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PubMed1999

Recruitment of SMRT/N-CoR-mSin3A-HDAC-repressing complexes is not a general mechanism for BTB/POZ transcriptional repressors: the case of HIC-1 and gammaFBP-B.

Hypermethylated in cancer (HIC-1), a new candidate tumor suppressor gene located in 17p13.3, encodes a protein with five C(2)H(2) zinc fingers and an N-terminal broad complex, tramtrack, and bric à brac/poxviruses and zinc-finger (BTB/POZ) domain found in actin binding proteins or transcriptional regulators involved in chromatin modeling. In the human B cell lymphoma (BCL-6) and promyelocityc leukemia (PLZF) oncoproteins, this domain mediates transcriptional repression through its ability to recruit a silencing mediator of retinoid and thyroid hormone receptor (SMRT)/nuclear receptor corepressor (N-CoR)-mSin3A-histone deacetylase (HDAC) complex, a mechanism shared with numerous transcription factors. HIC-1 appears unique because it contains a 13-aa insertion acquired late in evolution, because it is not found in its avian homologue, gammaF1-binding protein isoform B (gammaFBP-B), a transcriptional repressor of the gammaF-crystallin gene. This insertion, located in a conserved region involved in the dimerization and scaffolding of the BTB/POZ domain, mainly affects slightly the ability of the HIC-1 and gammaFBP-B BTB/POZ domains to homo- and heterodimerize in vivo, as shown by mammalian two-hybrid experiments. Both the HIC-1 and gammaFBP-B BTB/POZ domains behave as autonomous transcriptional repression domains. However, in striking contrast with BCL-6 and PLZF, both HIC-1 and gammaFBP-B similarly fail to interact with members of the HDAC complexes (SMRT/N-CoR, mSin3A or HDAC-1) in vivo and in vitro. In addition, a general and specific inhibitor of HDACs, trichostatin A, did not alleviate the HIC-1- and gammaFBP-B-mediated transcriptional repression, as previously shown for BCL-6. Taken together, our studies show that the recruitment onto target promoters of an HDAC complex is not a general property of transcriptional repressors containing a conserved BTB/POZ domain.

Animals↗
PubMed2003

Active repression of organizer genes by C-terminal domain of PV.1.

PV.1, a homeotic protein, ventralizes dorsal mesoderm and inhibits neuralization by mediating BMP-4 signaling in Xenopus embryo. In our previous report antimorphic PV.1 causes a secondary axis by inducing the ectopic organizer. We analyzed the structure of this transcription factor through domain level assessment. In a phenotype-inducing test, half of the N-terminus at the N-terminal side was unessential for inducing ventralization of embryos. We examined the transacting activity of several regions of PV.1 utilizing GAL4 hybrid system. The C-terminal region/GAL4DBD (DNA binding domain) exhibited strong repressive activity on a reporter gene (operator/promoter/reporter; Gal4-TK-luc) as much as the whole polypeptide/GAL4DBD, whereas the N-terminal region/GAL4DBD showed only modest repression. The results suggest that PV.1 functions as a transcriptional repressor and this repressive activity is localized mostly to the C-terminal region. Additional characterizations of N- and C-terminus with respect to the effects on the expression of other genes are described.

Animals↗
PubMed1991

Activation domains of stably bound GAL4 derivatives alleviate repression of promoters by nucleosomes.

GAL4 derivatives containing an activation domain alleviated repression of a promoter during nucleosome assembly. A GAL4 derivative lacking an activation domain stably bound the promoter during nucleosome assembly but was not sufficient to preserve promoter function. The activation domain of GAL4 derivatives was essential for preserving promoter function, and thus the transcriptional stimulatory activity attributable to these activation domains increased dramatically during nucleosome assembly. Furthermore, promoter-bound activation domains allowed the formation of preinitiation complexes after nucleosome assembly. Finally, GAL4 derivatives containing activation domains significantly stimulated transcription through bacterially produced yeast TFIID only from nucleosome-assembled templates. These data indicate that acidic activation domains stimulate transcription by enhancing the ability of basal transcription factors to compete with nucleosomes for occupancy of the promoter.

Animals↗
PubMed1995

A movable and regulable inactivation function within the central region of a temperature-sensitive p53 mutant.

p53 is the most frequently mutated gene in human cancer. Naturally occurring mutations of p53 are mainly located within a region containing residues 100-300 and are predominantly of missense type, resulting in loss of the protein's DNA binding activity. Here we show that this type of mutation also represses the p53 N-terminal activation domain. The repression activity is localized in the central region of mutant p53 containing residues 101-318. Interestingly, the central region of a temperature-sensitive mutant p53N247I possesses a movable and regulable inactivation function. It represses other activities present on the same polypeptide chain without strict regard to the configuration of that polypeptide only at the nonpermissive temperature (37 degrees C) and not at the permissive temperature (30 degrees C). Furthermore, this mutant p53 region exhibits no other activity, and its function is independent of endogenous p53 status.

Animals↗
PubMed2001

Identification of domains mediating transcription activation, repression, and inhibition in the paired-related homeobox protein, Prx2 (S8).

Despite the growing information concerning the developmental importance of the Prx2 protein, the structural determinants of Prx2 function are poorly understood. To gain insight into the transcription regulatory regions of the Prx2 protein, we generated a series of truncation mutants. Both the Prx2 response element (PRE) and a portion of the tenascin promoter, a downstream target of Prx2, were used as reporters in transient transfection assays. This analysis showed that a conserved domain (PRX), found in both Prx1 and Prx2, activated transcription in NIH 3T3 cells. This PRX domain, as well as other functional regions of Prx2, demonstrated both cell-specific and promoter-dependent transcriptional regulation. A second important region, the OAR (aristaless) domain, which is conserved among 35 Paired-type homeodomain proteins, was observed to inhibit transcription. Deletion of this element resulted in a 20-fold increase of transcription from the tenascin reporter in NIH 3T3 cells but not in C2C12 cells. The OAR domain did not function as a repressor in chimeric fusions with the Gal4 DNA binding domain in either cell type, characterizing it as an inhibitor instead of a repressor. These results give insight into the function of the Prx2 transcription factor while establishing the framework for comparison with the two isoforms of Prx1.

3T3 Cells↗
PubMed2003

Direct association between PU.1 and MeCP2 that recruits mSin3A-HDAC complex for PU.1-mediated transcriptional repression.

PU.1, a member of the Ets family of transcription factors, is implicated in hematopoietic cell differentiation through its interactions with other transcriptional factors and cofactors. To identify a novel protein(s) binding to PU.1, we carried out affinity purification using a column of Glutathione-Sepharose beads bound to GST-PU.1 fusion protein and isolated several individual proteins using murine erythroleukemia (MEL) cell extracts. Sequence analysis of these proteins revealed that one was MeCP2 a methyl CpG binding protein. GST-pull-down assay and immunoprecipitation assay showed that PU.1 bound directly to MeCP2 via its Ets domain and MeCP2 bound to PU.1 via either its amino terminal domain or trans-repression domain. MeCP2 repressed transcriptional activity of PU.1 on a reporter construct with trimerized PU.1 binding sites. This downregulation was recovered in the presence of histone deacetylase inhibitor, trichostatin A (TSA). MeCP2 was integrated in PU.1-mSin3A-HDAC complex but not in PU.1-CBP complex. Chromatin immunoprecipitation (ChIP) assays showed that PU.1 and MeCP2 were collocated at the PU.1 binding site on the reporter construct and the PU.1 binding site of the intervening sequence 2 (IVS2) region in the intron of the beta-globin gene, which has been proposed to regulate expression of the gene, in undifferentiated MEL cells. The complex disappeared from the region during the course of erythroid differentiation of MEL cells. Our results suggest that MeCP2 acts as a corepressor of PU.1 probably due to facilitating complex formation with mSin3A and HDACs.

Cell Differentiation↗
PubMed1979

Interaction between mutant alleles of araC of the Escherichia coli B/r L-arabinose operon.

Strains were constructed that contain mutational alterations affecting two distinct functional domains within the araC gene protein. The araCi (catabolite repression insensitivity) and araCh (catabolite repression hypersensitivity) mutations were used to alter the catabolite repression sensitivity domain, and mutation to D-fucose resistance was used to alter the inducer binding domain. araCh, D-fucose-resistant double mutants never exhibited constitutive ara operon expression, whereas all of the araCi, D-fucose-resistant double mutants did exhibit constitutivity. When L-arabinose was used as an inducer, most of the double mutants exhibited the sensitivity to catabolite repression associated with the araCi or araCh mutation. However, when D-fucose was used as an inducer, changes in sensitivity to catabolite repression were observed that were attributed to interactions between the two protein domains. The roles of catabolite activator protein and araC gene protein in the induction of the araBAD operon were discussed.

Alleles↗
PubMed1996

The regulatory domain of human heat shock factor 1 is sufficient to sense heat stress.

Heat shock factor (HSF) activates transcription in response to cellular stress. Human HSF1 has a central regulatory domain which can repress the activity of its activation domains at the control temperature and render them heat shock inducible. To determine whether the regulatory domain works in tandem with specific features of the HSF1 transcriptional activation domains, we first used deletion and point mutagenesis to define these activation domains. One of the activation domains can be reduced to just 20 amino acids. A GAL4 fusion protein containing the HSF 1 regulatory domain and this 20-amino-acid activation domain is repressed at the control temperature but potently activates transcription in response to heat shock. No specific amino acids in this activation domain are required for response to the regulatory domain; in particular, none of the potentially phosphorylated serine and threonine residues are required for heat induction, implying that heat-induced phosphorylation of the transcriptional activation domains is not required for induction. The regulatory domain is able to confer heat responsiveness to an otherwise completely heterologous chimeric activator that contains a portion of the VP16 activation domain, suggesting that the regulatory domain can sense heat in the absence of other portions of HSF1.

Amino Acid Sequence↗
PubMed1993

Regional repression of a Drosophila POU box gene in the endoderm involves inductive interactions between germ layers.

An induction process occurring between the mesodermal and the endodermal germ layers has recently been described in the regulation of the Drosophila homeotic gene labial (lab). We report here that proper spatial regulation of the Drosophila POU box gene pdm-1 products also involves interaction between these two germ layers. pdm-1 transcripts are initially present in both the anterior and the posterior endodermal midgut primordia. Upon fusion of the two primordia, transcripts disappear from two regions in the endoderm, a central domain and an anterior domain. The anterior repression domain of pdm-1 is independent of the expression of known homeotic genes and genes encoding secreted signalling molecules in the visceral mesoderm, both for its positioning and its repression. Repression in the central domain requires both the homeotic gene Ultrabithorax (Ubx) and the decapentaplegic (dpp) gene, which encodes a secreted protein. Both of these genes are also required for lab induction. However, the analysis of pdm-1 expression in various mutant backgrounds indicates that the regulation of lab and pdm-1 across germ layers is controlled by different genetic cascades. Our study indicates that dpp is not the signal that dictates central pdm-1 repression across germ layers and suggests that in the same midgut region, different signalling pathways result in the differential activation or repression of potential transcription factors.

Animals↗
PubMed2005

Conditional UAS-targeted repression in Drosophila.

The Gal4-UAS enhancer trap system is useful for driving gene expression in various tissues. A new tool that extends Gal4 technology is described here. A fusion protein containing the Gal4 binding domain and the repression domain of the isolator suppressor of hairy wing was placed under the control of a heat shock-inducible promoter. The construct mediates the conditional repression of genes located downstream of a UAS sequence. The repressive effects of the chimeric protein on fasII gene expression were tested by western-blot analysis and in brain sections of adult Drosophila. Owing to the increasing number of Gal4 and UAS transgenic lines, this versatile system will facilitate the study of gene function.

Animals↗
PubMed2004

Imprinting along the Kcnq1 domain on mouse chromosome 7 involves repressive histone methylation and recruitment of Polycomb group complexes.

Imprinted genes are clustered in domains, and their allelic repression is mediated by imprinting control regions. These imprinting control regions are marked by DNA methylation, which is essential to maintain imprinting in the embryo. To explore how imprinting is regulated in placenta, we studied the Kcnq1 domain on mouse distal chromosome 7. This large domain is controlled by an intronic imprinting control region and comprises multiple genes that are imprinted in placenta, without the involvement of promoter DNA methylation. We found that the paternal repression along the domain involves acquisition of trimethylation at Lys27 and dimethylation at Lys9 of histone H3. Eed-Ezh2 Polycomb complexes are recruited to the paternal chromosome and potentially regulate its repressive histone methylation. Studies on embryonic stem cells and early embryos support our proposal that chromatin repression is established early in development and is maintained in the placenta. In the embryo, however, imprinting is stably maintained only at genes that have promoter DNA methylation. These data underscore the importance of histone methylation in placental imprinting and identify mechanistic similarities with X-chromosome inactivation in extraembryonic tissues, suggesting that the two epigenetic mechanisms are evolutionarily linked.

Animals↗
PubMed1992

A common intermediary factor (p52/54) recognizing "acidic blob"-type domains is required for transcriptional activation by the Jun proteins.

The ability of the c-Jun protein, the main component of the transcription factor AP1, to interact directly or indirectly with the RNA polymerase II-initiation complex to activate transcription was investigated by in vivo transcription interference ("squelching") experiments. Coexpression of a Jun mutant lacking its DNA binding domain strongly represses the activity of wild-type c-Jun. Repression depends on the presence of the transactivation domains (TADs), suggesting that a limiting factor interacting with the TADs is essential to link Jun and the components of the transcriptional machinery. The activity of this intermediary factor(s) is restricted to TADs characterized by an abundance of negatively charged amino acids, as demonstrated by the abilities of the TADs of JunB, GAL4, and VP16 to repress c-Jun activity. Depending on the presence of the TADs of Jun, we found physical interaction between Jun and a cluster of three proteins with molecular masses of 52, 53, and 54 kDa (p52/54). Association between Jun and p52/54 is strongly reduced in the presence of VP16, suggesting that the two proteins compete for binding to p52/54. Transcription factors containing a different type of TAD (e.g., GHF1, estrogen receptor, or serum response factor) fail to inhibit Jun activity, suggesting that these proteins act through a different mechanism. We consider the requirement of Jun to interact with p52/54 utilized by other transcription factors a new mechanism in the regulation of transcription of Jun-dependent target genes.

Base Sequence↗
PubMed2003

Ventral dominance governs sequential patterns of gene expression across the dorsal-ventral axis of the neuroectoderm in the Drosophila embryo.

A nuclear concentration gradient of the maternal transcription factor Dorsal establishes three tissues across the dorsal-ventral axis of precellular Drosophila embryos: mesoderm, neuroectoderm, and dorsal ectoderm. Subsequent interactions among Dorsal target genes subdivide the mesoderm and dorsal ectoderm. Here we investigate the subdivision of the neuroectoderm by three conserved homeobox genes, ventral nervous system defective (vnd), intermediate neuroblasts defective (ind), and muscle segment homeobox (msh). These genes divide the ventral nerve cord into three columns along the dorsal-ventral axis. Sequential patterns of vnd, ind, and msh expression are established prior to gastrulation and evidence is presented that these genes respond to distinct thresholds of the Dorsal gradient. Maintenance of these patterns depends on cross-regulatory interactions, whereby genes expressed in ventral regions repress those expressed in more dorsal regions. This "ventral dominance" includes regulatory genes that are expressed in the mesectoderm and mesoderm. At least some of these regulatory interactions are direct. For example, the misexpression of vnd in transgenic embryos represses ind and msh, and the addition of Vnd binding sites to a heterologous enhancer is sufficient to mediate repression. The N-terminal domain of Vnd contains a putative eh1 repression domain that binds Groucho in vitro. Mutations in this domain diminish Groucho binding and also attenuate repression in vivo. We discuss the significance of ventral dominance with respect to the patterning of the vertebrate neural tube, and compare it with the previously observed phenomenon of posterior prevalence, which governs sequential patterns of Hox gene expression across the anterior-posterior axis of metazoan embryos.

Animals↗
PubMed2000

Four chromo-domain proteins of Schizosaccharomyces pombe differentially repress transcription at various chromosomal locations.

Transcription is repressed in regions of the fission yeast genome close to centromeres, telomeres, or the silent mating-type cassettes mat2-P and mat3-M. The repression involves the chromo-domain proteins Swi6 and Clr4. We report that two other chromo-domain proteins, Chp1 and Chp2, are also important for these position effects. Chp1 showed a specificity for centromeric regions. Its essentiality for the transcriptional repression of centromeric markers correlates with its importance for chromosome stability. Chp2 appeared more pleiotropic. Its effects on centromeric silencing were less pronounced than those of Chp1, and it participated in telomeric position effects and transcriptional silencing in the mating-type region. We also found that PolII-transcribed genes were repressed when placed in one of the Schizosaccharomyces pombe rDNA clusters, a situation analogous to that in the budding yeast Saccharomyces cerevisiae. Chp2, Swi6, Clr4, and, to a lesser extent, Chp1 participated in that repression.

Amino Acid Sequence↗
PubMed1994

Enhancer traps in the Drosophila bithorax complex mark parasegmental domains.

Eight P elements carrying a beta-galactosidase (lacZ) reporter have been mapped to sites within the Drosophila bithorax complex. The bithorax complex contains three homeotic genes, and at least nine regulatory regions which control their expression in successive parasegments of the fly. The enhancer traps inserted at the promoter of one of the genes, Ultrabithorax, express lacZ in patterns which mimic the Ultrabithorax protein pattern. Enhancer traps in the regulatory regions do not mimic the endogenous genes, but express lacZ globally in the relevant parasegments. Some P elements carry large DNA fragments upstream of the lacZ promoter but internal to the P element. In cases where these internal sequences specify a lacZ pattern, that pattern is generally suppressed when the element is inserted in the bithorax complex. In embryos mutant for genes of the Polycomb group, the lacZ expression from the enhancer traps spreads to all segments. Thus, the enhancer traps reveal parasegmental domains that are maintained by Polycomb-mediated repression. Such domains may be realized by parasegmental differences in chromatin structure.

Animals↗
PubMed2005

An allelic series reveals essential roles for FY in plant development in addition to flowering-time control.

The autonomous pathway functions to promote flowering in Arabidopsis by limiting the accumulation of the floral repressor FLOWERING LOCUS C (FLC). Within this pathway FCA is a plant-specific, nuclear RNA-binding protein, which interacts with FY, a highly conserved eukaryotic polyadenylation factor. FCA and FY function to control polyadenylation site choice during processing of the FCA transcript. Null mutations in the yeast FY homologue Pfs2p are lethal. This raises the question as to whether these essential RNA processing functions are conserved in plants. Characterisation of an allelic series of fy mutations reveals that null alleles are embryo lethal. Furthermore, silencing of FY, but not FCA, is deleterious to growth in Nicotiana. The late-flowering fy alleles are hypomorphic and indicate a requirement for both intact FY WD repeats and the C-terminal domain in repression of FLC. The FY C-terminal domain binds FCA and in vitro assays demonstrate a requirement for both C-terminal FY-PPLPP repeats during this interaction. The expression domain of FY supports its roles in essential and flowering-time functions. Hence, FY may mediate both regulated and constitutive RNA 3'-end processing.

Alleles↗
PubMed2003

Intersectin 1L guanine nucleotide exchange activity is regulated by adjacent src homology 3 domains that are also involved in endocytosis.

Intersectin 1L is a scaffolding protein involved in endocytosis that also has guanine nucleotide exchange activity for Cdc42. In the context of the full-length protein, the catalytic exchange activity of the DH domain is repressed. Here we use biochemical methods to dissect the mechanism for this inhibition. We demonstrate that the intersectin 1L SH3 domains, which bind endocytic proteins, directly inhibit the activity of the DH domain in assays for both binding and exchange of Cdc42. This inhibitory mechanism seems to act through steric hindrance of Cdc42 binding by an intramolecular interaction between the intersectin 1L SH3 domain region and the adjacent DH domain. Surprisingly, the mode of SH3 domain binding is other than through the proline peptide binding pocket. The dual role of the SH3 domains in endocytosis and repression of exchange activity suggests that the intersectin 1L exchange activity is regulated by endocytosis. We show that the endocytic protein, dynamin, competes for binding to the SH3 domains with the neural Wiskott-Aldrich Syndrome protein, an actin filament nucleation protein that is a substrate for activated Cdc42. Swapping of SH3 domain binding partners might act as a switch controlling the actin nucleation activity of intersectin 1L.

Actins↗
PubMed1991

Glucose repression in Saccharomyces cerevisiae is directly associated with hexose phosphorylation by hexokinases PI and PII.

Genetic and biochemical analyses showed that hexokinase PII is mainly responsible for glucose repression in Saccharomyces cerevisiae, indicating a regulatory domain mediating glucose repression. Hexokinase PI/PII hybrids were constructed to identify the supposed regulatory domain and the repression behavior was observed in the respective transformants. The hybrid constructs allowed the identification of a domain (amino acid residues 102-246) associated with the fructose/glucose phosphorylation ratio. This ratio is characteristic of each isoenzyme, therefore this domain probably corresponds to the catalytic domain of hexokinases PI and PII. Glucose repression was associated with the C-terminal part of hexokinase PII, but only these constructs had high catalytic activity whereas opposite constructs were less active. Reduction of hexokinase PII activity by promoter deletion was inversely followed by a decrease in the glucose repression of invertase and maltase. These results did not support the hypothesis that a specific regulatory domain of hexokinase PII exists which is independent of the hexokinase PII catalytic domain. Gene disruptions of hexokinases further decreased repression when hexokinase PI was removed in addition to hexokinase PII. This proved that hexokinase PI also has some function in glucose repression. Stable hexokinase PI overproducers were nearly as effective for glucose repression as hexokinase PII. This showed that hexokinase PI is also capable of mediating glucose repression. All these results demonstrated that catalytically active hexokinases are indispensable for glucose repression. To rule out any further glycolytic reactions necessary for glucose repression, phosphoglucoisomerase activity was gradually reduced. Cells with residual phosphoglucoisomerase activities of less than 10% showed reduced growth on glucose. Even 1% residual activity was sufficient for normal glucose repression, which proved that additional glycolytic reactions are not necessary for glucose repression. To verify the role of hexokinases in glucose repression, the third glucose-phosphorylating enzyme, glucokinase, was stably overexpressed in a hexokinase PI/PII double-null mutant. No strong effect on glucose repression was observed, even in strains with 2.6 U/mg glucose-phosphorylating activity, which is threefold increased compared to wild-type cells. This result indicated that glucose repression is only associated with the activity of hexokinases PI and PII and not with that of glucokinase.

Enzyme Repression↗