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PubMed1997

Studies on the mechanism of glucocorticoid-mediated repression from a negative glucocorticoid response element from the bovine prolactin gene.

Several models for repression of transcription by glucocorticoid hormone, some of which involve so-called negative glucocorticoid response elements (nGRE), have been suggested. In the cases where nGREs are required, the glucocorticoid receptor (GR) is thought to bind to the nGRE and interfere with transcriptional activation by positively acting transactivating factors. We have studied an nGRE from the bovine prolactin gene promoter (PRL3), which increases basal expression from a heterologous promoter in rat pituitary cells (GH3) and is repressed by glucocorticoids. Two proteins in addition to the GR were identified in pituitary cells to bind specifically to the PRL3 nGRE, one of which was the pituitary-specific transcription factor Pit-1/GHF-1. A mutation in the PRL3 nGRE, which destroyed Pit-1/GHF-1 binding, totally abolished the increased basal expression as well as glucocorticoid repression in transfected GH3 cells. A mutation in the binding site for the second protein, termed XTF, partially impaired basal activity but totally abrogated glucocorticoid repression. The same mutation had no effect on GR binding to the PRL3 nGRE. Mixing experiments with whole-cell extracts containing overexpressed GR from COS cells decreased the binding of both Pit-1/GHF-1 and XTF to the PRL3 element. However, Pit-1/GHF-1 displacement from the PRL3 element by the GR required XTF binding. Furthermore, GR binding to the PRL3 nGRE was required for glucocorticoid repression to occur, because a mutation of the GR binding site abolished the glucocorticoid effect. Moreover, the PRL3 nGRE was found to contain only half a palindromic GRE, allowing only one GR moiety to contact the DNA. These data demonstrate that the PRL3 nGRE is composite in nature and that the ability of the GR to repress transactivation by displacement requires an intermediary factor, XTF.

Animals↗
PubMed1993

Transcriptional repression by the Rb-related protein p107.

The transcription factor DRTF1/E2F is believed to play an important role in regulating cellular proliferation because it undergoes a series of periodic interactions with proteins that are known to be important regulators of the cell cycle, including the retinoblastoma gene product (pRb) and cyclin A. Furthermore, certain viral oncogene products, such as adenovirus E1a, disrupt these DRTF1/E2F complexes by sequestering the associated proteins. p107, a protein that is structurally related to pRb, also binds to DRTF1/E2F, and in this study we investigate the functional consequences of this interaction. We show that p107 can repress E2F binding site-dependent transcription and that the adenovirus E1a protein overcomes p107-mediated transcriptional repression. Two distinct but related proteins, pRb and p107, can therefore repress transcription driven by DRTF1/E2F, whereas the E1a protein overrides the repression. We also demonstrate that the transcription repressing properties of p107 and pRb are influenced by the cell type and by differentiation, because neither protein affects transcription in F9 embryonal carcinoma (EC) cells but both do so efficiently in differentiated derivatives. In this respect, the repressing activities of pRb and p107 inversely correlate with the presence of the cellular E1a-like activity previously documented in F9 EC cells. These data suggest that p107 and pRb exert their biological activities in some but not all cell types.

Adenovirus E1A Proteins↗
PubMed2002

Downstream E-box-mediated regulation of the human telomerase reverse transcriptase (hTERT) gene transcription: evidence for an endogenous mechanism of transcriptional repression.

Regulation of the hTERT gene encoding the telomerase catalytic subunit plays an important role in human cell senescence, immortalization, and carcinogenesis. By examining the activity of various deleted or mutated hTERT promoter fragments, we show that an E-box element downstream of the transcription initiation site is critical to differential hTERT transcription between the telomerase/hTERT-positive renal cell carcinoma cell line (RCC23) and its telomerase/hTERT-negative counterpart containing a transferred, normal chromosome 3 (RCC23+3). This E-box element mediated repression of hTERT transcription in RCC23+3 but not in RCC23. A copy number-dependent enhancement of the repression suggested active repression, rather than loss of activation, in RCC23+3. Endogenous expression levels of c-Myc or Mad1, which could activate or repress hTERT transcription when overexpressed, did not account for the differential hTERT transcription. Gel mobility shift assays identified the upstream stimulatory factors (USFs) as a major E-box-binding protein complex in both RCC23 and RCC23+3 and, importantly, detected an RCC23+3-specific, E-box-binding factor that was distinct from the USF and Myc/Mad families. The E-box-mediated repression was also active in normal human fibroblasts and epithelial cells and inactive in some, but not all, telomerase/hTERT-positive cancer cells. These findings provide evidence for an endogenous, repressive mechanism that actively functions in telomerase/hTERT-negative normal cells and becomes defective during carcinogenic processes, e.g., by an inactivation of the telomerase repressor gene on chromosome 3.

Cell Cycle Proteins↗
PubMed1988

a/Alpha-specific repression by MAT alpha 2.

The product of the MAT alpha 2 gene is a DNA-binding protein that acts as a repressor of two different sets of cell type-specific genes. In alpha cells, the alpha 2 protein represses the transcription of several a-specific genes. In a/alpha cells, the alpha 2 protein acts together with the product of the MATa1 gene, the a1 protein, to repress several genes used by haploids in the mating process. In addition to the mat alpha 2 mutations that result in defects in both types of regulation, other mat alpha 2 alleles have been described that result in defects in the repression of a-specific genes but that do not affect the ability of the alpha 2 and a1 proteins to interact to repress the haploid-specific genes. We report here the isolation of a new class of mat alpha 2 mutations that do not affect the ability of the alpha 2 protein to repress a-specific genes, but that interfere with the ability of the alpha 2 protein to interact with the a1 protein to repress the haploid-specific genes and establish the a/alpha cell type. These mutations may help determine the means by which the a1 protein interacts with alpha 2 to expand the set of genes under its control.

Amino Acid Sequence↗
PubMed1996

Repression of hybrid dysgenesis in Drosophila melanogaster by heat-shock-inducible sense and antisense P-element constructs.

Sets of sense and antisense P-element constructs controlled by a heat-shock-inducible promoter were tested for their ability to repress manifestations of P-element activity in vivo. As a group, the antisense constructs repressed pupal lethality, a somatic manifestation of P activity, and this repression was significantly enhanced by heat shock. Three of the 11 antisense constructs also repressed gonadal dysgenesis, a manifestation of P activity in the female germ line; however, none had any effect on P-element-mediated mutability in the male germ line. Among the 13 different heat-shock-inducible sense constructs that were tested, those containing the KP and DP elements were strong repressors of pupal lethality, gonadal dysgenesis and P-element-mediated mutability; however, individual lines carrying these constructs varied in their ability to repress each of these traits, presumably because of genomic position effects. With the exception of the sense construct that contained a complete P element, none of the sense or antisense constructs repressed a lacZ reporter gene driven by the P-element promoter. Overall, the experimental results suggest that in nature, P-element activity could be regulated by P-encoded polypeptides and by antisense P RNAs.

Animals↗
PubMed1998

The yeast protein complex containing cdc68 and pob3 mediates core-promoter repression through the cdc68 N-terminal domain.

Transcription of nuclear genes usually involves trans-activators, whereas repression is exerted by chromatin. For several genes the transcription mediated by trans-activators and the repression mediated by chromatin depend on the CP complex, a recently described abundant yeast nuclear complex of the Pob3 and Cdc68/Spt16 proteins. We report that the N-terminal third of the Saccharomyces cerevisiae Cdc68 protein is dispensable for gene activation but necessary for the maintenance of chromatin repression. The absence of this 300-residue N-terminal domain also decreases the need for the Swi/Snf chromatin-remodeling complex in transcription and confers an Spt- effect characteristic of chromatin alterations. The repression domain, and indeed the entire Cdc68 protein, is highly conserved, as shown by the sequence of the Cdc68 functional homolog from the yeast Kluyveromyces lactis and by database searches. The repression-defective (truncated) form of Cdc68 is stable but less active at high temperatures, whereas the known point-mutant form of Cdc68, encoded by three independent mutant alleles, alters the N-terminal repression domain and destabilizes the mutant protein.

Adenosine Triphosphatases↗
PubMed2000

Roles for the Saccharomyces cerevisiae SDS3, CBK1 and HYM1 genes in transcriptional repression by SIN3.

The Saccharomyces cerevisiae Sin3 transcriptional repressor is part of a large multiprotein complex that includes the Rpd3 histone deacetylase. A LexA-Sin3 fusion protein represses transcription of promoters with LexA binding sites. To identify genes involved in repression by Sin3, we conducted a screen for mutations that reduce repression by LexA-Sin3. One of the mutations identified that reduces LexA-Sin3 repression is in the RPD3 gene, consistent with the known roles of Rpd3 in transcriptional repression. Mutations in CBK1 and HYM1 reduce repression by LexA-Sin3 and also cause defects in cell separation and altered colony morphology. cbk1 and hym1 mutations affect some but not all genes regulated by SIN3 and RPD3, but the effect on transcription is much weaker. Genetic analysis suggests that CBK1 and HYM1 function in the same pathway, but this genetic pathway is separable from that of SIN3 and RPD3. The remaining gene from this screen described in this report is SDS3, previously identified in a screen for mutations that increase silencing at HML, HMR, and telomere-linked genes, a phenotype also seen in sin3 and rpd3 mutants. Genetic analysis demonstrates that SDS3 functions in the same genetic pathway as SIN3 and RPD3, and coimmunoprecipitation experiments show that Sds3 is physically present in the Sin3 complex.

Base Sequence↗
PubMed2003

Development of an siRNA-based method for repressing specific genes in renal organ culture and its use to show that the Wt1 tumour suppressor is required for nephron differentiation.

Wt1 is a tumour suppressor gene, mutation of which is a cause of Wilms' tumour, a childhood renal nephroblastoma. Wt1 is expressed in a rich pattern during renal development suggesting that it acts at three stages: determination of the kidney area, the differentiation of nephrons and maturation of glomeruli. Wt1-/- mice confirm that Wt1 is essential for the inception of kidney development; cells that ought to form kidneys die by apoptosis instead. Specific human WT1 mutations cause defects of glomerular maturation (Denys-Drash and Frasier syndromes), providing circumstantial evidence for action of Wt1 during glomerular maturation. There is, however, no genetic evidence for a function during nephron differentiation because this stage is never reached in Wt1-/- mice. We have therefore developed a novel technique, based on small interfering RNA (siRNA), to repress the expression of Wt1 and other specific genes at different stages of kidney development in culture. We find that early repression of Wt1 phenocopies the Wt1-/- mouse, but later repression prevents cells differentiating into nephrons and causes them instead to proliferate abnormally, possibly mimicking aspects of Wilms' tumour. In line with established hypotheses about genetic pathways that control kidney development, we find that repressing Pax2 using siRNAs represses Wt1 expression and blocks both bud growth and nephron differentiation, but that repressing Wnt4 blocks nephron differentiation without affecting Wt1 expression. As well as illuminating previously inaccessible aspects of Wt1 biology, our results suggest that siRNA in organ culture will be a powerful method for analyzing other developmental pathways and testing the effects of stage-specific loss of tumour suppressor genes.

Animals↗
PubMed1995

Three subunits of the RNA polymerase II mediator complex are involved in glucose repression.

Glucose triggers a complex response in yeast which includes induction and repression of a large number of genes. Glucose repression is in part mediated by the Mig1 repressor, a zinc finger protein that binds to the promoters of many glucose repressed genes. However, some genes that are required for gluconeogenic growth are also repressed by a Mig1-independent mechanism. We have isolated mutations in three genes that are involved in this Mig1-independent component of repression and cloned the genes by complementation. All three genes encode subunits of the recently discovered RNA polymerase II mediator complex. Two of them are yeast cyclin C and its associated kinase. Disruptions of the three genes have identical phenotypes with respect to glucose repression and show no synergism with each other. This suggests that these three subunits of the mediator complex function closely together in transmitting the transcriptional response to glucose.

Amino Acid Sequence↗
PubMed1996

In simple synthetic promoters YY1-induced DNA bending is important in transcription activation and repression.

Depending on promoter context, YY1 can activate or repress transcription, or provide a site for transcription initiation. To investigate whether the ability of YY1 to induce DNA bending influenced its ability to activate and repress transcription, simple synthetic promoters were constructed in which the YY1 binding site was inserted between the TATA box and either the NF1 or AP1 recognition sequences. In transient transfections of COS cells, the NF1YY1TATA and NF1RYY1TATA promoters exhibited a dramatic 15-20-fold increase in correctly initiated transcription. These promoters exhibited even larger 60-80-fold increases in transcription in HeLa cells. Neither multiple copies of the YY1 binding site alone, nor placement of a YY1 site upstream of the NF1 site activated transcription. Deletion of 4 bp between the NF1 and YY1 sites, which changes the phase of the DNA bends, abolished the 16-fold activation of transcription by NF1YY1TATA. Insertion of the YY1 site between the AP1 site and the TATA box decreased transcription approximately 3-fold. Replacing the YY1 binding site with an intrinsic DNA bending sequence mimicked this transcription repression. Sequences of similar length which do not bend DNA fail to repress AP1-mediated transcription. Gel mobility shift assays were used to show that binding of YY1 to its recognition sequence did not repress binding of AP1 to its recognition sequences. Our data indicate that YY1-induced DNA bending may activate and repress transcription by changing the spatial relationships between transcription activators and components of the basal transcription apparatus.

Animals↗
PubMed1996

Phosphorylation of p53 at the casein kinase II site selectively regulates p53-dependent transcriptional repression but not transactivation.

The p53 tumour suppressor protein is a potent transcription factor which plays a central role in the defence of cells against DNA damage and the propagation of malignant clones. We have previously shown that phosphorylation of serine 386 in mouse p53 by the growth- associated protein kinase, casein kinase II (CKII), plays an important role in the ability of p53 to block the proliferation of drug-resistant colonies. In this paper we show that blocking phosphorylation of serine 386 through an alanine substitution, or placing a constitutive negative charge at this position in the form of aspartate, had no significant influence on p53-dependent transcriptional activation of a promoter containing 13 copies of a p53 consensus binding sequence, or of the p21WAF1 promoter which is a natural target for p53. In contrast, the alanine mutant showed a weak reduction in the ability of p53 to repress expression from the c-fos promoter, which is a target for p53-dependent repression in vivo. Strikingly, when the repression of the SV40 early promoter was examined, a reduction in the repression capacity of up to 5-fold was observed. Moreover, repression of the SV40 promoter could be partially restored by aspartic acid substitution at the phosphorylation site. These data indicate that phosphorylation at a specific C-terminal site can selectively regulate p53-dependent repression, but not transactivation.

Animals↗
PubMed1997

HUB1, a novel Krüppel type zinc finger protein, represses the human T cell leukemia virus type I long terminal repeat-mediated expression.

We have shown that human T-cell leukemia virus type I (HTLV-I) gene expression is negatively regulated by the U5 repressive element (U5RE) of its long terminal repeat (LTR). To isolate factors binding to U5RE, we screened a cDNA expression library by south-western blotting with a U5RE probe. Screening 2 x10(6) clones gave a positive clone with a 3.8 kb insert encoding a novel 671 residue polypeptide, named HTLV-I U5RE binding protein 1 (HUB1), with five zinc finger domains and a Krüppel-associated box like domain; HUB1 may be related to a repressor belonging to the Krüppel type zinc finger protein. A 4.0 kb mRNA for HUB1 is ubiquitously expressed among all human tissues tested. HUB1 recognizes the TCCACCCC sequence as a core motif and exerts a strong repressive effect on HTLV-I LTR-mediated expression. A new repressive domain, named HUB1 repressive (HUR) domain, was identified, rather than the Krüppel-associated box like domain. The N-terminal region upstream of HUR domain seemed to be also indispensable to the repression. Thus, we propose that HUB1 is a new type repressor and plays an important role in the HTLV-I U5-mediated repression.

Amino Acid Sequence↗
PubMed1998

The LAZ3(BCL-6) oncoprotein recruits a SMRT/mSIN3A/histone deacetylase containing complex to mediate transcriptional repression.

Recent works demonstrated that some transcriptional repressors recruit histone deacetylases (HDACs) either through direct interaction, or as a member of a multisubunit repressing complex containing other components referred to as corepressors. For instance, the bHLH-Zip transcriptional repressors MAD/MXI recruit HDACs together with the mSIN3 corepressors, whereas unliganded nuclear receptors contact another corepressor, SMRT (or its relative N-CoR), which, in turn, associates with both mSIN3 and HDACs to form the repressor complex. Recently, we reported that SMRT also directly associates with LAZ3(BCL-6), a POZ/Zn finger transcriptional repressor involvedin the pathogenesis of non-Hodgkin lymphomas. However, whether LAZ3 recruits the HDACs-containing repression complex is currently unknown. We report here that LAZ3 associates with corepressor mSIN3A both in vivo and in vitro , and found that a central region, which harbours autonomous repression activity, is mainly responsible for this interaction. Conversely, the N-terminal half of mSIN3A is both necessary and sufficient to bind LAZ3. Moreover, we show that LAZ3 also interacts with an HDAC (HDAC-1) through its POZ domain in vitro while the immunoprecipitation of LAZ3 results in the coretention of an endogenous HDAC activity in vivo . Finally, inhibitors of HDACs significantly reduce the LAZ3-mediated repression. Taken together, we conclude that LAZ3 recruits a repressing complex containing SMRT, mSIN3A and a HDAC, and that its full repressing potential on transcription requires HDACs activity. Our results identify HDACs as molecular targets of LAZ3 oncogene and further strengthen the connection between aberrant chromatin acetylation and human cancers.

Acetylation↗
PubMed2004

The Saccharomyces cerevisiae TRT2 tRNAThr gene upstream of STE6 is a barrier to repression in MATalpha cells and exerts a potential tRNA position effect in MATa cells.

A growing body of evidence suggests that genes transcribed by RNA polymerase III exhibit multiple functions within a chromosome. While the predominant function of these genes is the synthesis of RNA molecules, certain RNA polymerase III genes also function as genomic landmarks. Transfer RNA genes are known to exhibit extra-transcriptional activities such as directing Ty element integration, pausing of replication forks, overriding nucleosome positioning sequences, repressing neighboring genes (tRNA position effect), and acting as a barrier to the spread of repressive chromatin. This study was designed to identify other tRNA loci that may act as barriers to chromatin-mediated repression, and focused on TRT2, a tRNA(Thr) adjacent to the STE6 alpha2 operator. We show that TRT2 acts as a barrier to repression, protecting the upstream CBT1 gene from the influence of the STE6 alpha2 operator in MATalpha cells. Interestingly, deletion of TRT2 results in an increase in CBT1 mRNA levels in MATa cells, indicating a potential tRNA position effect. The transcription of TRT2 itself is unaffected by the presence of the alpha2 operator, suggesting a hierarchy that favors assembly of the RNA polymerase III complex versus assembly of adjacent alpha2 operator-mediated repressed chromatin structures. This proposed hierarchy could explain how tRNA genes function as barriers to the propagation of repressive chromatin.

ATP-Binding Cassette Transporters↗
PubMed1993

Promoter-independent catabolite repression of the Bacillus subtilis gnt operon.

The mechanism underlying catabolite repression in Bacillus species remains unknown. A recent study of the promoter-independent catabolite repression of the gnt operon implicated a consensus sequence (ATTGAAAG) in catabolite repression in the genus Bacillus. The introduction of base-substitutions into the ATTGAAAG sequence in the chromosomal gnt operon affected catabolite repression of the gnt operon. Deletion analysis indicated that the ATTGAAAG sequence is probably part of a cis sequence necessary for the promoter-independent catabolite repression of the gnt operon. Furthermore, we subjected gnt transcripts synthesized with and without glucose to S1 nuclease and slot blotting analyses. The results indicated that the gnt transcripts decreased in the region (+93 to +203; +1, the transcription initiation nucleotide) only in the presence of glucose. Mechanisms underlying this promoter-independent catabolite repression are discussed.

Bacillus subtilis↗
PubMed2005

Polyglutamine represses cAMP-responsive-element-mediated transcription without aggregate formation.

Transcriptional dysregulation, particularly cAMP-responsive-element-mediated transcriptional repression, has been implicated in expanded polyglutamine diseases. However, it has not been clarified whether this transcriptional repression is a cause or result of neurodegeneration. Furthermore, the association between aggregates of expanded polyglutamine stretches and transcriptional repression is not clear. We established isogenic cell lines with polyglutamine stretches, which also expressed d2EGFP under the control of cAMP-responsive elements. In this system, the polyglutamine stretch repressed cAMP-responsive-element-mediated transcription without the formation of macroscopic expanded polyglutamine aggregates. Furthermore, aggregate formation did not have an adverse effect on the repression of transcriptional activity. The results demonstrated that the repression of cAMP-responsive-element-mediated transcription is an early event caused by a soluble form of polyglutamine stretch.

Cell Line↗
PubMed1993

Catabolite repression of beta-glucanase synthesis in Bacillus subtilis.

beta-Glucanase synthesis in Bacillus subtilis was repressed by glucose and other substrates of glycolysis. Experiments with different pts mutants showed that the phosphoenolpyruvate: sugar phosphotransferase system is not involved in carbon catabolite repression of beta-glucanase synthesis. Carbon catabolite repression of beta-glucanase synthesis was completely abolished in a ccpA mutant. An operator structure similar to those upstream of amyE and the xyl operon was found and was shown by site-directed mutagenesis to be the target for carbon catabolite repression of beta-glucanase synthesis. The presence of this operator on a multi-copy plasmid resulted in a reduced repression of both beta-glucanase and alpha-amylase synthesis. It seems likely that the gene encoding these enzymes are part of one regulon with respect to catabolite repression.

Bacillus subtilis↗
PubMed2001

A p-loop motif and two basic regions in the regulatory protein GvpD are important for the repression of gas vesicle formation in the archaeon Haloferax mediterranei.

DeltaD transformants containing all 14 gvp genes of Haloferax mediterranei required for gas vesicle formation except for gvpD are gas vesicle overproducers (Vac(++)), whereas DeltaD/D transformants containing the gvpD reading frame under ferredoxin promoter control on a second construct in addition to DeltaD did not form gas vesicles (Vac(-)). The amino acid sequence of GvpD indicates three interesting regions (a putative nucleotide-binding site called the p-loop motif, and two basic regions); these were altered by mutation, and the resulting GvpD(mut) proteins tested in DeltaD/D(mut) transformants for their ability to repress gas vesicle formation. The exchange of amino acids at conserved positions in the p-loop motif resulted in Vac(++) DeltaD/D(mut) transformants, indicating that these GvpD(mut) proteins were unable to repress gas vesicle formation. In contrast, a GvpD(mut) protein with an alteration of a non-conserved proline in the p-loop region (P41A) was still able to repress. The repressing function of the various GvpD proteins was also investigated at the promoter level of the gvpA gene. This promoter is only activated during the stationary phase, depending on the transcriptional activator protein GvpE. Whereas the Vac(++) DeltaD transformants contained very high amounts of gvpA mRNA predominantly in the stationary growth phase, the amount of this transcript was significantly reduced in the Vac(-) transformants DeltaD/D and DeltaD/D(P41A). In contrast, the Vac(++) DeltaD/D(mut) transformants harbouring GvpD(mut) with mutations at conserved positions in the p-loop motif contained large amounts of gvpA mRNA already during exponential growth, suggesting that this motif is important for the GvpD repressor function during this growth phase. The GvpD mutants containing mutations in the two basic regions were mostly defective in the repressing function. The GvpD(mut) protein containing an exchange of the three arginine residues 494RRR496 to alanine residues was able to repress gas vesicle formation. No gvpA mRNA was detectable in this transformant, demonstrating that this GvpD protein was acting as a strong repressor. All these results imply that the GvpD protein is able to prevent the GvpE-mediated gvpA promoter activation, and that the p-loop motif as well as the two basic regions are important for this function.

Amino Acid Motifs↗