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PubMed1996

A novel repressor, par-4, modulates transcription and growth suppression functions of the Wilms' tumor suppressor WT1.

The tumor suppressor WT1 represses and activates transcription. The loss and/or imbalance of the dual transcriptional activity of WT1 may contribute to Wilms' tumor. In this study, we identified par-4 (for prostate apoptosis response) as a WT1-interacting protein that itself functions as a transcriptional repressor. par-4 contains a putative leucine zipper domain and is specifically upregulated during apoptosis of prostate cells (S. F. Sells, D. P. Wood, Jr., S. S. Joshi-Barve, S. Muthukkumar, R. J. Jacob, S. A. Crist, S. Humphreys, and V. M. Rangnekar, Cell Growth Differ. 5:457-466, 1994). The leucine repeat domain of par-4 was shown to interact with the zinc finger DNA binding domain of WT1. Immunoprecipitation-Western blot (immunoblot) analyses demonstrated in vivo WT1-par-4 interactions. par-4 was ubiquitously expressed, and the protein was found in both the nucleus and the cytoplasm. Functionally, par-4 inhibited transcription activated by WT1, but not by the related protein EGR1. Inhibition of WT1-mediated transcription was dependent on the domain of par-4 that mediates its physical association with WT1. In addition, par-4 augmented WT1-mediated repression, possibly by contributing an additional repression domain. Consistent with these results, par-4 functioned as a transcriptional repressor when brought to a promoter via a heterologous DNA binding domain. Significantly, par-4, but not a mutant unable to interact with WT1, rescued growth suppression caused by WT1. Thus, we identified a novel repressor that modulates transcription as well as growth suppression functions of WT1.

Amino Acid Sequence↗
PubMed1990

Control of the initiation of homeotic gene expression by the gap genes giant and tailless in Drosophila.

The process of segmentation in Drosophila is controlled by both maternal and zygotic genes. Members of the gap class of segmentation genes play a key role in this process by interpreting maternal information and controlling the expression of pair-rule and homeotic genes. We have analyzed the pattern of expression of a variety of homeotic, pair-rule, and gap genes in tailless and giant gap mutants. tailless acts in two domains, one anterodorsal and one posterior. In its anterior domain tailless exerts a repressive effect on the expression of fushi tarazu, hunchback, and Deformed. In its posterior domain of action, tailless is responsible for the establishment of Abdominal-B expression and demarcating the posterior boundary of the initial domain of expression of Ultrabithorax. giant is an early zygotic regulator of the gap gene hunchback: in giant- embryos, alterations in the anterior domain of hunchback expression are visible by the beginning of cycle 14. giant also regulates the establishment of the expression patterns of Antennapedia and Abdominal-B. In particular, giant is the factor that controls the anterior limit of early Antennapedia expression.

Animals↗
PubMed1999

The PUMILIO-RNA interaction: a single RNA-binding domain monomer recognizes a bipartite target sequence.

Translational repression of hunchback (hb) mRNA in the posterior of the Drosophila embryo requires two copies of a bipartite sequence, the Nanos Response Element (NRE), located in the 3' untranslated region of the mRNA. The PUMILIO (PUM) protein is thought to bind the NREs and thereby repress hb translation. The RNA-binding domain of PUM defines an evolutionarily conserved family of RNA-binding proteins, the PUM-Homology Domain (PUM-HD) proteins, which have been identified in yeast, plants, and animals. The PUM RNA-binding domain, the Drosophila PUM-HD (DmPUM-HD), has been shown previously to recognize nucleotides in both the 5' and 3' halves of the NRE, suggesting that a dimer of PUM might recognize one NRE. Here, we analyze the RNA-binding affinity and stoichiometry of the DmPUM-HD and find that one DmPUM-HD monomer binds independently and with equal affinity to each NRE (KD approximately 0.5 nM). We detect no cooperative interactions between DmPUM-HD monomers bound at adjacent sites. Our results imply that a single DmPUM-HD protein recognizes nucleotides in both the 5' and 3' NRE half-sites. Based on our estimate of the intraembryonic concentration of PUM (>40 nM), we propose that in vivo nearly all NREs are occupied by a PUM monomer.

Animals↗
PubMed1993

Regulation of c-Src tyrosine kinase activity by the Src SH2 domain.

The protein-tyrosine kinase activity of pp60c-src (c-Src) is inhibited by phosphorylation of tyr527, within the c-Src c-terminal tail. Genetic and biochemical data have suggested that this negative regulation requires an intact Src homology 2 (SH2) domain. Since SH2 domains recognize phosphotyrosine, it is possible that these two non-catalytic domains associate, and thereby repress c-Src kinase activity. Consistent with this model, an isolated Src SH2 domain expressed in bacteria as a GST fusion protein bound in vitro to a synthetic phosphotyrosine-containing peptide modeled on the C-terminal 13 residues of the c-Src tail. Binding was absolutely dependent on phosphorylation of tyr527 in the tail peptide, and was modified by both the length and sequence of the peptide. Competition experiments indicated only a moderate binding affinity between the Src SH2 domain and the phosphorylated tail. A distinct phosphotyrosine-containing peptide previously identified as binding the Src SH2 domain with high affinity stimulated c-Src tyrosine kinase activity in vitro, possibly by competing with the endogenous tail phosphorylation site for binding to the SH2 domain. Indeed, this activation was competitively inhibited by purified bacterial Src SH2 domain. These data provide direct evidence that the c-Src tail has an intrinsic affinity for the Src SH2 domain, and suggest that such an interaction in the intact molecule contributes to maintaining c-Src in an inactive form.

3T3 Cells↗
PubMed1996

Translational repression dependent on the interaction of the Xenopus Y-box protein FRGY2 with mRNA. Role of the cold shock domain, tail domain, and selective RNA sequence recognition.

We have examined the determinants of the translational repression of mRNA by the Xenopus oocyte-specific Y-box protein FRGY2 using in vitro and in vivo assays. In vitro reconstitution of messenger ribonucleoprotein (mRNP) complexes demonstrates that the sequence-specific RNA-binding cold shock domain is not required for translational repression, whereas the RNA-binding C-terminal tail domain is essential. However, microinjection of reconstituted mRNPs into Xenopus oocytes demonstrates that although translational repression occurs in the absence of consensus RNA binding sequences for FRGY2, the presence of FRGY2 recognition elements within mRNA potentiates translational repression. Analysis of the in vivo assembly of mRNP shows that the cold shock domain alone is not stably incorporated into mRNP, whereas the C-terminal tail domain is sufficient for stable incorporation. We suggest that translational repression of mRNA by FRGY2 is favored by sequence-selective recognition of RNA sequences by the cold shock domain. However, translational repression in vitro and the assembly of mRNP in vivo requires the relatively nonspecific interaction of the C-terminal tail domain with mRNA. Thus two distinct domains of FRGY2 are likely to contribute to translational control.

Animals↗
PubMed2002

A Krüppel-associated box-zinc finger protein, NT2, represses cell-type-specific promoter activity of the alpha 2(XI) collagen gene.

Type XI collagen is composed of three chains, alpha 1(XI), alpha 2(XI), and alpha 3(XI), and plays a critical role in the formation of cartilage collagen fibrils and in skeletal morphogenesis. It was previously reported that the -530-bp promoter segment of the alpha 2(XI) collagen gene (Col11a2) was sufficient for cartilage-specific expression and that a 24-bp sequence from this segment was able to switch promoter activity from neural tissues to cartilage in transgenic mice when this sequence was placed in the heterologous neurofilament light gene (NFL) promoter. To identify a protein factor that bound to the 24-bp sequence of the Col11a2 promoter, we screened a mouse limb bud cDNA expression library in the yeast one-hybrid screening system and obtained the cDNA clone NT2. Sequence analysis revealed that NT2 is a zinc finger protein consisting of a Krüppel-associated box (KRAB) and is a homologue of human FPM315, which was previously isolated by random cloning and sequencing. The KRAB domain has been found in a number of zinc finger proteins and implicated as a transcriptional repression domain, although few target genes for KRAB-containing zinc finger proteins has been identified. Here, we demonstrate that NT2 functions as a negative regulator of Col11a2. In situ hybridization analysis of developing mouse cartilage showed that NT2 mRNA is highly expressed by hypertrophic chondrocytes but is minimally expressed by resting and proliferating chondrocytes, in an inverse correlation with the expression patterns of Col11a2. Gel shift assays showed that NT2 bound a specific sequence within the 24-bp site of the Col11a2 promoter. We found that Col11a2 promoter activity was inhibited by transfection of the NT2 expression vector in RSC cells, a chondrosarcoma cell line. The expression vector for mutant NT2 lacking the KRAB domain failed to inhibit Col11a2 promoter activity. These results demonstrate that KRAB-zinc finger protein NT2 inhibits transcription of its physiological target gene, suggesting a novel regulatory mechanism of cartilage-specific expression of Col11a2.

Amino Acid Sequence↗
PubMed1998

The Pumilio RNA-binding domain is also a translational regulator.

Posterior patterning in the Drosophila embryo requires the action of Nanos (Nos) and Pumilio (Pum), which collaborate to regulate the translation of maternal hunchback (hb) mRNA. Previous work demonstrated that Pum recognizes sites in the 3' UTR of hb mRNA. In this report, we first define the RNA-binding domain of Pum and then show that residues essential for translational repression are embedded within this domain. We also show that Nos and Pum can repress cap-independent translation from an internal ribosome entry site (IRES) in vivo, suggesting that they act downstream of the initial steps of normal, cap-dependent translation.

Amino Acid Sequence↗
PubMed2003

A novel domain within the DEAD-box protein DP103 is essential for transcriptional repression and helicase activity.

Members of the DEAD-box family of helicases, distinguished by a core characteristic sequence of Asp-Glu-Ala-Asp, are expressed in a wide range of prokaryotes and eukaryotes and exhibit diverse cellular functions, including DNA transcription, recombination and repair, RNA processing, translation, and posttranslational regulation. Although ubiquitous, the function of most DEAD-box proteins is unknown. We and others have recently cloned DP103, which harbors conserved DEAD-box, helicase, and ATPase domains in its N terminus. DP103 (also termed Gemin3 and DDX20) interacts with SF-1, SMN, EBNA2, and EBNA3C in mammalian cells. Here we demonstrate that a discrete domain within the nonconserved C-terminal region of DP103 directly interacts with SF-1. This domain exhibits an autonomous repression function and is necessary and sufficient for repressing the transcriptional activity of SF-1. Furthermore, intact DP103 exhibits helicase activity. Importantly, the C-terminal domain is obligatory but not sufficient for this unwinding activity of DP103. Together, our results support a novel paradigm for transcriptional repression and demonstrate the bifunctional role of the C-terminal domain of DP103.

Animals↗
PubMed2002

Methylation-dependent silencing at the H19 imprinting control region by MeCP2.

Methylation of CpG dinucleotides is correlated with transcriptional repression of genes, including imprinted genes. In the case of the imprinted H19 gene, a 2 kb imprinting control region (ICR) is subject to differential methylation, as it is methylated only on the silenced paternal allele. This region has previously been shown to act as a silencer element at the endogenous locus. The proteins that bind at the H19 differentially methylated domain (DMD) and mediate transcriptional silencing have yet to be identified, although a family of proteins containing a methyl-CpG-binding domain (MBD), of which MeCP2 is the best characterised, are obvious candidates. MeCP2 can bind to a single methylated CpG dinucleotide through its MBD and also contains a transcriptional repression domain (TRD). The TRD interacts with Sin3a and histone deacetylases (HDACs) in vivo, forming a repressive complex. Here we show that MeCP2 is recruited to the H19 DMD in vivo and can silence a reporter gene regulated by the H19 DMD in a methylation-dependent manner. This repression can be alleviated by deletion of the TRD from MeCP2 or by inhibition of HDAC activity. These data indicate that transcriptional silencing from the H19 ICR involves recruitment of MeCP2 and presumably an associated protein complex with deacetylase activity. This complex may also be recruited to the ICR in vivo, resulting in a compact, repressive chromatin structure capable of silencing the paternal H19 allele.

Animals↗
PubMed2003

The intracellular domain of teneurin-2 has a nuclear function and represses zic-1-mediated transcription.

Teneurin-2, a vertebrate homologue of the Drosophila pair-rule gene ten-m/odz, is revealed to be a membrane-bound transcription regulator. In the nucleus, the intracellular domain of teneurin-2 colocalizes with promyelocytic leukemia (PML) protein in nuclear bodies implicated in transcription control. Since Drosophila ten-m acts epistatically to another pair-rule gene opa, we investigated whether gene regulation by the mammalian opa homologue zic-1 was influenced by the intracellular domain of teneurin-2. We found that zic-mediated transcription from the apolipoprotein E promoter was inhibited. Release of the intracellular domain of teneurin-2 could be stimulated by homophilic interaction of the extracellular domain, and the intracellular domain was stabilized by proteasome inhibitors. We have previously shown that teneurin-2 is expressed by neurons belonging to the same functional circuit. Therefore, we hypothesize that homophilic interaction enables neurons to identify their targets and that the release of the intracellular domain of teneurin-2 provides them with a signal to switch their gene expression program from growth towards differentiation once the proper contact has been made.

Acetylcysteine↗
PubMed1999

A variety of RNA polymerases II and III-dependent promoter classes is repressed by factors containing the Krüppel-associated/finger preceding box of zinc finger proteins.

KRAB/FPB (Krüppel-associated/finger preceding box) domains are small, portable transcriptional repression motifs, encoded by hundreds of vertebrates C2-H2-type zinc finger genes. We report that KRAB/FPB domains feature an unprecedented, highly promiscuous DNA-binding dependent transcriptional repressing activity. Indeed, template bound chimeric factors containing KRAB/FPB modules actively repress in vivo the transcription of distinct promoter classes that depend on different core elements, recruit distinct basal transcriptional apparatuses and are transcribed either by RNA polymerase II or III. The promoter types repressed in transient assays in a dose- and DNA-binding dependent, but position- and orientation-independent manner, by GAL4-KRAB/FPB fusions include an RNA polymerase II-dependent small nuclear RNA promoter (U1) as well as RNA polymerase III-dependent class 2 (adenovirus VA1), class 3 (human U6) and atypical (human 7SL) promoters. Down-modulation of all of these templates depended on factors containing the A module of the KRAB/FPB domain. Data provide further insights into the properties and mode of action of this widespread repression motif, and support the notion that genes belonging to distinct classes may be repressed in vivo by KRAB/FPB containing zinc finger proteins. The exquisitely DNA-binding dependent transcriptional promiscuity exhibited by KRAB/FPB domains may provide a unique model system for studying the mechanism by which a promoter recruited repression motif can down-modulate a large variety of promoter types.

Animals↗
PubMed1993

Csk inhibition of c-Src activity requires both the SH2 and SH3 domains of Src.

The protein tyrosine kinase c-Src is negatively regulated by phosphorylation of Tyr527 in its carboxy-terminal tail. A kinase that phosphorylates Tyr527, called Csk, has recently been identified. We expressed c-Src in yeast to test the role of the SH2 and SH3 domains of Src in the negative regulation exerted by Tyr527 phosphorylation. Inducible expression of c-Src in Schizosaccharomyces pombe caused cell death. Co-expression of Csk counteracted this effect. Src proteins mutated in either the SH2 or SH3 domain were as lethal as wild type c-Src, but were insensitive to Csk, even though they were substrates for Csk in vivo. Peptide binding experiments revealed that Src proteins with mutant SH3 domains adopted a conformation in which the SH2 domain was not interacting with the tail. These data support the model of an SH2 domain-phosphorylated tail interaction repressing c-Src activity, but expand it to include a role for the SH3 domain. We propose that the SH3 domain contributes to the maintenance of the folded, inactive configuration of the Src molecule by stabilizing the SH2 domain-phosphorylated tail interaction. Moreover, the system we describe here allows for further study of the regulation of tyrosine kinases in a neutral background and in an organism amenable to genetic analysis.

Amino Acid Sequence↗
PubMed1995

Inserting the Ftz homeodomain into engrailed creates a dominant transcriptional repressor that specifically turns off Ftz target genes in vivo.

The Engrailed homeodomain protein is an 'active' or dominant transcriptional repressor in cultured cells. In contrast, the Fushi Tarazu homeodomain protein is an activator, both in cultured cells and in Drosophila embryos, where it activates several known target genes, including its own gene. This auto-activation has been shown to depend on targeting to a fushi tarazu enhancer by the Fushi Tarazu homeodomain. We combined Fushi Tarazu targeting and Engrailed active repression in a chimeric regulator, EFE. When EFE is ubiquitously expressed, it overrides endogenous Fushi Tarazu and causes a fushi tarazu mutant phenotype. Normal Fushi Tarazu target genes are affected as they are in fushi tarazu mutants. One such target gene is repressed by EFE even where Fushi Tarazu is not expressed, suggesting that the repression is active. This is confirmed by showing that the in vivo activity of EFE depends on a domain that is required for active repression in culture. A derivative that lacks this domain, while it cannot repress the endogenous fushi tarazu gene, can still reduce the activity of the fushi tarazu autoregulatory enhancer, suggesting that it competes with endogenous Fushi Tarazu for binding sites in vivo. However, this passive repression is much less effective than active repression.

Animals↗
PubMed1996

Mouse Sin3A interacts with and can functionally substitute for the amino-terminal repression of the Myc antagonist Mxi1.

Mxi1 is a basic region helix-loop-helix leucine zipper (bHLH/LZ) protein that, in association with Max, antagonizes Myc oncogenic activities. A possible mechanistic basis for Mxi1-mediated repression was provided by the recent demonstration that the repressive potential of Mxi1 correlates with its ability to physically associate with mSin3B, one of two mammalian homologues of the yeast transcriptional repressor SIN3. Here, we sought to characterize more fully the physical properties of the second homologue, mSin3A and to determine whether the recruitment of mSin3A by Mxi1 is indeed required for anti-Myc activity. Transient transfection of mammalian cells showed that the mSin3A protein can associate with the strong repressive isoform of Mxi1 (Mxi1-SR) and that, like other Myc superfamily members, both mSin3A and Mxi1-SR localize to the nucleus. From a developmental standpoint, a comparative analysis of Myc, Mxi1-SR and Sin3A expression during postnatal mouse development and in differentiating mouse erythroleukemia (MEL) cells revealed that dramatic and reciprocal changes in Myc and Mxi1-SR mRNA levels are accompanied by minimal stage-specific changes in mSin3A gene expression. This constant expression profile, coupled with the observation that over-expression of mSin3A does not augment the anti-Myc activity of Mxi1-SR in the rat embryo fibroblast (REF) transformation assay, suggests that mSin3A is not a limiting factor in the regulation of Myc superfamily function. Finally, a mSin3A-Mxi1 fusion protein, in which the amino terminal mSin3-interacting domain of Mxi1-SR was replaced with the full-length mSin3A, exhibited a level of repression activity equivalent to, or greater than, the level of repression obtained with Mxi1-SR. Taken together, these observations directly demonstrate that the amino-terminal repression domain of Mxi1-SR functions solely to recruit mSin3A and possibly other proteins like mSin3A and this association is necessary for the anti-Myc activity of Mxi1-SR.

Amino Acid Sequence↗
PubMed2001

Definition of a dioxin receptor mutant that is a constitutive activator of transcription: delineation of overlapping repression and ligand binding functions within the PAS domain.

The intracellular dioxin (aryl hydrocarbon) receptor is a ligand-activated transcription factor that mediates the adaptive and toxic responses to environmental pollutants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and structurally related congeners. Whereas the ligand-free receptor is characterized by its association with the molecular chaperone hsp90, exposure to ligand initiates a multistep activation process involving nuclear translocation, dissociation from the hsp90 complex, and dimerization with its partner protein Arnt. In this study, we have characterized a dioxin receptor deletion mutant lacking the minimal ligand-binding domain of the receptor. This mutant did not bind ligand and localized constitutively to the nucleus. However, this protein was functionally inert since it failed to dimerize with Arnt and to bind DNA. In contrast, a dioxin receptor deletion mutant lacking the minimal PAS B motif but maintaining the N-terminal half of the ligand-binding domain showed constitutive dimerization with Arnt, bound DNA, and activated transcription in a ligand-independent manner. Interestingly, this mutant showed a more potent functional activity than the dioxin-activated wild-type receptor in several different cell lines. In conclusion, the constitutively active dioxin receptor may provide an important mechanistic tool to investigate receptor-mediated regulatory pathways in closer detail.

Animals↗
PubMed2001

runt homology domain transcription factors (Runx, Cbfa, and AML) mediate repression of the bone sialoprotein promoter: evidence for promoter context-dependent activity of Cbfa proteins.

Expression of the bone sialoprotein (BSP) gene, a marker of bone formation, is largely restricted to cells in mineralized tissues. Recent studies have shown that the Cbfa1 (also known as Runx2, AML-3, and PEBP2alphaA) transcription factor supports commitment and differentiation of progenitor cells to hypertrophic chondrocytes and osteoblasts. This study addresses the functional involvement of Cbfa sites in expression of the Gallus BSP gene. Gel mobility shift analyses with nuclear extracts from ROS 17/2.8 osteoblastic cells revealed that multiple Cbfa consensus sequences are functional Cbfa DNA binding sites. Responsiveness of the 1.2-kb Gallus BSP promoter to Cbfa factors Cbfa1, Cbfa2, and Cbfa3 was assayed in osseous and nonosseous cells. Each of the Cbfa factors mediated repression of the wild-type BSP promoter, in contrast to their well known activation of various hematopoietic and skeletal phenotypic genes. Suppression of BSP by Cbfa factors was not observed in BSP promoters in which Cbfa sites were deleted or mutated. Expression of the endogenous BSP gene in Gallus osteoblasts was similarly downregulated by forced expression of Cbfa factors. Our data indicate that Cbfa repression of the BSP promoter does not involve the transducin-like enhancer (TLE) proteins. Neither coexpression of TLE1 or TLE2 nor the absence of the TLE interaction motif of Cbfa1 (amino acids 501 to 513) influenced repressor activity. However, removal of the C terminus of Cbfa1 (amino acids 362 to 513) relieved suppression of the BSP promoter. Our results, together with the evolutionary conservation of the seven Cbfa sites in the Gallus and human BSP promoters, suggest that suppressor activity by Cbfa is of significant physiologic consequence and may contribute to spatiotemporal expression of BSP during bone development.

Animals↗
PubMed2005

LvGroucho and nuclear beta-catenin functionally compete for Tcf binding to influence activation of the endomesoderm gene regulatory network in the sea urchin embryo.

In the sea urchin embryo, specification of the endomesoderm is accomplished by the activity of a network of regulatory genes in the vegetal hemisphere, called the endomesoderm gene regulatory network (GRN). The activation of this network is mediated primarily through the activity of the Wnt pathway, though details of pathway activation remain unclear. To gain further insight into control of endomesoderm GRN activation, we have identified a sea urchin homologue of the co-repressor Groucho (LvGroucho) that has been shown to antagonize beta-catenin/Tcf activation complexes during Wnt signaling in other systems. Groucho functions by recruiting the histone deacetylase Rpd3 to the DNA template via interaction with site-specific transcription factors, resulting in localized chromatin condensation and transcriptional silencing. Our results show that the LvGroucho protein localizes to all nuclei throughout embryonic development. Interaction assays demonstrate that LvGroucho interacts with Tcf via both the Q and the WD domains of the protein. LvGroucho interacts with Tcf to antagonize the expression of key endomesoderm regulatory genes. Assays demonstrate that LvGroucho and n beta-catenin functionally compete for binding to Tcf as a major mechanism by which the Tcf-control switch is regulated. Functional analysis of the N-terminal AES197 domain of LvGroucho shows that it is sufficient to recapitulate the function of full-length LvGroucho. This finding strongly supports the conclusion that the effects of LvGro overexpression are due primarily to its interactions with Tcf and not other Groucho interacting partners, since Tcf is the only protein present in the sea urchin known to interact with AES197. Because the Q domain is unable to bind Rpd3, it was expected to behave as a dominant negative LvGroucho. Unexpectedly, overexpression of the Q domain gave functional results similar to LvGroucho and the AES197 domain. This is the first evidence for an inherent repressive function for the Q domain alone. Together, our results indicate that LvGroucho functionally competes with beta-catenin for Tcf binding, and this competitive mechanism regulates one of the earliest steps in the initiation of the sea urchin endomesoderm GRN.

Amino Acid Sequence↗
PubMed1996

Two distinct upstream regulatory domains containing multicopy cellular transcription factor binding sites provide basal repression and inducible enhancer characteristics to the immediate-early IES (US3) promoter from human cytomegalovirus.

The US3 gene of human cytomegalovirus (HCMV) is expressed at immediate-early (IE) times in permissive HF cells, but not in nonpermissive rodent cells, and encodes several proteins that have been reported to have regulatory characteristics, although they are dispensable for growth in cell culture. Both spliced and unspliced forms of US3 IE transcripts are associated with the second of only two known large and complex upstream enhancer domains within the 229-kb HCMV genome, which we refer to as the IES cis-acting control region. Only the 260-bp proximal segment (from -313 to -55) of the 600-bp IES control domain, which contains multicopy NF-kappaB binding sites, proved to be necessary to transfer both high basal expression plus phorbol ester- and okadaic acid-inducible characteristics to heterologous promoters in transient assays in U-937 and K-562 cells. However, the IES control region contains a distinctive 280-bp distal domain, characterized by the presence of seven interspersed repeats of a 10-bp TGTCGCGACA palindromic consensus motif that encompasses a NruI site. This far upstream Nru repeat region (from -596 to -314) imparted up to 20-fold down-regulation effects onto strong basal heterologous promoters as well as onto the IES enhancer plus minimal promoter region in both U-937 and K-562 cells. Functional Nru repressor elements (NREs) could not be generated by multimerizing either the palindromic (P) Nru motifs alone or adjacent degenerate interrupted (I Nru motifs alone. However, multimerized forms of the combined P plus I elements reconstituted the full 20-fold cis-acting down-regulation phenotype of the intact NRE domain. The P and I forms of the Nru elements each bound independently and specifically to related cellular DNA-binding factors to form differently migrating A or B complexes, respectively, whereas the combined P plus I elements bound cooperatively to both the A and B complexes with high affinity. Interestingly, nuclear extracts from U-937, K-562, HeLa, and Vero cells all formed both the A and B NRE binding factor complexes, whereas those from HF cells produced only A complexes, and Raji, HL60, and BALB/c 3T3 cells lacked both types of binding factor complexes. The core pentameric CGACA and CGATA half sites present in both the P and I Nru motifs are related to recently described Drosophila chromosomal insulator binding sites. Therefore, in addition to its cis-repression or silencer characteristics, the NRE domain appears likely to act to shield adjacent segments of the viral genome from the chromatin-reorganizing effects of the IES-inducible enhancer. We speculate that differential expression and regulation of the IES enhancer-controlled US3 protein, either in concert with or separately from the major IE (MIE) enhancer-controlled IE1 and IE2 transactivator proteins, may play a critical role in determining HCMV permissiveness in some cell types and perhaps also in the establishment of or reactivation from latency.

Animals↗