Repressor Region Research Articles

Molecular cloning and characterization of three adult rat $beta;-globin gene promoters*1

We analyzed the promoter regions of the adult rat β (IIβ, IIIβ, and 0β)-globin genes. The results indicated that (1) the activities of the minimal promoters of these three genes are proportional to the gene expression levels in vivo, and (2) erythroid-specific repressor regions are located immediately upstream of the minimal promoter sequences and are regulated by the same transcription factor.

Molecular cloning and characterization of three adult rat β-globin gene promoters

We analyzed the promoter regions of the adult rat β (IIβ, IIIβ, and 0β)-globin genes. The results indicated that (1) the activities of the minimal promoters of these three genes are proportional to the gene expression levels in vivo, and (2) erythroid-specific repressor regions are located immediately upstream of the minimal promoter sequences and are regulated by the same transcription factor.

Characterization of functional elements in the neurofibromatosis (NF1) proximal promoter region

An essential requirement to understand how genes contribute to genetic disease is the thorough knowledge of the transcriptional regulation of gene expression. Here, we have characterized transcription factor binding sites within the type 1 neurofibromatosis (NF1) proximal regulatory region, and addressed the molecular mechanisms that regulate NF1 transcription. Overlapping regions of the NF1 proximal promoter have been cloned and characterized for use in luciferase reporter assays. These assays have identified a 500 bp region displaying activities up to 80-fold higher than control reporter levels. Mutations at putative CRE and SP1-binding sites immediately 5' to the transcription start site have dramatic effects that lead to a 70-90% decrease in reporter activity in all cell lines tested. Gelshift assays confirm binding of CREB and SP1/KLF family members to their putative recognition sequences, and provide the first evidence identifying functional sites likely involved in regulating NF1 transcription. These assays have also revealed a putative repressor region within the NF1 promoter region corresponding to CCCTC-rich sequences between the transcription and translation start sites. This work provides new information concerning the transcriptional regulation of the NF1 gene, and is the most thorough attempt to date to map functionally relevant regions within the NF1 proximal promoter region.

Differential expression of the antioxidant response element within the hNQO1 promoter in NSCLC versus SCLC

To determine whether the human (h) NQO1 (NAD(P)H: quinone oxidoreductase 1) gene contains DNA sequences that directly mediate its high and low expression in non-small cell lung carcinoma (NSCLC) and small cell lung carcinoma (SCLC), respectively, a series of deletion constructs spanning up to −4 kb of the 5 ′ flanking region of hNQO1 was used in transient transfection assays. The antioxidant response element (ARE) was found to be critical to the elevated expression of NQO1 in NSCLC. However, the ability of both heterologous and deletion promoter constructs to confer ARE responsiveness demonstrated that SCLC contains the necessary program/menu of transcription factors responsible to drive hNQO1 expression via the ARE. By examining the regulatory region of the hNQO1 gene, we identified a proximal repressor region between −259 and −131. These results provide the first evidence of a proximal repressor region exerting a negative role on the regulation of the hNQO1 promoter in SCLC.

Differential expression of the antioxidant response element within the hNQO1 promoter in NSCLC versus SCLC

To determine whether the human (h) NQO1 (NAD(P)H: quinone oxidoreductase 1) gene contains DNA sequences that directly mediate its high and low expression in non-small cell lung carcinoma (NSCLC) and small cell lung carcinoma (SCLC), respectively, a series of deletion constructs spanning up to −4 kb of the 5 ′ flanking region of hNQO1 was used in transient transfection assays. The antioxidant response element (ARE) was found to be critical to the elevated expression of NQO1 in NSCLC. However, the ability of both heterologous and deletion promoter constructs to confer ARE responsiveness demonstrated that SCLC contains the necessary program/menu of transcription factors responsible to drive hNQO1 expression via the ARE. By examining the regulatory region of the hNQO1 gene, we identified a proximal repressor region between −259 and −131. These results provide the first evidence of a proximal repressor region exerting a negative role on the regulation of the hNQO1 promoter in SCLC.

The MEF2 site is necessary for induction of the myosin light chain 2 slow promoter in overloaded regenerating plantaris muscle

Functional overload (OV) of the rat plantaris muscle results in a fast to slow change in muscle phenotype with induction of the slow contractile protein genes including myosin light chain 2 slow (MLC2s). To identify potential cis-acting DNA sites regulating MLC2s following ablation, plasmid constructs were transfected in vivo into regenerating overloaded plantaris muscles. Activity of the 270bp promoter (−270MLC2s) was increased in OV muscles at 28 days. Mutation of the MEF2 site (−270MEF2) knocked out the overload-induced activity of the promoter. Mutation of the Ebox (−270Ebox) resulted in an earlier induction with OV and mutation of the CACC site (–270CACC) resulted in increased activity in the CON PLN with OV induction detected by 21 days. These results demonstrate that the –270MLC2s promoter contains the elements necessary for expression of MLC2s in regenerating OV PLN. More importantly, mutation analysis of −270MLC2s promoter demonstrates that mechanical loading induced expression shares some common molecular mechanisms with slow nerve dependent model regulation. In these two models of physiological induction of MLC2s, the CACC site acts as a repressor region (on/off switch) and the MEF2 site acts to modulate quantitative expression.

Analysis of a repressor region in the human neuropeptide Y gene that binds Oct-1 and Pbx-1 in GT1-7 neurons

The mechanisms dictating the developmental expression of individual neuropeptides within the hypothalamus have not yet been elucidated. In this paper we have studied the cis-acting elements involved in the repression of neuropeptide Y (NPY) gene expression in a gonadotropin-releasing hormone (GnRH) neuronal cell model, GT1-7 cells. Using transient transfection of the human NPY 5 ′ regulatory region into the GT1-7 neurons, we have found a repressor region located between −867 and −1078. DNase I footprint analysis of this region revealed three specific protein binding elements. Further analysis of the region between −942 and −922 bp using electrophoretic mobility shift assays revealed that four different transcription factor-DNA complexes form with GT1-7 nuclear proteins, whereas only three complexes are detected using baby hamster kidney (BHK) cell nuclear extract. Mutation of the consensus binding sequence abolishes all complex formation on the −924/−922 oligonucleotide. Antibody supershift assays revealed that Oct-1 and Pbx-1 antibodies were able to eliminate the appearance of two specific complexes. Therefore we suggest that this region may be important for transcriptional repression of the NPY gene in a heterologous cell model, through complex, coordinate protein–protein interactions.

The Methyl-CpG-binding Protein MeCP2 Links DNA Methylation to Histone Methylation

DNA methylation plays an important role in mammalian development and correlates with chromatin-associated gene silencing. The recruitment of MeCP2 to methylated CpG dinucleotides represents a major mechanism by which DNA methylation can repress transcription. MeCP2 silences gene expression partly by recruiting histone deacetylase (HDAC) activity, resulting in chromatin remodeling. Here, we show that MeCP2 associates with histone methyltransferase activity in vivo and that this activity is directed against Lys(9) of histone H3. Two characterized repression domains of MeCP2 are involved in tethering the histone methyltransferase to MeCP2. We asked if MeCP2 can deliver Lys(9) H3 methylation to the H19 gene, whose activity it represses. We show that the presence of MeCP2 on nucleosomes within the repressor region of the H19 gene (the differentially methylated domain) coincides with an increase in H3 Lys(9) methylation. Our data provide evidence that MeCP2 reinforces a repressive chromatin state by acting as a bridge between two global epigenetic modifications, DNA methylation and histone methylation.

Interplay of TBP Inhibitors in Global Transcriptional Control

The TATA binding protein (TBP) is required for the expression of nearly all genes and is highly regulated both positively and negatively. Here, we use DNA microarrays to explore the genome-wide interplay of several TBP-interacting inhibitors in the yeast Saccharomyces cerevisiae. Our findings suggest the following: The NC2 inhibitor turns down, but not off, highly active genes. Autoinhibition of TBP through dimerization contributes to transcriptional repression, even at repressive subtelomeric regions. The TAND domain of TAF1 plays a primary inhibitory role at very few genes, but its function becomes widespread when other TBP interactions are compromised. These findings reveal that transcriptional output is limited in part by a collaboration of different combinations of TBP inhibitory mechanisms.

The murine COMP (cartilage oligomeric matrix protein) promoter contains a potent transcriptional repressor region

Objective A subgroup of patients with pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED) have been found to harbor mutations within the cartilage oligomeric matrix protein (COMP) gene. These two diseases are autosomal dominant disorders that are characterized by an early onset of osteoarthritis (OA). The COMP gene is expressed primarily in chondrocytes in articular cartilage as well as in tendon and ligament. Therefore, control over tissue specific COMP expression may be an important aspect in cartilage biology. To begin an analysis of the regulation of COMP expression, we have cloned, sequenced and characterized the entire genomic clone for mouse COMP that includes the COMP promoter.Methods and Results The COMP coding region spans 19 exons over approximately 8.4kb of DNA. The arrangement and size of the exons have a remarkable similarity to those of the human COMP genomic sequence, indicating a significant degree of genomic conservation. Analysis of a 453 basepair region of the putative COMP promoter reveals two strong transcriptional repressor elements located between position −356 and −304 and between −251 and −180, relative to the start site for transcription. These repressor elements down-regulate transcription from the promoter in a broad spectrum of cell lines. Removal of the repressor DNA sequence from the COMP promoter leads to significant enhancement in transcriptional activity, indicating that this region acts in a dominant manner to transcriptional activators located more proximal to the start site of transcription. This region also represses transcription when linked to a heterologous promoter.Conclusions This repressor region probably down-regulates transcription from the COMP promoter in vivo. It may help to repress transcription of COMP in non-cartilaginous tissues and/or may aid in the expression of COMP to the appropriate level in tissues such as cartilage, tendon and ligament.

Isolation and Characterization of a Human STAT1Gene Regulatory Element: INDUCIBILITY BY INTERFERON (IFN) TYPES I AND II AND ROLE OF IFN REGULATORY FACTOR-1

The transcription factor STAT1 plays a pivotal role in signal transduction of type I and II interferons (IFNs). STAT1 activation leads to changes in expression of key regulatory genes encoding caspases and cell cycle inhibitors. Deficient STAT1 expression in human cancer cells and virally mediated inhibition of STAT1 function have been associated with cellular resistance to IFNs and mycobacterial infection in humans. Thus, given the relative importance of STAT1, we isolated and characterized a human STAT1 intronic enhancer region displaying IFN-regulated activity. Functional analyses by transient expression identified a repressor region and type I and II IFN-inducible elements within the STAT1 enhancer sequence. A candidate IRF-E/GAS/IRF-E (IGI) sequence containing GAAANN nucleotide repeats was shown by gel shift assay to bind to IFN regulatory factor-1 (IRF-1), but not to IFN-stimulated gene factor-3 (ISGF-3) or STAT1-3. An additional larger IGI-binding complex containing IRF-1 was identified. Mutation of the GAAANN repeats within the IGI DNA element eliminated IRF-1 binding and the IFN-regulated activity of the STAT1 intronic enhancer region. Transfection of the IFN-resistant MM96 cell line to express increased levels of IRF-1 protein also elevated STAT1, STAT2, and p48/IRF-9 expression and enhanced cellular responsiveness to IFN-beta. Reciprocating regulation between IRF-1 and STAT1 genes and encoded proteins indicates that an intracellular amplifier circuit exists controlling cellular responsiveness to the IFNs.

Characterization of the cts4 repressor mutation in transposable bacteriophage Mu

Mu cts4 was isolated more than 30 years ago and was the first available thermoinducible derivative of transposable phage Mu. We have characterized the cts4 mutation and the corresponding mutant protein. Contrary to previously characterized thermoinducible Mu prophages (e.g., Mu cts62), Mu cts4 lysogenizes at reduced frequency even at 30 ∘ C. The cts4 mutation (Leu129Val) was located in this central repressor region. The cts4 protein was thermosensitive for operator DNA binding in vitro. Temperature-dependent changes in protein–protein cross-linking patterns in the absence of DNA were detected for purified wild type, cts62 and cts4 repressor proteins. The cts4 protein exhibited a subtly different electrophoretic profile, which became more marked at higher temperatures, from both the wild type and cts62. In addition the cts4 repressor generated a significantly different pattern of binding to DNA fragments carrying the early operator region. Consistent with the predicted involvement of the central leucine-rich region of the Mu repressor in the formation of multimeric forms, the cts4 mutation thus appeared to affect protein–protein interactions.

Epidermal Langerhans Cell-Targeted Gene Expression by a Dectin-2 Promoter

Despite their critical function as APCs for primary immune responses, dendritic cells (DC) and Langerhans cells (LC) have been rarely used as targets of gene-based manipulation because well-defined regulatory elements controlling LC/DC-specific expression have not been identified. Previously, we identified dectin-2, a C-type lectin receptor expressed selectively by LC-like XS cell lines and by LC within mouse epidermis. Because these characteristics raised the possibility that dectin-2 promoter may direct LC/DC-specific gene expression, we isolated a 3.2-kb nucleotide fragment from the 5'-flanking region of the dectin-2 gene (Dec2FR) and characterized its regulatory elements and the transcriptional activity using a luciferase (Luc) reporter system. The Dec2FR contains a putative TATA box and cis-acting elements, such as the IFN-stimulated response element, that drive gene expression specifically in XS cells. Dec2FR comprises repressor, enhancer, and promoter regions, and the latter two regions coregulate XS cell-specific gene expression. In transgenic mice bearing a Dec2FR-regulated Luc gene, the skin was the predominant site of Luc activity and LC were the exclusive source of such activity within epidermis. By contrast, other APCs (DC, macrophages, and B cells) and T cells expressed Luc activity close to background levels. We conclude that epidermal LC are targeted selectively for high-level constitutive gene expression by Dec2FR in vitro and in vivo. Our findings lay the foundation for use of the dectin-2 promoter in LC-targeted gene expression systems that may enhance vaccination efficacy and regulate immune responses.

Translational activation and repression by distinct elements within the 5'-UTR of ENaC alpha-subunit mRNA.

The rat amiloride-sensitive epithelial Na(+) channel (rENaC), the rate-limiting step in epithelial Na(+) transport, consists of three subunits, alpha, beta, and gamma. We hypothesized that alpha-rENaC translation is regulated via its 5'-untranslated region (UTR). Transient transfections of alpha-rENaC promoter-reporter constructs in representative epithelial cell lines demonstrated up to fivefold differences in activity among constructs containing different amounts of the alpha-rENaC 5'-UTR sequence. Differences in reporter protein activity did not parallel differences in reporter mRNA, demonstrating that 5'-UTR regulation must be at the level of translation. Specifically, translation was enhanced by a region extending from +53 to +211 bp downstream from the transcription start site and repressed by the region between +367 and +499 bp. Examination of the 5'-UTR sequence revealed an out-of-frame initiation codon within the repressive region, 43 bp upstream from the start of the alpha-rENaC open reading frame. Mutational analysis of this upstream start codon indicated that it plays, at most, a minor role in impeding translation both in vitro and in vivo, suggesting that additional mechanisms of translational regulation are operative.

The Phytochrome A-specific Signaling Intermediate SPA1 Interacts Directly with COP1, a Constitutive Repressor of Light Signaling inArabidopsis

SPA1 is a phytochrome A (phyA)-specific signaling intermediate that acts as a light-dependent repressor of photomorphogenesis in Arabidopsis seedlings. It contains a WD-repeat domain that shows high sequence similarity to the WD-repeat region of the constitutive repressor of light signaling, COP1. Here, using yeast two-hybrid and in vitro interaction assays, we show that SPA1 strongly and selectively binds to COP1. Domain mapping studies indicate that the putative coiled-coil domain of SPA1 is necessary and sufficient for binding to COP1. Conversely, similar deletion analyses of the COP1 protein suggest that SPA1 interacts with the presumed coiled-coil domain of COP1. To further investigate SPA1 function in the phyA signaling pathway, we tested whether SPA1, like COP1, mediates changes in gene expression in response to light. We show that spa1 mutations increase the photoresponsiveness of certain light-regulated genes within 2 h of light treatment. Taken together, the results suggest that SPA1 may function to link the phytochrome A-specific branch of the light signaling pathway to COP1. Hence, our data provide molecular support for the hypothesis that COP1 is a convergence point for upstream signaling pathways dedicated to individual photoreceptors.

The Y-box Binding Protein YB-1 Suppresses Collagen α1(I) Gene Transcription via an Evolutionarily Conserved Regulatory Element in the Proximal Promoter

Appropriate expression of collagen type I, a major component of connective tissue matrices, is dependent on tight transcriptional control and a number of trans-activating and repressing factors have been characterized. Here we identify the Y-box binding protein-1 (YB-1) as a novel repressor of the collagen type alpha1(I) (COL1A1) gene. Collagen type I mRNA and protein levels decreased upon overexpression of YB-1 by transfection in NRK fibroblasts. The human, rat, and mouse COL1A1 promoter -220/+115 contains three putative Y-boxes, one of these sites, designated collagen Y-box element (CYE), includes a Y-box plus an adjacent 3' inverted repeat. DNase-I footprinting and Southwestern blotting with fibroblast nuclear extract demonstrated binding of several nuclear proteins across the CYE, one of which was identified as YB-1. Recombinant YB-1 bound the CYE sequence in gel shift assays with a preference for single-stranded templates. The entire sequence (-88/-48) was required for high affinity binding. Complex formation of endogenous YB-1 with the CYE was established by supershift studies. COL1A1 promoter-reporter constructs were suppressed up to 80% by cotransfection with YB-1 in a variety of cell types. In addition, CYE conferred YB-1 responsiveness on two heterologous promoters further demonstrating the importance of this repressor region. Mung bean nuclease sensitivity analysis suggested that repression is most likely exerted through changes in DNA conformation.

Missense Mutations Cluster within the Carboxyl-Terminal Region of DAX-1 and Impair Transcriptional Repression

Missense Mutations Cluster within the Carboxyl-Terminal Region of DAX-1 and Impair Transcriptional Repression

Site-specific recognition of a 70-base-pair element containing d(GA)(n) repeats mediates bithoraxoid polycomb group response element-dependent silencing.

Polycomb group proteins act through Polycomb group response elements (PREs) to maintain silencing at homeotic loci. The minimal 1.5-kb bithoraxoid (bxd) PRE contains a region required for pairing-sensitive repression and flanking regions required for maintenance of embryonic silencing. Little is known about the identity of specific sequences necessary for function of the flanking regions. Using gel mobility shift analysis, we identify DNA binding activities that interact specifically with a multipartite 70-bp fragment (MHS-70) downstream of the pairing-sensitive sequence. Deletion of MHS-70 in the context of a 5.1-kb bxd Polycomb group response element derepresses maintenance of silencing in embryos. A partially purified binding activity requires multiple, nonoverlapping d(GA)(3) repeats for MHS-70 binding in vitro. Mutation of d(GA)(3) repeats within MHS-70 in the context of the 5.1-kb bxd PRE destabilizes maintenance of silencing in a subset of cells in vivo but gives weaker derepression than deletion of MHS-70. These results suggest that d(GA)(3) repeats are important for silencing but that other sequences within MHS-70 also contribute to silencing. Antibody supershift assays and Western analyses show that distinct isoforms of Polyhomeotic and two proteins that recognize d(GA)(3) repeats, the TRL/GAGA factor and Pipsqueak (Psq), are present in the MHS-70 binding activity. Mutations in Trl and psq enhance homeotic phenotypes of ph, indicating that TRL/GAGA factor and Psq are enhancers of Polycomb which have sequence-specific DNA binding activity. These studies demonstrate that site-specific recognition of the bxd PRE by d(GA)(n) repeat binding activities mediates PcG-dependent silencing.

The Identification of Prx1 Transcription Regulatory Domains Provides a Mechanism for Unequal Compensation by thePrx1 and Prx2 Loci

Transcription regulatory domains of the Prx1a and Prx1b homeoproteins were analyzed in transient transfection assays using artificial promoters as well as an established downstream target promoter (tenascin-c). Activation and repression domains were detected in their common amino end. In the carboxyl end of Prx1a an activation domain and an inhibition/masking region (OAR domain) were detected. The Prx1b isoform, generated by alternative splicing, does not contain these carboxyl activation or inhibition domains. Instead, the data demonstrate that the carboxyl tail of Prx1b contains a potent repressor region. This difference in the carboxyl tail accounts for a 45-fold difference observed in transcription regulatory activity between Prx1a and Prx1b. The data also support the likelihood that this difference between Prx1a and Prx1b is higher in the presence of still undetermined cofactors. DNA binding affinities of Prx1a, Prx1b, and a series of truncation mutants were also examined. The carboxyl tail of Prx1a, which inhibited transcription activation in the transfection assays, also inhibited DNA binding. These differences in biochemical function between Prx1a and Prx1b, as well as the recently described activities of Prx2, provide a mechanism for the unequal compensation between the Prx1 and Prx2 loci.

Distinct Enhancer Elements Control Hex Expression during Gastrulation and Early Organogenesis

In the mouse, embryological and genetic studies have indicated that two spatially distinct signalling centres, the anterior visceral endoderm and the node and its derivatives, are required for the correct patterning of the anterior neural ectoderm. The divergent homeobox gene Hex is expressed in the anterior visceral endoderm, in the node (transiently), and in the anterior definitive endoderm. Other sites of Hex expression include the liver and thyroid primordia and the endothelial cell precursors. We have used transgenic analysis to map the cis-acting regulatory elements controlling Hex expression during early mouse development. A 4.2-kb upstream region is important for Hex expression in the endothelial cell precursors, liver, and thyroid, and a 633-bp intronic fragment is both necessary and sufficient for Hex expression in the anterior visceral endoderm and the anterior definitive endoderm. These same regions drive expression in homologous structures in Xenopus laevis, indicating conservation of these regulatory regions in vertebrates. Analysis of the anterior visceral endoderm/anterior definitive endoderm enhancer identifies a repressor region that is required to downregulate Hex expression in the node once the anterior definitive endoderm has formed. This analysis also reveals that the initiation of Hex expression in the anterior visceral endoderm and axial mesendoderm requires common elements, but maintenance of expression is regulated independently in these tissues.