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PubMed2002

The T-box repressors TBX2 and TBX3 specifically regulate the tumor suppressor gene p14ARF via a variant T-site in the initiator.

The murine tumor suppressor p19(ARF) (p14(ARF) in humans) is thought to fulfill an important protective role in preventing primary cells from oncogenic transformation via its action in the p53 pathway. Several disease-implicated regulators of p19(ARF) are known to date, among which are the T-box genes TBX2, which resides on an amplicon in primary breast tumors, and TBX3, which is mutated in the human developmental disorder Ulnar-Mammary syndrome. Here we identify a variant T-site, matching 13 of 20 nucleotides of a consensus T-site, as the essential TBX2/TBX3-binding element in the human p14(ARF) promoter. Mutant analysis indicates that both the consensus T-box and a C-terminal conserved repression domain are essential for p14(ARF) repression. Whereas the core nucleotides required for interaction of the archetypal T-box protein Brachyury with a consensus T-site are conserved in the variant site, additional flanking nucleotides contribute to the specificity of TBX2 binding. This is illustrated by the inability of TBX1A or Xbra to activate via the variant p14(ARF) T-site. Importantly, this suggests a hitherto unsuspected level of specificity associated with T-box factors and corresponding recognition sites in regulating their target genes in vivo.

3T3 Cells↗
PubMed2001

Mutation spectrum in patients with Rett syndrome in the German population: Evidence of hot spot regions.

Mutations in the MECP2 (Methyl-CpG-binding protein) gene recently have been reported to cause Rett syndrome (RTT), an X-linked dominant neurodevelopmental disease. We investigated 125 sporadic cases of Rett syndrome by direct sequencing. Thirty different mutations were found in 97 patients with Rett syndrome. Seventeen mutations have not been described previously. We provide evidence for the existence of several hot spot regions and of a deletion-prone region located at the 3' most region of the gene. This latter region most probably forms secondary structures in vitro. Similar structures in vivo could explain the high frequency of deletions in this region. Nine of 10 recurrent mutations were located in either the methyl CpG binding domain (MBD) or in the transcriptional repression domain (TRD), and all missense mutations were located in one of these functionally important domains. There was a high frequency of more than 60% of truncating mutations (nonsense mutations along with frameshift mutations). One patient with a mild form of the disease and a normal head growth carries a novel c.27-6C>A mutation that causes a cryptic splice site in intron I resulting in a frameshift transcript. The detection rate in our collective was 77.6%. Our findings show that the majority of German Rett patients carry mutations in the MECP2 gene confirming the suggested locus homogeneity for the disease.

Base Sequence↗
PubMed1999

Identification of a human histone acetyltransferase related to monocytic leukemia zinc finger protein.

We describe here the identification and functional characterization of a novel human histone acetyltransferase, termed MORF (monocytic leukemia zinc finger protein-related factor). MORF is a 1781-residue protein displaying significant sequence similarity to MOZ (monocytic leukemia zinc finger protein). MORF is ubiquitously expressed in adult human tissues, and its gene is located at human chromosome band 10q22. MORF has intrinsic histone acetyltransferase activity. In addition to its histone acetyltransferase domain, MORF possesses a strong transcriptional repression domain at its N terminus and a highly potent activation domain at its C terminus. Therefore, MORF is a novel histone acetyltransferase that contains multiple functional domains and may be involved in both positive and negative regulation of transcription.

3T3 Cells↗
PubMed2000

The homeodomain protein Arix promotes protein kinase A-dependent activation of the dopamine beta-hydroxylase promoter through multiple elements and interaction with the coactivator cAMP-response element-binding protein-binding protein.

The differentiation and maintenance of a neurotransmitter phenotype is guided by the interaction of exogenous cues with intrinsic genetic machinery. For the noradrenergic phenotype, these influences combine to activate the expression of the catecholaminergic biosynthetic enzymes tyrosine hydroxylase and dopamine beta-hydroxylase (DBH). In this study, we evaluate the molecular mechanisms by which the transcription factor Arix/Phox2a contributes to DBH gene transcription. We have evaluated the contribution of individual homeodomain binding sites in the rat DBH promoter region and find that all are essential for both basal and cAMP-dependent protein kinase A (PKA)-stimulated transcription. Using mammalian one-hybrid and two-hybrid systems, we demonstrate that recruitment of Arix to the positions of homeodomain core recognition sites 1 and 2 at -153 to -166 of the DBH gene restores complete responsiveness of the promoter to PKA in SHSY-5Y neuroblastoma and HepG2 hepatoma cells. Intracellular Arix-Arix interactions are evident and may contribute to the interdependence of homeodomain binding sites. Analysis of functional domains of Arix reveals an N-terminal activation domain and a C-terminal repression domain. The N terminus of Arix contains an amino acid motif similar to a region in Brachyury and Pax9 transcription factors. The N-terminal activation domain of Arix interacts with the transcriptional co-activator, cAMP-response element-binding protein-binding protein, which potentiates transcription from the DBH promoter in a PKA-dependent manner. The present study supports the hypothesis that the paired-like homeodomain protein, Arix, acts as a critical phenotype-specific regulator of the DBH promoter by serving as an integrator of signal-dependent transcription activators within the network of the general transcription machinery.

Animals↗
PubMed2001

Characterization of a basic helix-loop-helix protein, ABF-1: nuclear localization, transcriptional properties, and interaction with Id-2.

The activated B-cell factor (ABF)-1 cDNA was initially isolated from Epstein-Barr virus (EBV)-infected B cells and codes for a DNA-binding protein belonging to the basic helix-loop-helix (bHLH) family of transcription factors. In this study, we characterized the nuclear localization signal of ABF-1, mapped two distinct transcriptional repression domains, and identified one ABF-1-interacting protein, Id-2. By examining the subcellular location of deletion mutants of ABF-1 fused to green fluorescent protein (GFP), critical regions involved in nuclear localization were determined. Analysis of GFP-tagged ABF-1 deletion mutants revealed two separate regions capable of directing nuclear localization. One region mapped to the N-terminal amino acids 71 to 103, whereas the second region localized to the C-terminal bHLH domain. Transient transfection of ABF-1 deletion mutants demonstrated that the N-terminal amino acids 1 to 40 and the bHLH domain function together to achieve maximum repression of E2A activity. Taken together, these results indicate that ABF-1 is a nuclear transcriptional repressor with two distinct regions that function in a synergistic fashion to attenuate E2A-mediated gene activation.

Basic Helix-Loop-Helix Proteins↗
PubMed1995

The amino-terminal domain of the prokaryotic enhancer-binding protein XylR is a specific intramolecular repressor.

The mechanism under which the signal-reception amino-terminal portion (A domain) of the prokaryotic enhancer-binding protein XylR controls the activity of the regulator has been investigated through complementation tests in vivo, in which the various protein segments were produced as independent polypeptides. Separate expression of the A domain repressed the otherwise constitutive activity of a truncated derivative of XylR deleted of its A domain (XylR delta A). Such inhibition was not released by m-xylene, the natural inducer of the system. Repression caused by the A domain was specific for XylR because it did not affect activation of the sigma 54 promoter PnifH by a derivative of its cognate regulator, NifA, deleted of its own A domain. The A domain was also unable to repress the activity of a NifA-XylR hybrid protein resulting from fusing two-thirds of the central domain of NifA to the carboxyl-terminal third of XylR, which includes its DNA-binding domain. The inhibitory effect caused by the A domain of XylR on XylR delta A seems, therefore, to result from specific interactions in trans between the two truncated proteins and not from mere hindering of an activating surface.

Bacterial Proteins↗
PubMed1999

RBP1 recruits both histone deacetylase-dependent and -independent repression activities to retinoblastoma family proteins.

Retinoblastoma (RB) tumor suppressor family proteins block cell proliferation in part by repressing certain E2F-specific promoters. Both histone deacetylase (HDAC)-dependent and -independent repression activities are associated with the RB "pocket." The mechanism by which these two repression functions occupy the pocket is unknown. A known RB-binding protein, RBP1, was previously found by our group to be an active corepressor which, if overexpressed, represses E2F-mediated transcription via its association with the pocket. We show here that RBP1 contains two repression domains, one of which binds all three known HDACs and represses them in an HDAC-dependent manner while the other domain functions independently of the HDACs. Thus, RB family members repress transcription by recruiting RBP1 to the pocket. RBP1, in turn, serves as a bridging molecule to recruit HDACs and, in addition, provides a second HDAC-independent repression function.

Binding Sites↗
PubMed1996

Alternative RNA splicing of the MLL gene in normal and malignant cells.

The human MLL (mixed-lineage leukemia or myeloid-lymphoid leukemia) gene belongs to the trithorax gene family of which the Drosophila trithorax (trx) gene is known to regulate homeotic genes through alternative RNA splicing. To test if such a splicing mechanism also operates in MLL, we evaluated mRNA transcripts from a large number of normal and malignant human cells, making use of RT-PCR, PCR cloning, DNA sequencing and Northern blot analysis. Our findings indicate that different cell types transcribe MLL mRNA species lacking exons that generally encode putative regulatory domains such as AT hooks (exon 3), repression domain (exon 6), zinc finger motifs (exon 8) and activation domain (exon 18). Such findings suggest that posttranscriptional regulation by alternative RNA splicing may play an important role in MLL gene expression and provides the rationale for a mechanism by which this gene, with multiple functional domains, could produce discrete protein products that may prove critical in the regulation of human homeobox genes.

Alternative Splicing↗
PubMed1998

Two phases of Hox gene regulation during early Xenopus development.

We have shown previously that fibroblast growth factor (FGF) signalling in posterior regions of the Xenopus embryo is required for the development of the trunk and tail via a molecular pathway that includes the caudal-related gene Xcad3 and the posterior Hox genes [1]. These results have been contested by the work of Kroll and Amaya [2], which shows that Xenopus embryos transgenic for a dominant-negative form of the FGF receptor (FGF-RI) express posterior Hox genes normally, leading these authors to suggest that the FGFs are not required for anteroposterior (A-P) patterning of the dorsal axis. In order to investigate the apparent discrepancy between these studies, we have produced Xenopus embryos transgenic for two inhibitors of the FGF/Caudal pathway: a kinase-deficient dominant-negative FGF receptor (XFD) [3]; and a domain-swapped form of Xcad3 (Xcad-EnR) in which the activation domain of Xcad3 is replaced by the repression domain of the Drosophila Engrailed protein. Both of these were introduced as fusions with the green fluorescent protein (GFP), which allows identification of non-mosaic transgenic embryos at early gastrula stages by simply looking for GFP fluorescence. Analysis of gene expression in embryos transgenic for these constructs indicated that the activation of posterior Hox genes during early neurula stages absolutely requires FGF signalling and transcriptional activation by Xcad3, while the maintenance of Hox gene expression in the trunk and tail during later development is independent of both FGF and Xcad.

Animals↗
PubMed2006

The polycystic kidney disease-1 gene is a target for p53-mediated transcriptional repression.

This study provides evidence that the tumor suppressor protein, p53, is a transcriptional repressor of PKD1. Kidneys of p53-null mice expressed higher Pkd1 mRNA levels than wild-type littermates; gamma-irradiation suppressed PKD1 gene expression in p53+/+ but not p53-/- cells; and chromatin immunoprecipitation assays demonstrated the binding of p53 to the PKD1 promoter in vivo. In transient transfection assays, p53 repressed PKD1 promoter activity independently of endogenous p21. Deletion analysis mapped p53-mediated repression to the proximal promoter region of PKD1. Mutations of the DNA binding or C-terminal minimal repression domains of p53 abolished its ability to repress PKD1. Moreover, trichostatin A, an inhibitor of histone deacetylase activity, attenuated p53-induced repression of the PKD1 promoter. These findings, together with previous reports showing that dedifferentiated Pkd1-deficient cells express lower p53 and p21 levels, suggest a model whereby PKD1 signaling activates the p53-p21 differentiation pathway. In turn, p53 cooperates with histone deacetylases to repress PKD1 gene transcription. Loss of a p53-mediated negative feedback loop in PKD1 mutant cells may therefore contribute to deregulated PKD1 expression and cystogenesis.

Animals↗
PubMed2005

Molecular cloning of a novel LOS2 gene from Capsella bursa-pastoris.

A new LOS2 gene was cloned from C. bursa-pastoris by rapid amplification of cDNA ends (RACE). The full-length cDNA of C. bursa-pastoris LOS2 gene (designated as Cblos2) was 1694 bp containing a 1332 bp open reading frame (ORF) encoding a 444 amino acid protein. The predicted CbLOS2 protein contained enolase-N domain, enolase domain, conserved putative DNA-binding and repression domains like LOS2 from A. thaliana. Bioinformatic analysis indicated that CbLOS2 had similarity with other enolase proteins. Cold acclimation assay revealed that Cblos2 expressed constitutively in C. bursa-pastoris and was involved in the cold acclimation process, implying CbLOS2 was a bi-functional enolase.

Acclimatization↗
PubMed2002

MBD3L1 and MBD3L2, two new proteins homologous to the methyl-CpG-binding proteins MBD2 and MBD3: characterization of MBD3L1 as a testis-specific transcriptional repressor.

Methylation of cytosines at CpG dinucleotides is essential for mammalian development. MeCP2, MBD1, MBD2, MBD3, and MBD4 comprise a family of proteins that contain a methyl-CpG binding domain (MBD). Except for MBD4, these proteins are involved in gene silencing imposed by methylated DNA. We have identified a human gene that codes for a protein that is 42% identical to MBD3 and 38% identical to MBD2 but lacks the methyl-CpG binding domain. The recombinant protein does not bind to methylated DNA in vitro. The corresponding mouse Mbd3L1 gene was also cloned. The MBD3L1 gene is expressed specifically in testis. During spermatogenesis, expression of MBD3L1 is observed only in round spermatids, suggesting a role for the gene product in the postmeiotic stages of male germ cell development. The MBD3L1 protein is localized to discrete areas in the nucleus and contains an N-terminal transcriptional repression domain. This repression is independent of histone deacetylase inhibition. A homologue of MBD3L1, MBD3L2, was also identified and cloned. Expression of MBD3L2 was found in germ cell tumors and some somatic tissues. These novel proteins may function as counterparts of MBD2 and/or MBD3 in developmental stage-specific transcriptional repression.

Amino Acid Sequence↗
PubMed2004

Characterization of the isoforms of MOVO zinc finger protein, a mouse homologue of Drosophila Ovo, as transcription factors.

We previously described two isoforms (MOVO-A and -B) of a novel zinc finger protein MOVO, a mouse homologue of Drosophila Ovo protein. Here, we isolated cDNA encoding the third isoform MOVO-C, which had a transactivation domain and zinc finger domain, but lacked an N-terminal potential repression domain that was present in MOVO-A. Three isoform mRNAs were expressed highly in mouse testis and also in the ovary at lower levels. The structural analyses of the isolated Movo gene and mRNAs demonstrated that three different Movo transcripts were differentially processed to generate three isoforms. Major mRNA species encoded MOVO-B with a zinc finger domain alone, and minor mRNA species encoded MOVO-A (potential repressor) and MOVO-C (potential activator). To assign MOVO to a transcriptional factor, we characterized DNA-binding and transactivation properties. Random oligonucleotide selection, electrophoretic mobility shift assay and footprinting indicated that MOVO bound to the sequence, 5'-G(G/C/T)GGGGG-3'. These motifs were found in the 5'-flanking regions of Movo and other testis-specific genes. Nuclear proteins binding to this motif were detected in mouse testis, and the expression of MOVO mRNA was restricted in spermatocytes. The luciferase assay demonstrated that MOVO-C activated Movo promoter and MOVO-A repressed it, but MOVO-B had no effects. Mutated MOVO-binding motifs in the Movo promoter reduced the luciferase activity. All the isoforms had no effects on SV40 promoter without MOVO-binding motifs. MOVO-A partially rescued oogenesis of a Drosophila ovo mutant. These results suggest that MOVO isoforms are transcription factors to regulate genes carrying the MOVO-binding motifs in the testis.

Amino Acid Sequence↗
PubMed2003

Identification and characterization of Grainyhead-like epithelial transactivator (GET-1), a novel mammalian Grainyhead-like factor.

LMO-4 is an LIM-only factor that is highly expressed in many epithelial cells, including those of the epidermis and hair follicles. Because LMOs may function by interacting with DNA-binding proteins, we have used the yeast two-hybrid system to screen mouse skin libraries for LMO-4-interacting DNA-binding proteins. In this screen, we isolated a novel LMO-4-interacting factor highly related to the Drosophila gene Grainyhead. Grainyhead is epidermally expressed and carries out important functions in cuticular formation in the fly embryo. With the identification of this novel mammalian Grainyhead-like gene, referred to as Grainyhead-like epithelial transactivator 1 (GET-1), the known members of the mammalian Grainyhead-like gene family are extended to six, falling into two classes based on sequence homology. Of interest, the expression pattern of GET-1 is similar to that of Drosophila Grainyhead with highest expression in the somatic ectoderm/epidermis, but GET-1 is additionally expressed in epithelial cells of gastrointestinal, genitourinary, and respiratory tracks. The GET-1 protein localizes to the nucleus and binds to at least one Grainyhead DNA-binding site. The GET-1 DNA-binding domain maps to a region containing homology to the Drosophila Grainyhead DNA-binding domain. GET-1 homodimerizes in solution by means of a short C-terminally located domain that is homologous to other Grainyhead-like genes. A short domain located between amino acids 100 and 190, which bears no homology to known transactivation domains, is sufficient to confer transactivation to the heterologous GAL4 DNA-binding domain. In addition, GET-1 appears to contain repression domains consistent with the observation that Grainyhead and other mammalian Grainyhead-like genes can act both as activators and repressors. These data suggest that GET-1 is a transcriptional regulator that may perform important functions in epithelial tissues of mammals.

Adaptor Proteins, Signal Transducing↗
PubMed2004

MLL/GRAF fusion in an infant acute monocytic leukemia (AML M5b) with a cytogenetically cryptic ins(5;11)(q31;q23q23).

More than 30 fusions involving the MLL gene at 11q23 have been reported in acute myeloid leukemia (AML). Some of these chimeras are rather common, such as MLL/MLLT3(AF9), but many are quite rare, with some, for example, MLL/GRAF, described only in a single case. The MLL/GRAF fusion, in which the reciprocal hybrid was not expressed, suggesting that the former transcript was the leukemogenic one, was detected in a juvenile myelomonocytic leukemia with a t(5;11)(q31;q23). Here, we report a second case--an infant acute monocytic leukemia (AML M5b)--with an MLL/GRAF fusion. By conventional G-banding, the karyotype was normal. However, Southern blot and fluorescence in situ hybridization analyses revealed that MLL was rearranged and that the 5' part of the MLL gene was inserted into 5q in the vicinity of 5q31, which harbors GRAF. Reverse-transcriptase polymerase chain reaction (PCR) showed that exon 9 of MLL was fused in-frame with exon 19 of GRAF. Extralong genomic PCR with subsequent sequence analysis demonstrated that the breakpoints occurred in intron 9 of MLL, nine base pairs (bp) downstream from exon 9, and in intron 18 of GRAF, 117 bp downstream from exon 18. A 6-bp insertion (ACACTC) of unknown origin was present at the junction. The putative MLL/GRAF fusion protein would retain the AT-hook DNA-binding domain, the DNA methyl transferase motif, the transcription repression domain of MLL, and the SH3 domain of GRAF. As expected, the reciprocal GRAF/MLL was neither expressed nor generated at the genomic level as a consequence of the ins(5;11)(q31;q23q23). On the basis of the now-reported two cases with MLL/GRAF, we conclude that this transcript--but not the reciprocal one--characterizes a rare genetic subgroup of infant AML.

Blotting, Southern↗
PubMed1999

The t(8;21) fusion protein, AML1/ETO, transforms NIH3T3 cells and activates AP-1.

The 8;21 translocation is the most common cytogenetic abnormality in human acute myelogenous leukemia, joining the AML1 gene on chromosome 21, to the ETO gene on chromosome 8, forming the AML1/ETO fusion gene. The AMLI/ETO fusion protein has been shown to function mainly as a transcriptional repressor of AML1 target genes and to block AML1 function in vitro and in vivo. However, AML1/ETO can also activate the BCL-2 promoter and cause enhanced hematopoietic progenitor self-renewal in vitro, suggesting gain-of-functions unique to the fusion protein. We used NIH3T3 cells to determine the transforming capacity of AML1/ETO, and to further characterize its mechanism of action. Expression of AML1/ETO in NIH3T3 cells caused cell-type specific cell death, and cellular transformation, characterized by phenotypic changes, anchorage-independent growth, and tumor formation in nude mice. In contrast, neither expression of AML1A, AML1B or ETO altered the normal growth pattern of the cells. To investigate the mechanism of transformation by AML1/ETO, we analysed the levels of activated, phosphorylated c-Jun (ser63) and other constituents of the AP-1 complex, in the presence of various AML1/ETO related proteins. Expression of AML1/ETO increased the level of c-Jun-P (ser63), and activated AP-1 dependent transcription, which was inhibited by expression of a dominant-negative c-Jun protein. Mutational analysis revealed that the runt homology domain (RHD) and a C-terminal transcriptional repression domain in AML1/ETO are required for transformation, activation of c-Jun and increased AP-1 activity. These results establish the transforming potential of the t(8;21) fusion protein and link this gain-of-function property to modulation of AP-1 activity.

3T3 Cells↗
PubMed1996

A novel mesoderm inducer, Madr2, functions in the activin signal transduction pathway.

A functional assay to clone mouse mesoderm inducers has identified the mouse gene Mad related 2 (Madr2). Madr2 induces dorsal mesoderm from Xenopus ectoderm and can mimic the organizer in recruiting neighboring cells into a second axis. By analyzing the expression of a lacZ/Madr2 fusion protein, we find Madr2 confined to the nucleus in the deep, anterior cells of the second axis, whereas in epidermal and more posterior cells the protein is cytoplasmically localized. This context-dependent nuclear localization suggests that in certain regions of the embryo, Madr2 responds to a localized signal and amplifies this signal to form the second axis. Furthermore, although Madr2 remains unlocalized in ectodermal explants, addition of activin enhances the concentration of Madr2 in the nucleus. Significantly, a functional lacZ fusion to a carboxy-terminal portion of Madr2 is nuclear localized even in the absence of activin. This indicates that Madr2 contains a domain that can activate downstream components and a repressive domain that anchors the protein in the cytoplasm. Nuclear localization of Madr2 in response to activin, and the activin-like phenotypes induced by overexpression of Madr2, indicate that Madr2 is a signal transduction component that mediates the activity of activin.

Activins↗
PubMed2005

Scaffold attachment factor B1 functions in development, growth, and reproduction.

Scaffold attachment factor B1 (SAFB1) is a multifunctional protein that can bind both DNA and RNA and is involved in RNA processing and stress response. In addition, SAFB1 contains a transcriptional repression domain and can bind certain hormone receptors and repress their activity. To assess the role of SAFB1 in vivo, we generated SAFB1 mutant mice through targeted deletion in embryonic stem cells. While viable homozygous mutant (SAFB1-/-) mice were obtained, genotypic distribution indicated that homozygous deficiency resulted in both prenatal and neonatal lethality. Mice lacking SAFB1 exhibited dwarfism, as a result of in utero growth retardation, and had low serum insulin-like growth factor 1 (IGF1) levels. In agreement with the previous characterization of SAFB1 as a corepressor for hormone receptors, we found that SAFB1-/- mice displayed dramatic defects in the development and function of the reproductive system. Male SAFB1 null mice were infertile, apparently because of low circulating levels of testosterone. SAFB1-/- testes were small and showed progressive degeneration of the germinal epithelium, increased apoptosis of germ cells, and Leydig cell hyperplasia. SAFB-/- female mice were subfertile and showed progressive infertility, in part because of defects in oviductal transport and reduced numbers of follicles. Immortalized SAFB1-/- mouse embryonic fibroblasts showed cell-intrinsic defects including increased transcriptional estrogen receptor alpha activity and enhanced responsiveness to IGF1. Together, these in vivo findings establish a critical role for SAFB1 in development, growth regulation, and reproduction.

Animals↗