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At least 397 records · Page 22Linked to original sources
PubMed2003

Regulated recruitment of HP1 to a euchromatic gene induces mitotically heritable, epigenetic gene silencing: a mammalian cell culture model of gene variegation.

Heterochromatin protein 1 (HP1) is a key component of constitutive heterochromatin in Drosophila and is required for stable epigenetic gene silencing classically observed as position effect variegation. Less is known of the family of mammalian HP1 proteins, which may be euchromatic, targeted to expressed loci by repressor-corepressor complexes, and retained there by Lys 9-methylated histone H3 (H3-MeK9). To characterize the physical properties of euchromatic loci bound by HP1, we developed a strategy for regulated recruitment of HP1 to an expressed transgene in mammalian cells by using a synthetic, hormone-regulated KRAB repression domain. We show that its obligate corepressor, KAP1, can coordinate all the machinery required for stable gene silencing. In the presence of hormone, the transgene is rapidly silenced, spatially recruited to HP1-rich nuclear regions, assumes a compact chromatin structure, and is physically associated with KAP1, HP1, and the H3 Lys 9-specific methyltransferase, SETDB1, over a highly localized region centered around the promoter. Remarkably, silencing established by a short pulse of hormone is stably maintained for >50 population doublings in the absence of hormone in clonal-cell populations, and the silent transgenes in these clones show promoter hypermethylation. Thus, like variegation in Drosophila, recruitment of mammalian HP1 to a euchromatic promoter can establish a silenced state that is epigenetically heritable.

3T3 Cells↗
PubMed2001

Sharp, an inducible cofactor that integrates nuclear receptor repression and activation.

A yeast two-hybrid screen using the conserved carboxyl terminus of the nuclear receptor corepressor SMRT as a bait led to the isolation of a novel human gene termed SHARP (SMRT/HDAC1 Associated Repressor Protein). SHARP is a potent transcriptional repressor whose repression domain (RD) interacts directly with SMRT and at least five members of the NuRD complex including HDAC1 and HDAC2. In addition, SHARP binds to the steroid receptor RNA coactivator SRA via an intrinsic RNA binding domain and suppresses SRA-potentiated steroid receptor transcription activity. Accordingly, SHARP has the capacity to modulate both liganded and nonliganded nuclear receptors. Surprisingly, the expression of SHARP is itself steroid inducible, suggesting a simple feedback mechanism for attenuation of the hormonal response.

Amino Acid Motifs↗
PubMed2005

AREB1 is a transcription activator of novel ABRE-dependent ABA signaling that enhances drought stress tolerance in Arabidopsis.

ABSCISIC ACID-RESPONSIVE ELEMENT BINDING PROTEIN1 (AREB1) (i.e., ABF2) is a basic domain/leucine zipper transcription factor that binds to the abscisic acid (ABA)-responsive element (ABRE) motif in the promoter region of ABA-inducible genes. Here, we show that expression of the intact AREB1 gene on its own is insufficient to lead to expression of downstream genes under normal growth conditions. To overcome the masked transactivation activity of AREB1, we created an activated form of AREB1 (AREB1DeltaQT). AREB1DeltaQT-overexpressing plants showed ABA hypersensitivity and enhanced drought tolerance, and eight genes with two or more ABRE motifs in the promoter regions in two groups were greatly upregulated: late embryogenesis abundant class genes and ABA- and drought stress-inducible regulatory genes. By contrast, an areb1 null mutant and a dominant loss-of-function mutant of AREB1 (AREB1:RD) with a repression domain exhibited ABA insensitivity. Furthermore, AREB1:RD plants displayed reduced survival under dehydration, and three of the eight greatly upregulated genes were downregulated, including genes for linker histone H1 and AAA ATPase, which govern gene expression and multiple cellular activities through protein folding, respectively. Thus, these data suggest that AREB1 regulates novel ABRE-dependent ABA signaling that enhances drought tolerance in vegetative tissues.

Abscisic Acid↗
PubMed1995

Direct regulation of the ATPase activity of the transcriptional activator DmpR by aromatic compounds.

The NtrC-like regulator DmpR controls transcription from the dmp operon that encodes the enzymes for catabolism of phenol and some related aromatic compounds. DmpR activates transcription from the sigma 54-dependent dmp-operon promoter in the presence of pathway substrates or structural analogues in the growth medium. Using affinity-purified DmpR and a truncated derivative, we show here that aromatic compounds directly activate the ATPase activity of this protein in vitro, and that the amino-terminal domain represses this activity in the absence of an aromatic ligand. In order to dissect the activation process, derivatives of DmpR exhibiting single amino acid changes were isolated and their effector-dependence and specificity profiles were analysed in vivo. The mechanistic implications of the phenotypes of these mutants are discussed.

Adenosine Triphosphatases↗
PubMed2004

The tumor suppressor HIC1 (hypermethylated in cancer 1) is O-GlcNAc glycosylated.

HIC1 (hypermethylated in cancer 1) is a transcriptional repressor containing five Krüppel-like C(2)H(2) zinc fingers and an N-terminal dimerization and autonomous repression domain called BTB/POZ. Here, we demonstrate that full-length HIC1 proteins are modified both in vivo and in vitro with O-linked N-acetylglucosamine (O-GlcNAc). This is a highly dynamic glycosylation found within the cytosolic and the nuclear compartments of eukaryotes. Analysis of [(3)H]Gal-labeled tryptic peptides indicates that HIC1 has three major sites for O-GlcNAc glycosylation. Using C-terminal deletion mutants, we have shown that O-GlcNAc modification of HIC1 proteins occurred preferentially in the DNA-binding domain. Nonglycosylated and glycosylated forms of full-length HIC1 proteins separated by wheat germ agglutinin affinity purification, displayed the same specific DNA-binding activity in electrophoretic mobility shift assays proving that the O-GlcNAc modification is not directly implicated in the specific DNA recognition of HIC1. Intriguingly, N-terminal truncated forms corresponding to BTB-POZ-deleted proteins exhibited a strikingly differential activity, as the glycosylated truncated forms are unable to bind DNA whereas the unglycosylated ones do. Electrophoretic mobility shift assays performed with separated pools of glycosylated and unglycosylated forms of a construct exhibiting only the DNA-binding domain and the C-terminal tail of HIC1 (residues 399-714) and supershift experiments with wheat germ agglutinin or RL-2, an antibody raised against O-GlcNAc residues, fully corroborated these results. Interestingly, these truncated proteins are O-GlcNAc modified in their C-terminal tail (residues 670-711) and not in the DNA-binding domain, as for the full-length proteins. Thus, the O-GlcNAc modification of HIC1 does not affect its specific DNA-binding activity and is highly sensitive to conformational effects, notably its dimerization through the BTB/POZ domain.

Acetylglucosamine↗
PubMed1994

Functional characterization of Xenopus laevis p53: evidence of temperature-sensitive transactivation but not of repression.

We have investigated the effect of Xenopus laevis p53 (Xp53) on transcription from a variety of promoters which are regulated by mouse p53 using a chloramphenicol acetyltransferase reporter system. Although Xp53 transactivated promoters that are up-regulated by mouse p53, it was unable to cause repression. This ability to transactivate gene expression was dependent on a temperature of 32 degrees C, and activity was lost at 37 degrees C. Temperature-sensitive transactivation was correlated with temperature-dependent binding of Xp53 to the adenovirus E1B58K protein. Despite the marked loss of transcriptional activation and binding to E1B58K at 37 degrees C, Xp53 was still capable of binding simian virus 40 large T antigen and inhibiting simian virus 40 origin-dependent DNA replication. These data show that Xp53 is temperature sensitive for N-terminal activities and suggest that the transactivation and repression "domains" of p53 are distinct.

Adenovirus E1B Proteins↗
PubMed2003

Sequestration of p53 in the cytoplasm by adenovirus type 12 E1B 55-kilodalton oncoprotein is required for inhibition of p53-mediated apoptosis.

The adenovirus E1B 55-kDa protein is a potent inhibitor of p53-mediated transactivation and apoptosis. The proposed mechanisms include tethering the E1B repression domain to p53-responsive promoters via direct E1B-p53 interaction. Cytoplasmic sequestration of p53 by the 55-kDa protein would impose additional inhibition on p53-mediated effects. To investigate further the role of cytoplasmic sequestration of p53 in its inhibition by the E1B 55-kDa protein we systematically examined domains in both the Ad12 55-kDa protein and p53 that underpin their colocalization in the cytoplasmic body and show that the N-terminal transactivation domain (TAD) of p53 is essential for retaining p53 in the cytoplasmic body. Deletion of amino acids 11 to 27 or even point mutation L22Q/W23S abolished the localization of p53 to the cytoplasmic body, whereas other parts of TAD and the C-terminal domain of p53 are dispensable. This cytoplasmic body is distinct from aggresome associated with overexpression of some proteins, since it neither altered vimentin intermediate filaments nor associated with centrosome or ubiquitin. Formation of this structure is sensitive to mutation of the Ad12 55-kDa protein. Strikingly, mutation S476/477A near the C terminus of the Ad12 55-kDa protein eliminated the formation of the cytoplasmic body. The equivalent residues in the Ad5 55-kDa protein were shown to be critical for its ability to inhibit p53. Indeed, Ad12 55-kDa mutants that cannot form a cytoplasmic body can no longer inhibit p53-mediated effects. Conversely, the Ad12 55-kDa protein does not suppress p53 mutant L22Q/W23S-mediated apoptosis. Finally, we show that E1B can still sequester p53 that contains the mitochondrial import sequence, thereby potentially preventing the localization of p53 to mitochondria. Thus, cytoplasmic sequestration of p53 by the E1B 55-kDa protein plays an important role in restricting p53 activities.

Adenovirus E1B Proteins↗
PubMed1992

Transcriptional repression by a novel member of the bZIP family of transcription factors.

We describe here a novel member of the bZIP family of DNA-binding proteins, designated E4BP4, that displays an unusual DNA-binding specificity which overlaps that of the activating transcription factor family of factors. When expressed in a transient transfection assay with a suitable reporter plasmid, E4BP4 strongly repressed transcription in a DNA-binding-site-dependent manner. Examination of a series of deletion mutants revealed that sequences responsible for the repressing potential of E4BP4 lie within the carboxyl-terminal region of the protein. No similarity was found between this region and the repressing domains of other known eukaryotic transcriptional repressors.

Amino Acid Sequence↗
PubMed1996

Unliganded thyroid hormone receptor alpha can target TATA-binding protein for transcriptional repression.

Unliganded human thyroid hormone receptor alpha (hTR alpha) can repress transcription by inhibiting the formation of a functional preinitiation complex (PIC) on promoters bearing thyroid hormone receptor (TR)-binding elements. Here we demonstrate that hTR alpha directly contacts the TATA-binding protein (TBP) and that preincubation of hTR alpha with TBP completely alleviates TR-mediated repression in vitro. Using stepwise preassembled PICs, we show that hTR alpha targets either the TBP/TFIIA or the TBP/TFIIA/TFIIB steps of PIC assembly for repression. We also show that the repression domain of hTR alpha maps to the C-terminal ligand-binding region and that direct TR-TBP interactions can be inhibited by thyroid hormone. Together, these results suggest a model in which unliganded hTR alpha contacts promoter-bound TBP and interferes with later steps in the initiation of transcription.

Amino Acid Sequence↗
PubMed1998

Nuclear receptor DAX-1 recruits nuclear receptor corepressor N-CoR to steroidogenic factor 1.

The orphan nuclear receptor steroidogenic factor 1 (SF-1) is a critical developmental regulator in the urogenital ridge, because mice targeted for disruption of the SF-1 gene lack adrenal glands and gonads. SF-1 was recently shown to interact with DAX-1, another orphan receptor whose tissue distribution overlaps that of SF-1. Naturally occurring loss-of-function mutations of the DAX-1 gene cause the human disorder X-linked adrenal hypoplasia congenita (AHC), which resembles the phenotype of SF-1-deficient mice. Paradoxically, however, DAX-1 represses the transcriptional activity of SF-1, and AHC mutants of DAX-1 lose repression function. To further investigate these findings, we characterized the interaction between SF-1 and DAX-1 and found that their interaction indeed occurs through a repressive domain within the carboxy terminus of SF-1. Furthermore, we demonstrate that DAX-1 recruits the nuclear receptor corepressor N-CoR to SF-1, whereas naturally occurring AHC mutations of DAX-1 permit the SF-1-DAX-1 interaction, but markedly diminish corepressor recruitment. Finally, the interaction between DAX-1 and N-CoR shares similarities with that of the nuclear receptor RevErb and N-CoR, because the related corepressor SMRT was not efficiently recruited by DAX-1. Therefore, DAX-1 can serve as an adapter molecule that recruits nuclear receptor corepressors to DNA-bound nuclear receptors like SF-1, thereby extending the range of corepressor action.

Adrenal Gland Diseases↗
PubMed1998

Characterization of ABF-1, a novel basic helix-loop-helix transcription factor expressed in activated B lymphocytes.

Proteins of the basic helix-loop-helix (bHLH) family are required for a number of different developmental pathways, including neurogenesis, lymphopoiesis, myogenesis, and sex determination. Using a yeast two-hybrid screen, we have identified a new bHLH transcription factor, ABF-1, from a human B-cell cDNA library. Within the bHLH region, ABF-1 shows a remarkable conservation with other HLH proteins, including tal-1, NeuroD, and paraxis. Its expression pattern is restricted to a subset of lymphoid tissues, Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines, and activated human B cells. ABF-1 is capable of binding an E-box element either as a homodimer or as a heterodimer with E2A. Furthermore, a heterodimeric complex containing ABF-1 and E2A can be detected in EBV-immortalized lymphoblastoid cell lines. ABF-1 contains a transcriptional repression domain and is capable of inhibiting the transactivation capability of E47 in mammalian cells. ABF-1 represents the first example of a B-cell-restricted bHLH protein, and its expression pattern suggests that ABF-1 may play a role in regulating antigen-dependent B-cell differentiation.

Adenovirus E2 Proteins↗
PubMed2000

The ETO protein disrupted in t(8;21)-associated acute myeloid leukemia is a corepressor for the promyelocytic leukemia zinc finger protein.

The ETO protein was originally identified by its fusion to the AML-1 transcription factor in translocation (8;21) associated with the M2 form of acute myeloid leukemia (AML). The resulting AML-1-ETO fusion is an aberrant transcriptional regulator due to the ability of ETO, which does not bind DNA itself, to recruit the transcriptional corepressors N-CoR, SMRT, and Sin3A and histone deacetylases. The promyelocytic leukemia zinc finger (PLZF) protein is a sequence-specific DNA-binding transcriptional factor fused to retinoic acid receptor alpha in acute promyelocytic leukemia associated with the (11;17)(q23;q21) translocation. PLZF also mediates transcriptional repression through the actions of corepressors and histone deacetylases. We found that ETO is one of the corepressors recruited by PLZF. The PLZF and ETO proteins associate in vivo and in vitro, and ETO can potentiate transcriptional repression by PLZF. The N-terminal portion of ETO forms complexes with PLZF, while the C-terminal region, which was shown to bind to N-CoR and SMRT, is required for the ability of ETO to augment transcriptional repression by PLZF. The second repression domain (RD2) of PLZF, not the POZ/BTB domain, is necessary to bind to ETO. Corepression by ETO was completely abrogated by histone deacetylase inhibitors. This identifies ETO as a cofactor for a sequence-specific transcription factor and indicates that, like other corepressors, it functions through the action of histone deactylase.

Acute Disease↗
PubMed2004

Influence of MECP2 gene mutation and X-chromosome inactivation on the Rett syndrome phenotype.

To date, approximately 200 different mutations in the MECP2 gene have been identified. We analyzed the entire coding sequence of the MECP2 gene and the X-chromosome inactivation pattern in 42 sporadic cases of Rett syndrome. Of the 42 patients, 30 had pathogenic mutations, including 14 different mutations: 9 missense mutations, 4 nonsense mutations, and 1 frameshift mutation. One was a novel mutation (S134P). There was a tendency for patients who had a nonsense mutation in the transcriptional repression domain region to show earlier onset of regression and more severe language retardation than patients with a mutation in the methyl-CpG binding domain region. However, the parameters of clinical severity were variable among patients with the same type of mutation, depending on the pattern of X-chromosome inactivation. This study suggests that the X-chromosome inactivation pattern can modify the phenotype of Rett syndrome, which is primarily determined by the type and site of MECP2 gene mutation.

Age of Onset↗
PubMed2003

Activation of the knirps locus links patterning to morphogenesis of the second wing vein in Drosophila.

The adjacent knirps (kni) and knirps-related (knrl) genes encode functionally related zinc finger transcription factors that collaborate to initiate development of the second longitudinal wing vein (L2). kni and knrl are expressed in the third instar larval wing disc in a narrow stripe of cells just anterior to the broad central zone of cells expressing high levels of the related spalt genes. Here, we identify a 1.4 kb cis-acting enhancer element from the kni locus that faithfully directs gene expression in the L2 primordium. We find that three independent ri alleles have alterations mapping within the L2-enhancer element and show that two of these observed lesions eliminate the ability of the enhancer element to direct gene expression in the L2 primordium. The L2 enhancer can be subdivided into distinct activation and repression domains. The activation domain mediates the combined action of the general wing activator Scalloped and a putative locally provided factor, the activity of which is abrogated by a single nucleotide alteration in the ri(53j) mutant. We also find that misexpression of genes in L2 that are normally expressed in veins other than L2 results in abnormal L2 development. These experiments provide a mechanistic basis for understanding how kni and knrl link AP patterning to morphogenesis of the L2 vein by orchestrating the expression of a selective subset of vein-promoting genes in the L2 primordium.

Alleles↗
PubMed2004

KNUCKLES (KNU) encodes a C2H2 zinc-finger protein that regulates development of basal pattern elements of the Arabidopsis gynoecium.

Flowers of the parthenocarpic knuckles mutant are conditionally male sterile and contain ectopic stamens and carpels that originate from placental tissue within developing gynoecia. The mutation was mapped to a 123 Kb interval on chromosome 5 using molecular markers. All aspects of the knuckles phenotype could be complemented by a genomic fragment from the region which contained the annotated MAC12.2 gene. A guanine to adenine transition within a predicted C2H2 zinc finger-encoding region of MAC12.2 causes the second of two critical zinc-binding cysteine residues to be replaced by a tyrosine. Transgenic plants in which translational fusions of the GUS reporter to KNUCKLES were driven by the presumptive KNUCKLES promoter indicate that the gene is expressed first in developing carpel primordia, and later in stamens and ovules of flower buds. In situ hybridization experiments showed a broader pattern of transcript localization, suggesting that post-transcriptional regulatory mechanisms may limit KNUCKLES protein accumulation and localization. Based on genetic evidence and the presence of a carboxy-terminal motif demonstrated by others to function as an active repression domain, we propose that KNUCKLES might function as a transcriptional repressor of cellular proliferation that regulates floral determinacy and relative size of basal pattern elements along the proximo-distal axis of the developing Arabidopsis gynoecium.

Amino Acid Sequence↗
PubMed1997

A role for Siamois in Spemann organizer formation.

The vertebrate body plan is specified in the early embryo through the inductive influence of the organizer, a special region that forms on the dorsalmost side of the embryo at the beginning of gastrulation. In Xenopus, the homeobox gene Siamois is activated prior to gastrulation in the area of organizer activity and is capable of inducing a secondary body axis when ectopically expressed. To elucidate the function of endogeneous Siamois in dorsoventral axis formation, we made a dominant repressor construct (SE) in which the Siamois homeodomain was fused to an active repression domain of Drosophila engrailed. Overexpression of 1-5 pg of this chimeric mRNA in the early embryo blocks axis development and inhibits activation of dorsal, but not ventrolateral, marginal zone markers. At similar expression levels, SE proteins with altered DNA-binding specificity do not have the same effect. Coexpression of mRNA encoding wild-type Siamois, but not a mutated Siamois, restores dorsal development to SE embryos. Furthermore, SE strongly blocks axis formation triggered by beta-catenin but not by the organizer product noggin. These results suggest that Siamois function is essential for beta-catenin-mediated formation of the Spemann organizer, and that Siamois acts prior to noggin in specifying dorsal development.

Animals↗
PubMed1999

Spatially regulated SpEts4 transcription factor activity along the sea urchin embryo animal-vegetal axis.

Because the transcription of the SpHE gene is regulated cell-autonomously and asymmetrically along the maternally determined animal-vegetal axis of the very early sea urchin embryo, its regulators provide an excellent entry point for investigating the mechanism(s) that establishes this initial polarity. Previous studies support a model in which spatial regulation of SpHE transcription relies on multiple nonvegetal positive transcription factor activities (Wei, Z., Angerer, L. M. and Angerer, R. C. (1997) Dev. Biol. 187, 71-78) and a yeast one-hybrid screen has identified one, SpEts4, which binds with high specificity to a cis element in the SpHE regulatory region and confers positive activation of SpHE promoter transgenes (Wei, Z., Angerer, R. C. and Angerer, L. M. (1999) Mol. Cell. Biol. 19, 1271-1278). Here we demonstrate that SpEts4 can bind to the regulatory region of the endogenous SpHE gene because a dominant repressor, created by fusing SpEts4 DNA binding and Drosophila engrailed repression domains, suppresses its transcription. The pattern of expression of the SpEts4 gene is consistent with a role in regulating SpHE transcription in the nonvegetal region of the embryo during late cleavage/early blastula stages. Although maternal transcripts are uniformly distributed in the egg and early cleaving embryo, they rapidly turn over and are replaced by zygotic transcripts that accumulate in a pattern congruent with SpHE transcription. In addition, in vivo functional tests show that the SpEts4 cis element confers nonvegetal transcription of a beta-galactosidase reporter gene containing the SpHE basal promoter, and provide strong evidence that the activity of this transcription factor is an integral component of the nonvegetal transcriptional regulatory apparatus, which is proximal to, or part of, the mechanism that establishes the animal-vegetal axis of the sea urchin embryo.

Animals↗
PubMed2025

Unidirectional recruitment between MeCP2 and KSHV-encoded LANA revealed by CRISPR/Cas9 recruitment assay.

Kaposi's sarcoma-associated herpesvirus (KSHV, HHV-8) is associated with several human malignancies. During latency, the viral genomes reside in the nucleus of infected cells as large non-integrated plasmids, known as episomes. To ensure episome maintenance, the latency protein LANA tethers the viral episomes to the cell chromosomes during cell division. Directional recruitment of protein complexes is critical for the proper function of many nuclear processes. To test for recruitment directionality between LANA and cellular proteins, we directed LANA via catalytically inactive Cas9 (dCas9) to a repeat sequence to obtain easily detectable dots. Then, the recruitment of nuclear proteins to these dots can be evaluated. We termed this assay CRISPR-PITA for Protein Interaction and Telomere Recruitment Assay. Using this protein recruitment assay, we found that LANA recruits its known interactors ORC2 and SIN3A. Interestingly, LANA was unable to recruit MeCP2, but MeCP2 recruited LANA. Both LANA and histone deacetylase 1 (HDAC1) interact with the transcriptional-repression domain (TRD) and the methyl-CpG-binding domain (MBD) of MeCP2. Similar to LANA, HDAC1 was unable to recruit MeCP2. While heterochromatin protein 1 (HP1), which interacts with the N-terminal of MeCP2, can recruit MeCP2. We propose that available interacting domains force this recruitment directionality. We hypothesized that the tandem repeats in the SunTag may force MeCP2 dimerization and mimic the form of DNA-bound MeCP2. Indeed, providing only the tandem epitopes of SunTag allows LANA to recruit MeCP2 in infected cells. Therefore, CRISPR-PITA revealed the rules of unidirectional recruitment and allowed us to break this directionality.

Humans↗