL contains a novel type of RNA polymerase II interaction domain that is capable of repressing polymerase activity in promoter-specific transcription. Remarkably, the ELL translocation that is found in patients with AML results in the deletion of exactly this functional domain. Here we report that the EAP30 subunit of the ELL complex has sequence homology to the Saccharomyces cerevisiae SNF8, whose genetic analysis suggests its involvement in the derepression of gene expression. Remarkably, EAP30 can interact with ELL and derepress ELL's inhibitory activity in vitro. This finding may reveal a key role for EAP30 in the pathogenesis of human leukemia.

Amino Acid Sequence↗
PubMed1996

Zik1, a transcriptional repressor that interacts with the heterogeneous nuclear ribonucleoprotein particle K protein.

The heterogeneous nuclear ribonucleoprotein particle (hnRNP) K protein is comprised of multiple modular domains that serve to engage a diverse group of molecular partners including DNA, RNA, the product of the proto-oncogene vav, and tyrosine and serine/threonine kinases. To identify additional K protein molecular partners and to further understand its function, we used a fragment of K protein as a bait in the yeast two-hybrid screen. The deduced primary structure of one of the positive clones revealed a novel zinc finger protein, hereby denoted as Zik1. In addition to the nine contiguous zinc fingers in the C terminus, Zik1 contains a KRAB-A domain thought to be involved in transcriptional repression. Zik1 and K protein bound in vitro and co-immunoprecipitated from cell extracts indicating that in vivo their interaction is direct. Expression of Gal4 DNA-binding domain-Zik1 fusion protein repressed a gene promoter bearing Gal4-binding elements, indicating that from cognate DNA elements Zik1 is a transcriptional repressor. The known diverse nature of K protein molecular interactions and now the identification of a K protein partner that is a transcriptional repressor lends support to the notion that K protein is a remarkably versatile molecule that may be acting as a docking platform to facilitate communication among molecules involved in signal transduction and gene expression.

Amino Acid Sequence↗
PubMed1996

Activation domain-mediated enhancement of activator binding to chromatin in mammalian cells.

DNA binding by transcriptional activators is typically an obligatory step in the activation of gene expression. Activator binding and subsequent steps in transcription are repressed by genomic chromatin. Studies in vitro have suggested that overcoming this repression is an important function of some activation domains. Here we provide quantitative in vivo evidence that the activation domain of GAL4-VP16 can increase the affinity of GAL4 for its binding site on genomic DNA in mammalian cells. Moreover, the VP16 activation domain has a much greater stimulatory effect on expression from a genomic reporter gene than on a transiently transfected reporter gene, where factor binding is more permissive. We found that not all activation domains showed a greater activation potential in a genomic context, suggesting that only some activation domains can function in vivo to alleviate the repressive effects of chromatin. These data demonstrate the importance of activation domains in relieving chromatin-mediated repression in vivo and suggest that one way they function is to increase binding of the activator itself.

3T3 Cells↗
PubMed1994

Imaginal disc silencers from Ultrabithorax: evidence for Polycomb response elements.

Silencers from the Drosophila homeotic gene Ultrabithorax (Ubx) require hunchback (hb) and Polycomb (Pc) to suppress the activity of embryonic enhancers outside the Ubx domain. Embryonic silencing is initiated by hb protein which binds to the silencers to repress Ubx, thereby defining the Ubx domain. Here, we study silencing during subsequent development by examining expression patterns in imaginal discs conferred by individual Ubx fragments and pair-wise combinations thereof. We find that fragments which mediate silencing in anterior regions of imaginal discs contain embryonic silencers and hb target sites. One exception to this is a fragment called BXD which is not under hb control itself, but whose silencing activity depends on combination with fragments containing hb protein binding sites. Since silencing by BXD also requires Pc function, this suggests that BXD contains target sites for Pc or for Pc-like proteins. We propose that stable silencing of Ubx is achieved through cooperation between hb and Pc target sites.

Animals↗
PubMed1998

Identification of YY1 sequences necessary for association with the nuclear matrix and for transcriptional repression functions.

YY1 is a zinc finger-containing transcription factor that can both repress and activate transcription. YY1 appears to use multiple mechanisms to carry out its diverse functions. Recently, it was observed that YY1 can exist in multiple nuclear compartments. In addition to being present in the nuclear extract fraction, YY1 is also a component of the nuclear matrix. We show that YY1 can be sequestered in vivo into a high-molecular-weight complex and can be dislodged from this complex either by treatment with formamide or by incubation with an oligonucleotide containing the YY1 DNA binding site sequence. By transfecting plasmids expressing various YY1 deletion constructs and subsequent nuclear fractionation, we have identified sequences necessary for association with the nuclear matrix. These sequences (residues 256-340) co-localized with those necessary for in vivo sequestration of YY1 into the high-molecular-weight complex. We have also characterized YY1 sequences necessary for repression of activated transcription (residues 333-371) and those necessary for masking of the YY1 transactivation domain (residues 371-397). Sequences that repress activated transcription partially overlap YY1 sequences necessary for association with the nuclear matrix. However, these sequences are distinct from those that appear to mask the YY1 transactivation domain. The potential role of nuclear matrix association in controlling YY1 function is discussed.

3T3 Cells↗
PubMed2001

Long-range repression by multiple polycomb group (PcG) proteins targeted by fusion to a defined DNA-binding domain in Drosophila.

A tethering assay was developed to study the effects of Polycomb group (PcG) proteins on gene expression in vivo. This system employed the Su(Hw) DNA-binding domain (ZnF) to direct PcG proteins to transposons that carried the white and yellow reporter genes. These reporters constituted naive sensors of PcG effects, as bona fide PcG response elements (PREs) were absent from the constructs. To assess the effects of different genomic environments, reporter transposons integrated at nearly 40 chromosomal sites were analyzed. Three PcG fusion proteins, ZnF-PC, ZnF-SCM, and ZnF-ESC, were studied, since biochemical analyses place these PcG proteins in distinct complexes. Tethered ZnF-PcG proteins repressed white and yellow expression at the majority of sites tested, with each fusion protein displaying a characteristic degree of silencing. Repression by ZnF-PC was stronger than ZnF-SCM, which was stronger than ZnF-ESC, as judged by the percentage of insertion lines affected and the magnitude of the conferred repression. ZnF-PcG repression was more effective at centric and telomeric reporter insertion sites, as compared to euchromatic sites. ZnF-PcG proteins tethered as far as 3.0 kb away from the target promoter produced silencing, indicating that these effects were long range. Repression by ZnF-SCM required a protein interaction domain, the SPM domain, which suggests that this domain is not primarily used to direct SCM to chromosomal loci. This targeting system is useful for studying protein domains and mechanisms involved in PcG repression in vivo.

ATP-Binding Cassette Transporters↗
PubMed1993

Combinatorial interactions between positive and negative cis-acting elements control spatial patterns of 4CL-1 expression in transgenic tobacco.

The phenylpropanoid enzyme 4-coumarate:coenzyme A ligase (4CL) plays a key role in linking general phenylpropanoid metabolism to end-product specific biosynthetic pathways. During vascular system and floral organ differentiation, the parsley 4CL-1 gene is expressed in a restricted set of tissues and cell types where 4CL activity is required to supply precursors for the synthesis of diverse phenylpropanoid-derived products such as lignin and flavonoids. In order to localize cis-acting elements which specify complex patterns of 4CL-1 expression, we analyzed the expression of internally deleted promoter fragment-GUS fusions in tobacco plants and parsley protoplasts. Elements located between -244 and -78 were required for most aspects of developmentally regulated expression. Within this region, three separate promotor domains containing partially redundant cis-elements directed vascular-specific expression when combined with a TATA-proximal domain. A negative cis-acting element which represses phloem expression was revealed in one of the domains and appears to be responsible for restricting vascular expression to the xylem. Distinct but overlapping promoter domain combinations were required for expression in floral organs, suggesting that different combinations of cis-acting elements may direct expression in different organs. Gel retardation assays were used to demonstrate the formation of DNA-protein complexes between factors present in nuclear extracts of parsley tissue culture cells and various tobacco organs and a 4CL-1 promoter fragment. Competition experiments showed that complex formation required the presence of a 42 bp promoter domain shown to be critical for 4CL-1 expression in vascular and floral tissues. The results are discussed in light of the coordinate expression of 4CL and other phenylpropanoid genes.

Base Sequence↗
PubMed2005

The crystal structure of a partial mouse Notch-1 ankyrin domain: repeats 4 through 7 preserve an ankyrin fold.

Folding and stability of proteins containing ankyrin repeats (ARs) is of great interest because they mediate numerous protein-protein interactions involved in a wide range of regulatory cellular processes. Notch, an ankyrin domain containing protein, signals by converting a transcriptional repression complex into an activation complex. The Notch ANK domain is essential for Notch function and contains seven ARs. Here, we present the 2.2 A crystal structure of ARs 4-7 from mouse Notch 1 (m1ANK). These C-terminal repeats were resistant to degradation during crystallization, and their secondary and tertiary structures are maintained in the absence of repeats 1-3. The crystallized fragment adopts a typical ankyrin fold including the poorly conserved seventh AR, as seen in the Drosophila Notch ANK domain (dANK). The structural preservation and stability of the C-terminal repeats shed a new light onto the mechanism of hetero-oligomeric assembly during Notch-mediated transcriptional activation.

Amino Acid Sequence↗
PubMed2004

The influence of neural signal transduction on EEC gene expression under consideration of chromatin, following myenteric ablation (review).

Significant numerical and spatial changes in 5-HT i.r. cells, CCK i.r. I-cells, glucagon and glicentin i.r. I-cells, somatostatin i.r. D-cells and neurotensin i.r. N-cells occur after a 98% myenteric ablation in the rat. Signal transduction from G-protein-coupled crypt cell receptors (m2, m3; VCAP1 and 2, CAP1; Y2, Y5, Y4) stimulates a cAMP-responsive transcription machinery in which phosphorylation of the cAMP-responsive elements (e.g. CREB) is the first step in initiation of transcription. A DNA pre-initiation complex (PIC), consisting of DNA transcription activators, general activators (TFIID, IIA, IIB, IIF, IIE, II-I and IIH), at least 14 different TAFIIs and CBP/300 coactivators which contain multiple enzymatic activities, associated with the central TBP (TATA-binding protein), which together bind to the RNA-polymerase II holoenzyme disrupts chromatin blockade over the promoter with or without the intervention of activated chromatin remodeling factors. CBP/p300 contains several highly conserved domains e.g., KIX, whose methylation by CARM-1 represses CREB transcription activation, but the bromo-binding domain of CBP increases CREB transcription. A similar positive/negative switch occurs in the regulation of gastrointestinal hormones by transcription factors, from Myc/Max to Mad/Max + corepressor mSin3A, during terminal differentiation of the cell. From these observations we conclude that the primary targets for neural signals are factors of the basal DNA transcriptional apparatus, whose promoter factors then activate chromatin induction, which facilitates transcription positively or negatively.

Animals↗
PubMed2002

Repression of hepatocyte nuclear factor 4alpha tumor suppressor p53: involvement of the ligand-binding domain and histone deacetylase activity.

Tumor suppressor p53 is known to inhibit transactivation by certain nuclear receptors, and overexpressed p53 is known to correlate with poor differentiation in liver cancer. Therefore, we investigated whether wild-type p53 might also affect the function of hepatocyte nuclear factor 4alpha1 (HNF4alpha1), an orphan receptor required for liver differentiation. Our results show that HNF4alpha1-mediated transactivation is repressed by p53 but that the mechanism of repression is not due to inhibition of HNF4alpha1 DNA binding. Rather, transfections with Gal4 fusion constructs indicate that the repression is via the ligand-binding domain of HNF4alpha1. Furthermore, we found that p53 in human embryonic kidney whole-cell extracts preferentially bound the ligand-binding domain of HNF4alpha1 and that the activation function 2 region was required for the binding. Competition for coactivator CREB binding protein could not entirely account for the repression but trichostatin A, an inhibitor of histone deacetylase activity, could reverse p53-mediated repression of HNF4alpha1. In contrast, p53-mediated repression of transcriptional activation of the same promoter by another transcriptional activator, CCAAT/enhancer-binding protein-alpha, could not be reversed by the addition of trichostatin A. These results suggest that p53, like other transcriptional repressors, inhibits transcription by multiple mechanisms, one of which involves interaction with the ligand-binding domain and recruitment of histone deacetylase activity.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗
PubMed2005

CHF1/Hey2 suppresses SM-MHC promoter activity through an interaction with GATA-6.

The bHLH transcription factor CHF1/Hey2 has been previously shown to regulate neointimal formation after vascular injury, but the mechanisms have not been fully elucidated. The zinc-finger protein GATA-6 has also been shown to regulate vascular smooth-muscle phenotype through regulation of smooth-muscle contractile protein gene expression. To address the potential mechanisms by which CHF1/Hey2 regulates vascular smooth-muscle phenotype switching, we investigated the effect of CHF1/Hey2 on GATA-6-dependent smooth-muscle myosin heavy chain promoter activity. When cotransfected into NIH3T3 cells, CHF1/Hey2 reduced GATA-6-dependent activation of the promoter by 90%. Exogenous p300 was not sufficient to overcome this repression effect, demonstrating that the inhibitor effect did not involve coactivation by p300. Coimmunoprecipitation studies demonstrated that CHF1/Hey2 interacts directly with GATA-6. Mutational analysis demonstrated that the bHLH domain is required for transcriptional repression. Our findings highlight an important transcriptional mechanism by which CHF1/Hey2 may affect smooth-muscle cell phenotype.

Animals↗
PubMed1991

Concentration-dependent transcriptional activation or repression by Krüppel from a single binding site.

One of the gap class of segmentation genes, Krüppel (Kr), is required for normal thorax and abdominal development of the Drosophila embryo. Its gene product, a zinc-finger type protein, forms a bell-shape concentration gradient in a central position of the blastoderm. Genetic and molecular studies suggested that the Kr protein (KR) may act both as a positive and as a negative regulator of transcription on several other genes of the zygotic segmentation hierarchy. We have examined the regulatory potential of Kr by a series of cotransfection experiments in the Drosophila Schneider cell line system. Different doses of Kr expression plasmid were tested for their ability to drive reporter gene expression mediated by a single 11-base pair KR in vitro binding site common to several putative Kr target genes. Our results show that low amounts of Kr expression plasmid lead to transcriptional activation, whereas high amounts result in repression. Distinct portions of KR other than the DNA-binding domain are required for gene activation and repression, suggesting that KR itself can act as a concentration-dependent positive and negative regulator of transcription.

Animals↗
PubMed1997

Expression of human heat shock transcription factors 1 and 2 in HeLa cells and yeast.

We have examined reporter gene (beta-gal) expression directed by human heat shock transcription factors 1 and 2 (HSF1 and HSF2) in HeLa cells and in yeast (Saccharomyces cerevisiae). Transcriptional activation domains of both HSFs were mapped to the C-termini using chimeric proteins containing the GAL4 DNA binding domain (GAL4-DBD). Deletion analysis of HSF1 largely confirmed the mapping and expression pattern of activation domain 2 (AD2) previously reported by Green et al (1995) with the exception of the contribution of the oligomerization domain (hydrophobic region A) to basal repression in yeast, but not in HeLa cells. In addition, a C-terminal activation domain for HSF2 (amino acids 397 to 536) was identified by analysis in yeast. In contrast to HSF1, full length HSF2 and the isolated activation domain of HSF2 showed little activity in HeLa cells. Analysis of point mutations generated by low fidelity PCR within AD2 of HSF1 indicated that hydrophobic and charged amino acids in addition to proline, serine and threonine make critical contributions to transcriptional activity. Co-expression of GAL4-DBD fusions with AD2 of HSF1 and the C-terminal activation domain of HSF2 showed no evidence of synergism in the activation of transcription. Wild-type human HSF1 and HSF2 were both able to substitute for the endogenous yeast HSF under normal growth conditions.

Cell Line↗
PubMed1997

Silencing of RNA polymerases II and III-dependent transcription by the KRAB protein domain of KOX1, a Krüppel-type zinc finger factor.

The so-called KRAB domain, which is present in about one third of the vertebrate Kruppel-type zinc finger factors, has previously been shown to inhibit transcription in cis when tethered to promoter regions. Here we analyze this effect with fusions of the KRAB domain derived from KOX1/ZNF10 zinc finger protein to the heterologous DNA binding domains of both LexA and GAL4 factors. In transfected human cells, repression of reporter gene transcription is observed not only from proximal promoter positions, but also when KRAB is tethered to DNA at a remote position more than 1.8 kb downstream of the initiation site of transcription. Furthermore, KRAB-mediated silencing over short and long distances is not restricted to RNA polymerase II, since transcription by RNA polymerase III is also repressed. However, transcription by RNA polymerase I and by phage T7 RNA polymerase in mammalian cells are not significantly influenced by the KRAB domain. These latter results may indicate that repression by the KRAB domain, at least under our assay conditions, involves specific inhibition of some component(s) of RNA polymerase II and III transcription, rather than inducing some gross physical alteration of template chromatin structure.

Binding Sites↗
PubMed2000

CBP (CREB binding protein) integrates NF-kappaB (nuclear factor-kappaB) and glucocorticoid receptor physical interactions and antagonism.

Nuclear factor-kappaB (NF-kappaB) and the glucocorticoid receptor (GR) are transcription factors with opposing actions in the modulation of immune/inflammatory responses. NF-kappaB induces the expression of proinflammatory genes, while GR suppresses immune function in part by suppressing expression of the same genes. Previously, we demonstrated that physiological antagonism between NF-kappaB and GR is due to a mutual transcriptional antagonism that requires the p65 subunit of NF-kappaB and multiple domains of GR (1). To elucidate the mechanism(s) of NF-kappaB p65 and GR transcriptional antagonism, we analyzed the interactions of wild-type p65 and p65 RHD (rel homology domain, a dominant negative mutant of p65 which lacks a transactivation domain) with GR. We show that p65RHD blocks p65-mediated transactivation, yet does not block the repression of GR transactivation by p65, indicating that transcriptional activity by p65 is not required to repress GR function. Both p65 and p65 RHD physically interact with GR, but only intact p65 represses GR-mediated signaling, implicating the p65 transactivation domain in the transcriptional repression of GR. To further characterize p65-GR interactions, we examined the role of the transcriptional co-integrator CREB binding protein (CBP) in their mutual antagonism. GR-mediated repression of p65 transactivation and p65-mediated repression of GR transactivation, as well as the physical interaction between NF-kappaB and GR, are enhanced by CBP. GR bound to the antagonist RU 486, although transcriptionally inactive, retains the ability to repress p65 transactivation. However, CBP does not physically interact with antagonist-bound GR and does not enhance its repressive effect on p65. These data suggest that CBP functions as an integrator of p65/GR physical interaction, rather than as a limiting cofactor for which p65 and GR compete.

Animals↗