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PubMed1997

Physical and functional interactions between the transcriptional inhibitors Id3 and ITF-2b. Evidence toward a novel mechanism regulating muscle-specific gene expression.

We have used an interaction cloning strategy to identify an inhibitory isoform of the ITF-2 transcription factor, ITF-2b, that interacts with the transcriptional inhibitor Id3/HLH462. The interaction was confirmed in vitro, and inside intact myogenic C2C12 cells. As expected, overexpression of either Id3/HLH462 or ITF-2b effectively inhibited the activation of the muscle-specific creatine kinase promoter by the myogenic transcription factor MyoD. However, when overexpressed simultaneously, ITF-2b and Id3/HLH462 counteracted each other's inhibitory effect to produce a reduced overall inhibition. Moreover, while ITF-2b inhibited the creatine kinase promoter, it acted as a weak transactivator on an artificial promoter consisting of three tandem copies of the consensus myogenic factor DNA binding site. Further investigation indicated that the ITF-2b/MyoD heterodimer bound to its specific DNA binding site in vitro, and the DNA binding was effectively blocked by Id3/HLH462. Additional analysis revealed the presence of transcripts for both the activating (ITF-2a) and inhibitory (ITF-2b) isoforms in differentiating C2C12 cultures, suggesting that both isoforms might participate in regulating the differentiation process. Taken together, this study reveals a more complex pattern of regulatory interactions involving the helix-loop-helix proteins than was previously anticipated.

Actins↗
PubMed1992

Structure and expression of the rice mitochondrial apocytochrome b gene (cob-1) and pseudogene (cob-2).

Rice mitochondrial DNA contains an intact copy and a pseudogene copy of a apocytochrome b gene (cob-1 and cob-2, respectively). Using primer extension and capping analyses, the transcriptional start site has been mapped; an 11-base motif at the transcription start site closely matches the consensus promoter motifs proposed for maize, wheat and soybean mitochondrial genes. Although both copies are identical in the 5' upstream region and through most of the coding region, only cob-1-specific mRNA is detected on RNA gel-blots. Run-on transcription analysis indicates, however, that both cob-1 and cob-2 mRNAs are synthesized in vivo but less cob-2 is accumulated. At its mapped 3' terminus the cob-1 transcript possesses a sequence that could fold into a double stem-loop structure. The possible roles of a double stem-loop structure in mitochondrial gene expression are discussed.

Apoproteins↗
PubMed2006

Solution structure of the apical stem-loop of the human hepatitis B virus encapsidation signal.

Hepatitis B virus (HBV) replication is initiated by HBV RT binding to the highly conserved encapsidation signal, epsilon, at the 5' end of the RNA pregenome. Epsilon contains an apical stem-loop, whose residues are either totally conserved or show rare non-disruptive mutations. Here we present the structure of the apical stem-loop based on NOE, RDC and (1)H chemical shift NMR data. The (1)H chemical shifts proved to be crucial to define the loop conformation. The loop sequence 5'-CUGUGC-3' folds into a UGU triloop with a CG closing base pair and a bulged out C and hence forms a pseudo-triloop, a proposed protein recognition motif. In the UGU loop conformations most consistent with experimental data, the guanine nucleobase is located on the minor groove face and the two uracil bases on the major groove face. The underlying helix is disrupted by a conserved non-paired U bulge. This U bulge adopts multiple conformations, with the nucleobase being located either in the major groove or partially intercalated in the helix from the minor groove side, and bends the helical stem. The pseudo-triloop motif, together with the U bulge, may represent important anchor points for the initial recognition of epsilon by the viral RT.

5' Untranslated Regions↗
PubMed1998

Somatic hypermutation introduces insertions and deletions into immunoglobulin V genes.

During a germinal center reaction, random mutations are introduced into immunoglobulin V genes to increase the affinity of antibody molecules and to further diversify the B cell repertoire. Antigen-directed selection of B cell clones that generate high affinity surface Ig results in the affinity maturation of the antibody response. The mutations of Ig genes are typically basepair substitutions, although DNA insertions and deletions have been reported to occur at a low frequency. In this study, we describe five insertion and four deletion events in otherwise somatically mutated VH gene cDNA molecules. Two of these insertions and all four deletions were obtained through the sequencing of 395 cDNA clones (approximately 110,000 nucleotides) from CD38+IgD- germinal center, and CD38-IgD- memory B cell populations from a single human tonsil. No germline genes that could have encoded these six cDNA clones were found after an extensive characterization of the genomic VH4 repertoire of the tonsil donor. These six insertions or deletions and three additional insertion events isolated from other sources occurred as triplets or multiples thereof, leaving the transcripts in frame. Additionally, 8 of 9 of these events occurred in the CDR1 or CDR2, following a pattern consistent with selection, and making it unlikely that these events were artifacts of the experimental system. The lack of similar instances in unmutated IgD+CD38- follicular mantle cDNA clones statistically associates these events to the somatic hypermutation process (P = 0.014). Close scrutiny of the 9 insertion/deletion events reported here, and of 25 additional insertions or deletions collected from the literature, suggest that secondary structural elements in the DNA sequences capable of producing loop intermediates may be a prerequisite in most instances. Furthermore, these events most frequently involve sequence motifs resembling known intrinsic hotspots of somatic hypermutation. These insertion/deletion events are consistent with models of somatic hypermutation involving an unstable polymerase enzyme complex lacking proofreading capabilities, and suggest a downregulation or alteration of DNA repair at the V locus during the hypermutation process.

Amino Acid Sequence↗
PubMed1997

Backbone dynamics of homologous fibronectin type III cell adhesion domains from fibronectin and tenascin.

BACKGROUND: Fibronectin type III domains are found as autonomously-folded domains in a large variety of multidomain proteins, including extracellular matrix proteins. A subset of these domains employ an Arg-Gly-Asp (RGD) tripeptide motif to mediate contact with cell-surface receptors (integrins). This motif mediates protein-protein interactions in a diverse range of biological processes, such as in tissue development, would healing and metastasis. The molecular basis for affinity and specificity of cell adhesion via type III domains has not been clearly established. The tenth type III domain from fibronectin (FNfn10) and the third type III domain from tenascin-C (TNfn3) have 27% sequence identity and share the same overall protein fold, but present the RGD motifs in different structural contexts. The dynamical properties of the RGD motifs may affect the specificity and affinity of the FNfn10 and TNfn3 domains. Structure-dynamics correlations for these structurally homologous proteins may reveal common molecular features which are important to the dynamical properties of proteins. RESULTS: The intramolecular dynamics of the protein backbones of FNfn10 and TNfn3 have been studied by 15N nuclear spin relaxation. The FG loop in FNfn10, which contains the RGD motif, exhibits extensive flexibility on picosecond to nanosecond timescales, but motions on microsecond to millisecond timescales are not observed. The equivalent region in TNfn3 is as rigid as regular elements of secondary structure. The CC' loop also is more flexible on picosecond-nanosecond timescales in FNfn10 than in TNfn3. Conformational exchange, reflecting flexibility on microsecond-millisecond timescales, is observed in beta strands A and B of both FNfn10 and TNfn3. CONCLUSIONS: Comparison of the structures of the FNfn10 and TNfn3 reveals several features related to their different dynamical properties. The larger amplitude motions of loops in FNfn10 are consistent with the hypothesis that flexibility of these regions facilitates induced-fit recognition of fibronectin by multiple receptors. Similarly, the more rigid loops of TNfn3 may reflect greater specificity for particular integrins. The correlations observed between structural features and dynamical properties of the homologous type III domains indicate the influence of hydrogen bonding and hydrophobic packing on dynamical fluctuations in proteins.

Amino Acid Sequence↗
PubMed1995

Correlation of antiviral activity with beta-turn types for V3 synthetic multibranched peptides from HIV-1 gp120.

SPC3 is a synthetic multibranched peptide containing eight HIV-1 gp120 V3 loop GPGRAF motifs. SPC3 inhibits HIV-1 infection in human lymphocytes and macrophages, while the monomer counterpart of SPC3, i.e., the GPGRAF peptide, has no effect. Circular dichroism (CD) of these molecules in phosphate buffer, pH 7, and in a water solution containing 50% trifluoroethanol (TFE) showed significant differences. In TFE, the inactive monomer has a CD spectrum associated to type II beta-turn (class B spectrum), while SPC3 has a class C CD spectrum associated to type I beta-turn. To investigate the structure--function relationship, SPC3 analogs were built in solid-phase synthesis, and their activity and structures were compared to SPC3. Analogs having respectively two and four GPGRAF motifs show that polymerization is associated with these structural changes. Analogs with eight motifs but differing in their sequence show also that the sequence is important to stabilize a type I beta-turn structure. The activity tests of these analogs show a remarkable correlation between the antiviral activity and their ability to exhibit a class C CD spectrum associated to type I beta-turn. Taking in account CD results, a model was made using energy minimization and dynamics, which shows that, for SPC3, a model with motifs in a type I beta-turn structure is favored compared to one with a type II beta-turn. These data suggest that the SPC3 antiviral activity is related to the structure of the GPGRAF motif in the polymer, with special emphasis on the presence of a type I beta-turn structure.

Amino Acid Sequence↗
PubMed1994

The mixotope: a combinatorial peptide library as a T cell and B cell immunogen.

We report a new approach in peptide vaccine strategy based on combinatorial synthesis. A library of 7.5 x 10(5) related peptides, termed mixotope, was derived from the sequence of the third hypervariable domain (V3 loop) of the human immunodeficiency virus (HIV) envelope protein. This preparation induced a strong immune response in all syngeneic and outbred rodents tested. The response directed against the mixotope included antibodies, CD4+ T helper cells (TH1 and TH2) and CD8+ T cells. In rodents immunized with the mixotope, the T cell response directed against individual V3 peptide sequences (BRU, MN, RF, SF2, and ELI) as measured by T cell proliferation and interleukin (IL)-2 production, was found to be major histocompatibility complex haplotype-dependent. However, additional experiments performed in mice indicated that selectivity was less restrictive when using IL-3 secretion to explore T cell activation. This combinatorial antigen could be considered as a series of agretopic motifs framing a multiplicity of closely related epitopes for T cell recognition and able to elicit a T cell and B cell repertoire. This new construct may therefore provide a basis for the design of future vaccine strategies.

AIDS Vaccines↗
PubMed1990

Transcriptional activation domain of the muscle-specific gene-regulatory protein myf5.

The human muscle determination factor myf5, like MyoD and other members of the family of skeletal muscle-specific regulatory proteins, contains a highly conserved putative helix-loop-helix domain. In MyoD this motif is required for the initiation of myogenesis in C3H mouse 10T1/2 fibroblasts and other non-muscle cells as well as for transcriptional activation of muscle genes. High affinity DNA binding of MyoD to regulatory DNA elements in muscle genes requires the formation of heterodimers with ubiquitous helix-loop-helix proteins such as E12 or E47. To investigate the potential of myf5 as a transcription factor, we have fused the GAL4 DNA-binding domain to various parts of the myf5 protein and analysed the transactivation of a GAL4 reporter plasmid. Here we report that myf5 contains an intrinsic transcriptional activation domain which is distinct from the helix-loop-helix motif. The predominant transactivating effect is associated with the C-terminal half of the myf5 molecule. High-affinity sequence-specific DNA binding of myf5 also requires hetero-oligomeric association with the enhancer-binding protein E12 to confer muscle-specific transactivation.

Animals↗
PubMed1998

Dopamine D1B receptor chimeras reveal modulation of partial agonist activity by carboxyl-terminal tail sequences.

NNC 01-0012, a second-generation benzazepine compound, pharmacologically differentiates multiple vertebrate D1 receptor subtypes (D1A, D1B, D1C, and D1D) and displays high selectivity and affinity for dopamine D1C receptors. Functionally, whereas NNC 01-0012 acts as a full or poor antagonist at D1C and D1A receptor-mediated cyclic AMP production, respectively, it exhibits partial agonist activity at the D1B receptor. To define some of the structural motifs that regulate the pharmacological and functional differentiation of vertebrate dopamine D1 receptors by NNC 01-0012, a series of receptor chimeras were constructed in which the divergent carboxyl-terminal (CT) receptor tails were replaced with the corresponding sequences of D1A, D1B, or D1C receptors. Substitution of the vertebrate D1B carboxyl-terminal-tail at position Tyr345 with carboxyl-terminal-tail sequences of the D1A receptor abolished the partial agonist activity of NNC 01-0012 without affecting dopamine-stimulated cyclic AMP accumulation. At vertebrate D1B/D1CcT-tail receptor mutants, however, the intrinsic activity of the partial agonist NNC 01-0012 (10 microM) was markedly enhanced (approximately 60% relative to 10 microM dopamine) with no concomitant alteration in the molecule's ligand binding affinity or constitutive activity of the chimeric receptor. Similar results were obtained with other benzazepines such as SKF-38393 and SCH-23390, which act as partial agonists at vertebrate D1B receptors. Substitution of D1A and D1C receptor carboxyl-terminal tails with sequences encoded by the D1B receptor carboxyl-terminal tail did not, however, produce receptors with functional characteristics significantly different from wild type. Taken together, these data clearly suggest that in addition to well-characterized domains and amino acid residues in the third cytoplasmic loop, partial agonist activity at the D1B receptor is modulated by sequence-specific motifs within the carboxyl-terminal tail, a region that may underlie the possible structural basis for functionally divergent roles of multiple dopamine D1-like receptors.

Animals↗
PubMed2002

Common origin and evolution of glycosyltransferases using Dol-P-monosaccharides as donor substrate.

On the basis of the analysis of 64 glycosyltransferases from 14 species we propose that several successive duplications of a common ancestral gene, followed by divergent evolution, have generated the mannosyltransferases and the glucosyltransferases involved in asparagine-linked glycosylation (ALG) and phosphatidyl-inositol glycan anchor (PIG or GPI), which use lipid-related donor and acceptor substrates. Long and short conserved peptide motifs were found in all enzymes. Conserved and identical amino acid positions were found for the alpha 2/6- and the alpha 3/4-mannosyltransferases and for the alpha 2/3-glucosyltransferases, suggesting unique ancestors for these three superfamilies. The three members of the alpha 2-mannosyltransferase family (ALG9, PIG-B, and SMP3) and the two members of the alpha 3-glucosyltransferase family (ALG6 and ALG8) shared 11 and 30 identical amino acid positions, respectively, suggesting that these enzymes have also originated by duplication and divergent evolution. This model predicts a common genetic origin for ALG and PIG enzymes using dolichyl-phospho-monosaccharide (Dol-P-monosaccharide) donors, which might be related to similar spatial orientation of the hydroxyl acceptors. On the basis of the multiple sequence analysis and the prediction of transmembrane topology we propose that the endoplasmic reticulum glycosyltransferases using Dol-P-monosaccharides as donor substrate have a multispan transmembrane topology with a first large luminal conserved loop containing the long motif and a small cytosolic conserved loop containing the short motif, different from the classical type II glycosyltransferases, which are anchored in the Golgi by a single transmembrane domain.

Amino Acid Motifs↗
PubMed1995

Probing the solution structure of the DNA-binding protein Max by a combination of proteolysis and mass spectrometry.

A simple biochemical method that combines enzymatic proteolysis and matrix-assisted laser desorption ionization mass spectrometry was developed to probe the solution structure of DNA-binding proteins. The method is based on inferring structural information from determinations of protection against enzymatic proteolysis, as governed by solvent accessibility and protein flexibility. This approach was applied to the study of the transcription factor Max--a member of the basic/helix-loop-helix/zipper family of DNA-binding proteins. In the absence of DNA and at low ionic strengths, Max is rapidly digested by each of six endoproteases selected for the study, results consistent with an open and flexible structure of the protein. At physiological salt levels, the rates of digestion are moderately slowed; this and the patterns of cleavage are consistent with homodimerization of the protein through a predominantly hydrophobic interface. In the presence of Max-specific DNA, the protein becomes dramatically protected against proteolysis, exhibiting up to a 100-fold reduction in cleavage rates. Over a 2-day period, both complete and partial proteolysis of the Max-DNA complex is observed. The partial proteolytic fragmentation patterns reflect a very high degree of protection in the N-terminal and helix-loop-helix regions of the protein, correlating with those expected of a stable dimer bound to DNA at its basic N-terminals. Less protection is seen at the C-terminal where a slow, sequential proteolytic cleavage occurs, correlating to the presence of a leucine zipper. The results also indicate a high affinity of Max for its target DNA that remains high even when the leucine zipper is proteolytically removed. In addition to the study of the helix-loop-helix protein Max, the present method appears well suited for a range of other structural biological applications.

Amino Acid Sequence↗
PubMed1996

Molecular cloning of a myoD-like gene from the parasitic nematode, Trichinella spiralis.

The infective larval stage of the nematode Trichinella spiralis is an intracellular parasite of skeletal muscle cells. Infection with T. spiralis results in dedifferentiation of the host cell and the formation of a host/parasite complex. A gene encoding a T. spiralis Helix-Loop-helix (HLH) protein with homology to the myogenic transcription factor, MyoD, and to the Caenorhabditis elegans protein, CeMyoD, has been identified and partially characterized. The tsmyd-1 gene is expressed constitutively during the muscle-larval and adult stages. A purified recombinant Tsmyd-1 protein, expressed in Escherichia coli, binds to a high affinity mouse MyoD DNA binding site in vitro. The present study describes the first HLH gene to be identified in Trichinella.

Amino Acid Sequence↗
PubMed2004

NMR structures of loop B RNAs from the stem-loop IV domain of the enterovirus internal ribosome entry site: a single C to U substitution drastically changes the shape and flexibility of RNA.

The 5'-untranslated region of positive-strand RNA viruses harbors many cis-acting RNA structural elements that are important for various viral processes such as replication, translation, and packaging of new virions. Among these is loop B RNA of the stem-loop IV domain within the internal ribosomal entry site (IRES) of enteroviruses, including Poliovirus type 1 (PV1). Studies on PV1 have shown that specific recognition of loop B by the first KH (hnRNP K homology) domain of cellular poly(rC)-binding protein 2 (PCBP2) is essential for efficient translation of the viral mRNA. Here we report the NMR solution structures of two representative sequence variants of enteroviral loop B RNA. The two RNA variants differ at only one position (C vs U) within a six-nucleotide asymmetric internal loop sequence that is the binding site for the PCBP2 KH1 domain. Surprisingly, the two RNAs are drastically different in the overall shape and local dynamics of the bulge region. The RNA with the 5'-AUCCCU bulge sequence adopts an overall L shape. Its bulge nucleotides, especially the last four, are highly flexible and not very well defined by NMR. The RNA with the 5'-AUUCCU bulge sequence adopts an overall U shape, and its bulge sequence exhibits only limited flexibility. A detailed analysis of the two RNA structures and their dynamic properties, as well as available sequence data and known KH domain-RNA complex structures, not only provides insights into how loop B RNA might be recognized by the PCBP2 KH1 domain but also suggests a possible correlation between structural flexibility and pre-existing structural features for protein recognition.

5' Untranslated Regions↗
PubMed1996

E-box sequence and context-dependent TAL1/SCL modulation of basic helix-loop-helix protein-mediated transcriptional activation.

TAL1/SCL is a basic helix-loop-helix (bHLH) oncoprotein that is expressed in several cell lines including many hematolymphoid cells, but not in T- and B-lineage cells. The TAL1 gene was originally discovered as being transcriptionally activated by chromosomal rearrangements in T-cell acute lymphoblastic leukemia (T-ALL). Here we have shown that TAL1 and the ubiquitously expressed murine bHLH transcription factor ALF1 formed heterodimers that, compared with ALF1 homodimers, had a more restricted E-box specificity and bound preferentially to the glucocorticoid-responsive E-box (Egre) motif (AACAGATGGT). Overexpression of the dominant inhibitory HLH protein Id1 in NIH3T3 cells reduced the transcriptional activity mediated by ALF1 homodimers, whereas the transcriptional activity mediated by TAL1/ALF1 heterodimers was resistant to Id overexpression. Our results show that ALF1 may serve as a dimerization partner for the bHLH oncoprotein TAL1 and form a complex with a distinctive DNA binding property. These findings support the hypothesis that the leukemic characteristics of the TAL1 oncoprotein could be mediated by activation of a set of target genes as heterodimeric complexes with ubiquitously expressed bHLH transcription factors such as ALF1 and that a principal role of TAL1 might be to neutralize an Id-mediated inactivation.

3T3 Cells↗
PubMed1993

Identification and cloning of the CHL4 gene controlling chromosome segregation in yeast.

A collection of chl mutants characterized by decreased fidelity of chromosome transmission and by minichromosome nondisjunction in mitosis was examined for the ability to maintain nonessential dicentric plasmids. In one of the seven mutants analyzed, chl4, dicentric plasmids did not depress cell division. Moreover, nonessential dicentric plasmids were maintained stably without any rearrangements during many generations in the chl4 mutant. The rate of mitotic heteroallelic recombination in the chl4 mutant was not increased compared to that in an isogenic wild-type strain. Analysis of the segregation of a marked chromosome indicated that sister chromatid nondisjunction and sister chromatid loss contributed equally to chromosome malsegregation in the chl4 mutant. A genomic clone of CHL4 was isolated by complementation of the chl4-1 mutation and was physically mapped to the right arm of chromosome IV near the SUP2 gene. Nucleotide sequence analysis of CHL4 clone revealed a 1.4-kb open reading frame coding for a 53-kD predicted protein which does not have homology to published proteins. A strain containing a null allele of CHL4 is viable under standard growth conditions but has a temperature-sensitive phenotype (conditional lethality at 36 degrees). We suggest that the CHL4 gene is required for kinetochore function in the yeast Saccharomyces cerevisiae.

Amino Acid Sequence↗
PubMed1996

E2A basic-helix-loop-helix transcription factors are negatively regulated by serum growth factors and by the Id3 protein.

Id3, a member of the Id multigene family of dominant negative helix-loop-helix transcription factors, is induced sharply in murine fibroblasts by serum growth factors. To identify relevant targets of Id3 activity, the yeast two-hybrid system was used to identify proteins that dimerize with Id3. Four murine cDNAs were identified in the screen, all of which encode helix-loop-helix proteins: E12, E47, ALF1 and Id4. Co-immunoprecipitation assays confirm that Id3 interacts with E12, E47 and two alternative splice products of ALF1 in vitro. Id3 disrupts DNA binding by these proteins in vitro and blocks transcriptional activation by these factors in cultured murine cells. Additionally, Id3 shows evidence of interacting with the related proteins E2-2 and MyoD, but not c-Myc. These results suggest that Id3 can function as a general negative regulator of the basic-helix-loop-helix family of transcription factors exemplified by the 'E' proteins and MyoD. Although it was previously suspected that E2A is constitutively expressed, our data indicate that E2A is induced in quiescent fibroblasts, by growth factor withdrawal but not by contact inhibition of cell proliferation. These observations extend the role of Id3 in the functional antagonism of E2A-class transcription factors, and suggest that E2A proteins may mediate growth inhibition.

3T3 Cells↗
PubMed2000

A tetratricopeptide repeat half-site in the aryl hydrocarbon receptor is important for DNA binding and trans-activation potential.

Similar to certain unliganded steroid hormone receptor complexes, the unliganded aryl hydrocarbon receptor has been shown to consist of a multimeric core complex that includes the 90-kDa heat shock protein (hsp90) and the immunophilin-like hepatitis B X-associated protein 2 (XAP2). Immunophilins and XAP2 associated with these complexes bind to the carboxyl-terminal end of hsp90 through an interaction with their tetratricopeptide repeat (TPR) domains. The consensus TPR binding motif contains two domains, A and B. Recently, the carboxyl terminus of XAP2 has been shown to contain a highly conserved TPR domain that is required for the assembly of XAP2 with both hsp90 and AhR. A search of the murine AhR sequence identified domain B (A-F-A-P) of the consensus TPR sequence directly adjacent to the carboxyl-terminal side of the helix-loop-helix region of the murine and human AhR. We hypothesized that this conserved domain B region may be involved with mediating interactions between either AhR-hsp90, AhR-XAP2, and/or AhR-AhR nuclear translocator protein. Site-directed mutagenesis of the amino-terminal alanine residue of this region to an aspartic acid (A78D) completely inhibited 2,3,7, 8-tetrachloro-p-dioxin (TCDD) -dependent activation of a xenobiotic response element (XRE) driven gene expression construct in transfected COS-1 and BP8 cells. The A82F mutation caused a 40 to 50% decrease in TCDD-dependent activation. The inability of A78D and the reduction of A82F to trans-activate XRE-driven reporter activity did not result from impaired AhR-XAP2-hsp90 interactions, TCDD-dependent AhR translocation to the nucleus, or AhR-AhR nuclear translocator protein interactions. In vitro DNA binding analysis demonstrated that loss of trans-activation potential by the A78D mutation resulted from impaired XRE binding. This study underscores the potential importance of AhR mutations that occur naturally outside of known functional domains.

Amino Acid Sequence↗
PubMed1999

Silencing of the Epstein-Barr virus latent membrane protein 1 gene by the Max-Mad1-mSin3A modulator of chromatin structure.

The tumor-associated latent membrane protein 1 (LMP1) gene in the Epstein-Barr virus (EBV) genome is activated by EBV-encoded proteins and cellular factors that are part of general signal transduction pathways. As previously demonstrated, the proximal region of the LMP1 promoter regulatory sequence (LRS) contains a negative cis element with a major role in EBNA2-mediated regulation of LMP1 gene expression in B cells. Here, we show that this silencing activity overlaps with a transcriptional enhancer in an LRS sequence that contains an E-box-homologous motif. Mutation of the putative repressor binding site relieved the repression both in a promoter-proximal context and in a complete LRS context, indicating a functional role of the repressor. Gel retardation assays showed that members of the basic helix-loop-helix transcription factor family, including Max, Mad1, USF, E12, and E47, and the corepressor mSin3A bound to the E-box-containing sequence. The enhancer activity correlated with the binding of USF. Moreover, the activity of the LMP1 promoter in reporter constructs was upregulated by overexpression of USF1 and USF2a, and the transactivation was inhibited by the concurrent expression of Max and Mad1. This suggests that Max-Mad1-mediated anchorage of a multiprotein complex including mSin3A and histone deacetylases to the E-box site constitutes the basis for the repression. Removal of acetyl moieties from histones H3 and H4 should result in a chromatin structure that is inaccessible to transcription factors. Accordingly, inhibition of deacetylase activity with trichostatin A induced expression of the endogenous LMP1 gene in EBV-transformed cells.

Base Sequence↗