Repressor Region Research Articles

The transcriptional repressor even-skipped interacts directly with TATA-binding protein.

The Drosophila homeodomain protein Even-skipped (Eve) has previously been shown to function as a sequence-specific transcriptional repressor, and in vitro and in vivo experiments have shown that the protein can actively block basal transcription. However, the mechanism of repression is not known. Here, we present evidence establishing a direct interaction between Eve and the TATA-binding protein (TBP). Using cotransfection assays with minimal basal promoters whose activity can be enhanced by coexpression of TBP, we found that Eve could efficiently block, or squelch, this enhancement. Squelching did not require Eve DNA-binding sites on the reporter plasmids but was dependent on the presence of the Eve repression domain. Further support for an in vivo interaction between the Eve repression domain and TBP was derived from a two-hybrid-type assay with transfected cells. Evidence that Eve and TBP interact directly was provided by in vitro binding assays, which revealed a specific protein-protein interaction that required an intact Eve repression domain and the conserved C terminus of TBP. The Eve homeodomain was also required for these associations, suggesting that it may function in protein-protein interactions. We also show that a previously characterized artificial repression region behaves in a manner similar to that of the Eve repression domain, including its ability to squelch TBP-enhanced expression in vivo and to bind TBP specifically in vitro. Our results suggest a model for transcriptional repression that involves an interaction between Eve and TBP.

Repression of the Id2 (inhibitor of differentiation) gene promoter during exit from the cell cycle.

The Id2 gene is one of several "Id-like" genes which encode helix-loop-helix proteins which dimerize with basic helix-loop-helix proteins and inhibit binding to the DNA enhancer element known as an E box. By repressing the DNA binding activity of basic helix-loop-helix proteins, Id proteins inhibit transcription of tissue-specific genes in myoblasts, hematopoietic precursor cells, and other types of undifferentiated cells. Serum starvation results in the disappearance of Id gene transcripts in most types of cultured cells, and often induces differentiation of these cells. In order to gain some insight into this process, we have analyzed Id2 promoter function in the glioma cell line U87Y. We have isolated 300 base pairs of Id2 promoter sequence which is sufficient to repress the activity of a reporter gene in serum-starved U87Y cells, but induces the activity of the reporter gene when the cells are stimulated with fresh serum. Two regions within this 300 base pair sequence contain repressor elements; deletion of either region results in increased promoter activity. Both repressor regions serve as binding sites for a protein present in extracts from serum-starved U87Y cells but not in serum-stimulated cells.

The NF-κB-Like Site in the TNF-α Repressor Element Is Essential for Its Repressor Function

We have previously identified a repressor element in the TNF-α promoter (−280 to −172) by deletion analysis. When this 108 bp repressor element was placed in front of a heterologous promoter containing an NF-κB binding site, less repression was observed. When this element was dimerized and placed in front of the positive element (−125 to −102) of the TNF-α promoter, instead of repression, activation was observed. There is an NF-κB-like site in the 108 bp repressor region (−211 to −202) and our gel retardation analysis showed that this site and a known NF-κB binding site both could compete for one of the specific protein complexes formed on the 108 bp probe. To test the functionality of this NF-κB-like site, we mutagenized the critical GGGG sequence to ATCC. Contrary to our prediction, such a mutation blocked the repressor function of the 108 bp element. This suggests that the NF-κB-like site is an essential sequence for the repressor function of the 108 bp repressor element.

Characterization of the mouse tartrate-resistant acid phosphatase (TRAP) gene promoter.

Tartrate-resistant acid phosphatase (TRAP) is an iron-binding protein that is highly expressed in osteoclasts. To characterize the regulation of TRAP gene expression, progressive 5' and 3' deletions of a 1.8 kb fragment containing the 5'-flanking sequence were fused to a luciferase reporter gene. Two nonoverlapping regions of this 1.8 kb fragment had promoter activity. The upstream promoter (P1) was located within the region from -881 bp to -463 bp relative to the ATG, while the downstream promoter (P2) was located between -363 bp to -1 bp in a region we have previously shown to be an intron in transcripts originating from the upstream promoter. A putative repressor region for the P2 promoter at -1846 bp to -1240 bp and a putative enhancer region at -962 bp to -881 bp relative to the ATG were identified. PCR analysis of promoter-specific transcription of the TRAP gene in various murine tissues showed that both promoters were active in several tissues. Transferrin-bound iron increased P1 promoter activity 2.5-fold and hemin decreased P1 promoter activity, but neither had any effect on P2 activity. These data show that the transcriptional regulation of the TRAP gene is complex and that iron may play a key role in TRAP gene regulation.

Alternatively Processed Isoforms of Cellular Nucleic Acid-binding Protein Interact with a Suppressor Region of the Human β-Myosin Heavy Chain Gene

Analysis of a series of human beta-myosin heavy chain (MHC) constructs with progressive deletions in the 5'-flanking region has localized a strong positive element at positions -298/277 with a repressor region located immediately upstream at -332/-300 (Flink, I. L., Edwards, J. G., Bahl, J. J., Liew, C.-C., Sole, M., and Morkin, E. (1992) J. Biol. Chem. 267, 9917-9924). A 49-base pair restriction fragment containing the suppressor element was used to screen a cardiac expression library. The 0.65-kilobase pair cDNA identified by this procedure was similar in sequence, except for the absence of a 21-base pair region encoding seven amino acids, to cellular nucleic acid-binding protein (CNBP), a 19-kDa zinc finger DNA-binding protein isolated earlier from liver, which may be involved in negative regulation of cholesterol biosynthesis (Rajavashisth, T. B., Taylor, A. K., Andalibi, A., Svenson, K. L., and Lusis, A. J. (1989) Science 245, 640-643). An additional clone identical to the one originally found in liver, referred to as CNBP alpha, was isolated from the cardiac library by hybridization screening. Gel mobility shift analysis indicated that CNBP alpha and CNBP beta isoforms preferentially interact with single-stranded DNA corresponding to the proximal and distal regions of the suppressor. When cotransfected with a beta-MHC reporter construct, CNBP alpha repressed activity in a dosage-dependent manner, whereas repression was not observed with the shorter construct (CNBP beta). Cotransfection of a combination of CNBP alpha and CNBP beta repressed reporter activity to an extent similar to cotransfection with CNBP alpha alone, suggesting that CNBP beta is not translationally active under these conditions. The results of RNase protection assays and genomic sequencing indicated that the alpha and beta isoforms are formed by alternative use of 5' donor sites within a single exon. These results suggest that CNBP isoforms may modulate the activity of the beta-MHC gene by interaction with a repressor region.

Identification of transactivation and repression functions of the dioxin receptor and its basic helix-loop-helix/PAS partner factor Arnt: inducible versus constitutive modes of regulation.

Gene regulation by dioxins is mediated via the dioxin receptor, a ligand-dependent basic helix-loop-helix (bHLH)/PAS transcription factor. The latent dioxin receptor responds to dioxin signalling by forming an activated heterodimeric complex with a specific bHLH partner, Arnt, an essential process for target DNA recognition. We have analyzed the transactivating potential within this heterodimeric complex by dissecting it into individual subunits, replacing the dimerization and DNA-binding bHLH motifs with heterologous zinc finger DNA-binding domains. The uncoupled Arnt chimera, maintaining 84% of Arnt residues, forms a potent and constitutive transcription factor. Chimeric proteins show that the dioxin receptor also harbors a strong transactivation domain in the C terminus, although this activity was silenced by inclusion of 82 amino acids from the central ligand-binding portion of the dioxin receptor. This central repression region conferred binding of the molecular chaperone hsp90 upon otherwise constitutive chimeras in vitro, indicating that hsp90 has the ability to mediate a cis-repressive function on distant transactivation domains. Importantly, when the ligand-binding domain of the dioxin receptor remained intact, the ability of this hsp90-binding activity to confer repression became conditional rather than irreversible. Our data are consistent with a model in which crucial activities of the dioxin receptor, such as dimerization with Arnt and transactivation, are conditionally repressed by the central ligand- and-hsp90-binding region of the receptor. In contrast, the Arnt protein appears to be free from any repressive activity. Moreover, within the context of the dioxin response element (xenobiotic response element), the C terminus of Arnt conferred a potent, dominating transactivation function onto the native bHLH heterodimeric complex. Finally, the relative transactivation potencies of the individual dioxin receptor and Arnt chimeras varied with cell type and promoter architecture, indicating that the mechanisms for transcriptional activation may differ between these two subunits and that in the native complex the transactivation pathway may be dependent upon cell-specific and promoter contexts.

Identification of transactivation and repression functions of the dioxin receptor and its basic helix-loop-helix/PAS partner factor Arnt: inducible versus constitutive modes of regulation.

Gene regulation by dioxins is mediated via the dioxin receptor, a ligand-dependent basic helix-loop-helix (bHLH)/PAS transcription factor. The latent dioxin receptor responds to dioxin signalling by forming an activated heterodimeric complex with a specific bHLH partner, Arnt, an essential process for target DNA recognition. We have analyzed the transactivating potential within this heterodimeric complex by dissecting it into individual subunits, replacing the dimerization and DNA-binding bHLH motifs with heterologous zinc finger DNA-binding domains. The uncoupled Arnt chimera, maintaining 84% of Arnt residues, forms a potent and constitutive transcription factor. Chimeric proteins show that the dioxin receptor also harbors a strong transactivation domain in the C terminus, although this activity was silenced by inclusion of 82 amino acids from the central ligand-binding portion of the dioxin receptor. This central repression region conferred binding of the molecular chaperone hsp90 upon otherwise constitutive chimeras in vitro, indicating that hsp90 has the ability to mediate a cis-repressive function on distant transactivation domains. Importantly, when the ligand-binding domain of the dioxin receptor remained intact, the ability of this hsp90-binding activity to confer repression became conditional rather than irreversible. Our data are consistent with a model in which crucial activities of the dioxin receptor, such as dimerization with Arnt and transactivation, are conditionally repressed by the central ligand- and-hsp90-binding region of the receptor. In contrast, the Arnt protein appears to be free from any repressive activity. Moreover, within the context of the dioxin response element (xenobiotic response element), the C terminus of Arnt conferred a potent, dominating transactivation function onto the native bHLH heterodimeric complex. Finally, the relative transactivation potencies of the individual dioxin receptor and Arnt chimeras varied with cell type and promoter architecture, indicating that the mechanisms for transcriptional activation may differ between these two subunits and that in the native complex the transactivation pathway may be dependent upon cell-specific and promoter contexts.

Multiple regulatory elements direct the complex expression pattern of the Drosophila segmentation gene paired

The paired (prd) gene of Drosophila belongs to the pair-rule class of segmentation genes involved in establishing the metameric organization of the Drosophila body plan. The complex expression pattern of prd has previously been shown to depend upon a number of segmentation genes, including gap and pair-rule genes. In an attempt to characterize and analyze the regulatory regions necessary and sufficient for prd expression, we have identified an 18-kb genomic fragment, consisting of the transcribed portion of prd and 10 kb of 5′- and 5 kb of 3′- flanking region, that is able to rescue prd mutant embryos to full viability. Analysis of a series of prd-lacZ fusion constructs containing progressively reduced lengths of prd 5′-flanking sequences delimits different cis-regulatory regions. The entire 5′-flanking region directs fusion gene expression in a pattern similar, but not identical, to the endogenous prd protein pattern. This 10-kb fragment contains both activator and repressor regions that mediate the establishment of the seven-stripe prd pattern, as well as the splitting into anterior and posterior stripes for the 14-stripe expression phase. The prd intron in combination with a minimal upstream region (0.15 kb) is able to direct low levels of prd-lacZ fusion gene expression in stripes. Information for expression of the anterior dorsal spot and of the early seven-stripe pattern is located downstream of the prd coding region. We propose that regulation of prd by pair-rule and gap gene products is mediated by upstream and downstream cis-regulatory elements. Regulation during separate but overlapping phasesof expression by separable regulatory regions might be a general characteristic of segmentation genes.

Multiple positive and negative cis-acting elements of the STA2 gene regulate glucoamylase synthesis in Saccharomyces cerevisiae

Expression of the glucoamylase-encoding gene ( STA2) in Saccharomyces cerevisiae was previously shown to be regulated transcriptionally by both positive and negative factors. The objective of this work was to identify the cis-acting elements responsible for STA2 transcriptional activation as well as the transcriptional repressor effects of STA10 and MATa/MATα. We identified two upstream activation regions ( UAS). Three repressor regions responsive to STA10-mediated repression were identified, as well as two regions for down-regulation of STA2 expression. MATa/MATα repression appears to effect STA2 expression either downstream from the translational start site or, indirectly, since no functional al/α2-responsive sequence was identified in the promoter region.

The global transcriptional regulators, SSN6 and TUP1, play distinct roles in the establishment of a repressive chromatin structure.

Repression of a-cell specific gene expression in yeast alpha cells requires MAT alpha 2 and MCM1, as well as two global repressors, SSN6 and TUP1. Previous studies demonstrated that nucleosomes positioned adjacent to the alpha 2/MCM1 operator in alpha cells directly contribute to repression. To investigate the possibility that SSN6 and TUP1 provide a link between MAT alpha 2/MCM1 and neighboring histones, nucleosome locations were examined in ssn6 and tup1 alpha cells. In both cases, nucleosome positions downstream of the operator were disrupted, and the severity of the disruption correlated with the degree of derepression. Nevertheless, the observed changes in chromatin structure were not dependent on transcription. Our data strongly indicate that SSN6 and TUP1 directly organize repressive regions of chromatin.

Mapping and Mutagenesis of the Amino-Terminal Transcriptional Repression Domain of the Drosophila Krüppel Proteint†

We previously demonstrated that the Drosophila Krüppel protein is a transcriptional repressor with separable DNA-binding and transcriptional repression activities. In this study, the minimal amino (N)-terminal repression region of the Krüppel protein was defined by transferring regions of the Krüppel protein to a heterologous DNA-binding protein, the lacI protein. Fusion of a predicted alpha-helical region from amino acids 62 to 92 in the N terminus of the Krüppel protein was sufficient to transfer repression activity. This putative alpha-helix has several hydrophobic surfaces, as well as a glutamine-rich surface. Mutants containing multiple amino acid substitutions of the glutamine residues demonstrated that this putative alpha-helical region is essential for repression activity of a Krüppel protein containing the entire N-terminal and DNA-binding regions. Furthermore, one point mutant with only a single glutamine on this surface altered to lysine abolished the ability of the Krüppel protein to repress, indicating the importance of the amino acid at residue 86 for repression. The N terminus also contained an adjacent activation region localized between amino acids 86 and 117. Finally, in accordance with predictions from primary amino acid sequence similarity, a repression region from the Drosophila even-skipped protein, which was six times more potent than that of the Krüppel protein in the mammalian cells, was characterized. This segment included a hydrophobic stretch of 11 consecutive alanine residues and a proline-rich region.

Mapping and mutagenesis of the amino-terminal transcriptional repression domain of the Drosophila Krüppel protein.

We previously demonstrated that the Drosophila Krüppel protein is a transcriptional repressor with separable DNA-binding and transcriptional repression activities. In this study, the minimal amino (N)-terminal repression region of the Krüppel protein was defined by transferring regions of the Krüppel protein to a heterologous DNA-binding protein, the lacI protein. Fusion of a predicted alpha-helical region from amino acids 62 to 92 in the N terminus of the Krüppel protein was sufficient to transfer repression activity. This putative alpha-helix has several hydrophobic surfaces, as well as a glutamine-rich surface. Mutants containing multiple amino acid substitutions of the glutamine residues demonstrated that this putative alpha-helical region is essential for repression activity of a Krüppel protein containing the entire N-terminal and DNA-binding regions. Furthermore, one point mutant with only a single glutamine on this surface altered to lysine abolished the ability of the Krüppel protein to repress, indicating the importance of the amino acid at residue 86 for repression. The N terminus also contained an adjacent activation region localized between amino acids 86 and 117. Finally, in accordance with predictions from primary amino acid sequence similarity, a repression region from the Drosophila even-skipped protein, which was six times more potent than that of the Krüppel protein in the mammalian cells, was characterized. This segment included a hydrophobic stretch of 11 consecutive alanine residues and a proline-rich region.

Mediation of suppression of c‐fos transcription in rasT24‐transformed rat cells by a cis‐acting represser element

Prolonged expression of activated ras mutants resulted in both neoplastic transformation and suppression of serum-induced c-fos expression in Rat1 fibroblasts. Expression of other serum-inducible genes, including c-jun and beta-actin, was not suppressed in ras-transformed Rat1 cells, indicating that these effects are specific for c-fos and that growth-factor signal transduction pathways remain essentially intact. Run-on transcription studies indicated that c-fos transcription was blocked at the level of initiation in these cells. Transient transfection studies using 360 bp from the wild-type c-fos promoter as well as a series of mutated c-fos promoter fragments linked to the chloramphenicol acetyltransferase gene indicated that repression of c-fos was mediated by approximately 49 bp immediately upstream of the dyad symmetry element (DSE). Deletion of this region, referred to as the upstream repressor region (URR), restored serum inducibility to the c-fos promoter in ras-transformed cells. In contrast, suppression of c-fos transcription was not affected by either deletion of 240 bp between the DSE and the TATA element or by base-substitution mutations that inactive the ternary complex factor and fos-AP-1-like binding sites. In addition, in vitro competition studies indicated that ras-transformed cells express one or more repressor factors that interact with as-yet-unidentified elements within the c-fos promoter (possibly the URR) and block serum induction of c-fos. These findings suggest that prolonged expression of activated ras results in the activation of one or more as-yet-unidentified proteins that suppress transcription of the c-fos gene by interacting with the URR.

Functional dissection of the yeast Cyc8-Tup1 transcriptional co-repressor complex.

DNA-binding repressor proteins mediate regulation of yeast genes by cell type (Mcm1/alpha 2 and a1/alpha 2), glucose (Mig1) and oxygen (Rox1) (refs 1-4 respectively). An unusual feature of all these regulatory pathways is that transcriptional repression requires two physically associated proteins that do not bind DNA Cyc8(Ssn6) and Tup1. The Cyc8-Tup1 complex has been proposed to be a co-repressor that is recruited to target promoters by pathway-specific DNA-binding proteins, but the specific functions of the individual proteins are unknown. Here we show that when it is bound upstream of a functional promoter through the LexA DNA-binding domain, Tup1 represses transcription in the absence of Cyc8. Deletion analysis indicates that Tup1 contains at least two non-overlapping transcriptional repression regions with minimal primary sequence similarity, and a separable Cyc8-interaction domain. These Tup1 domains, which do not include the beta-transducin motifs, are necessary and partially sufficient for Tup1 function. We suggest that Tup1 performs the repression function of the Cyc8-Tup1 co-repressor complex, and that Cyc8 serves as a link with the pathway-specific DNA-binding proteins.

Mutations in the repressor region ( rr) of the hepatitis B virus enhancer I (HBV-EI) in interferon-resistant patients with chronic hepatitis B (CHB). Abnormal interactions of nuclear extracts (NEX) with the mutated rr

Mutations in the repressor region ( rr) of the hepatitis B virus enhancer I (HBV-EI) in interferon-resistant patients with chronic hepatitis B (CHB). Abnormal interactions of nuclear extracts (NEX) with the mutated rr

Identification of two repressor regions within the plasminogen activator inhibitor 2 (PAI-2) gene promoter

Identification of two repressor regions within the plasminogen activator inhibitor 2 (PAI-2) gene promoter

Effect of alpha-interferon (α-IFN) on the binding activity of nuclear factors to the repressor region ( rr) of the HBV enhancer I (HBV-EI) in patients with chronic hepatitis B (CHB)

Effect of alpha-interferon (α-IFN) on the binding activity of nuclear factors to the repressor region ( rr) of the HBV enhancer I (HBV-EI) in patients with chronic hepatitis B (CHB)

Cell cycle regulation of a human cyclin-like gene encoding uracil-DNA glycosylase.

The predicted amino acid sequence of a human cDNA encoding uracil-DNA glycosylase activity shows striking similarity to the cyclin protein family. To characterize the expression of this DNA repair enzyme, we have isolated the corresponding genomic clone. This gene is contained within 4.2 kilobases and is composed of only two exons. Sequence analysis of the upstream region shows that it contains two cell cycle box (CCB) regulatory elements which are also found in yeast cyclin genes. Deletional analysis of the promoter reveals the presence of a repressor region located from position -812 to -603. An inverted CCB element (alpha-CCB) and an SP1-like binding site are contained within this region. When uracil-DNA glycosylase mRNA levels are examined in vivo, a 2-3-fold increase is associated with G1 phase in both HeLa S3 and WI38 cells. To examine the role of the 209-base pair repressor region in mediating cell cycle regulation, this fragment was used in gel shift assays with cellular extracts prepared from various stages of the cell cycle. Several specific complexes are formed during S and G2 phases which are not present during M and G1 phases. Two of the complexes are the result of alpha-CCB binding as they can be specifically disrupted by the addition of an oligonucleotide containing the alpha-CCB binding site. Immunoprecipitation studies reveal that uracil-DNA glycosylase protein levels are also elevated during G1 phase. Additionally, we show that the 36-kDa uracil-DNA glycosylase protein is turned over during the course of one cell cycle. These results demonstrate coordinate regulation of uracil-DNA glycosylase at both the transcriptional and the post-transcriptional levels as a function of the cell cycle.

A repressor region in the human β-myosin heavy chain gene that has a partial position dependency

Expression of the human β-myosin heavy chain (βMHC) gene was studied by transient assay in culture and in situ by direct injection of plasmids into adult rat hearts. In this report we describe a unique repressor region located −326/−309 (5′-TTGGTGGTCGTGGTCAGT-3′) of the human βMHC gene that is conserved among the rat, rabbit, and human βMHC genes. This sequence conferred repression onto heterologous promoters when the sequence was located 5′ but not 3′ to the promoters. This partial positional dependency suggests that the factor may act by limiting the binding of enhancers, located more proximally, to their DNA binding sites.

Activation and Suppression of a Cryptic Promoter in the Intron of the Human Melanoma‐associated ME491 Antigen Gene

A deletion mutant of the human melanoma‐associated ME491 antigen gene starting at the first intron (λR31) differentially mediates the antigen expression depending on the cell type. Cryptic promoter activity residing in a 270‐base‐pair (bp) fragment of the first intron was examined by primer extension analysis and recombinant chloramphenicol acetyltransferase (CAT) assay. The cryptic promoter, further localized within a 153‐bp fragment (fr153BN), exerted its effect in Ltk‐ and H‐ras‐transformed NIH3T3 (3T3‐Hras) but not in parental NIH3T3 cells. The results suggested that the cryptic promoter was associated with a novel ras‐responsive positive regulatory element, since fr153BN did not contain an AP‐1‐binding sequence motif, known as the rew‐responsive enhancer element. The cryptic promoter activity of fr153BN was suppressed by an upstream 121‐bp fragment (fr121SB) which contained a consensus sequence motif for binding of a repressor protein, GC factor, and regions showing sequence similarity with putative cis‐acting repressor elements found in the vimentin gene. The degree of the suppression was greater in 3T3‐Hras than in Ltk‐ cells. These positive and negative regulatory elements may be differentially involved in the regulation of ME491 antigen expression depending on the cell type.