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PubMed2000

The interaction of the carboxyl terminus-binding protein with the Smad corepressor TGIF is disrupted by a holoprosencephaly mutation in TGIF.

The homeodomain protein TGIF represses transcription in part by recruiting histone deacetylases. TGIF binds directly to DNA to repress transcription or interacts with TGF-beta-activated Smads, thereby repressing genes normally activated by TGF-beta. Loss of function mutations in TGIF result in holoprosencephaly (HPE) in humans. One HPE mutation in TGIF results in a single amino acid substitution in a conserved PLDLS motif within the amino-terminal repression domain. We demonstrate that TGIF interacts with the corepressor carboxyl terminus-binding protein (CtBP) via this motif. CtBP, which was first identified by its ability to bind the adenovirus E1A protein, interacts both with gene-specific transcriptional repressors and with a subset of polycomb proteins. Efficient repression of TGF-beta-activated gene responses by TGIF is dependent on interaction with CtBP, and we show that TGIF is able to recruit CtBP to a TGF-beta-activated Smad complex. Disruption of the PLDLS motif in TGIF abolishes the interaction of CtBP with TGIF and compromises the ability of TGIF to repress transcription. Thus, at least one HPE mutation in TGIF appears to prevent CtBP-dependent transcriptional repression by TGIF, suggesting an important developmental role for the recruitment of CtBP by TGIF.

Alcohol Oxidoreductases↗

PubMed2001

Intracellular localization of the Ret finger protein depends on a functional nuclear export signal and protein kinase C activation.

The Ret finger protein (RFP) was identified initially as an oncogene product and belongs to a family of proteins that contain a tripartite motif consisting of a RING finger, a B box, and a coiled-coil domain. RFP represses transcription by interacting with Enhancer of Polycomb and is localized to the cytoplasm or nucleus depending on the cell type. Here, we have identified the nuclear export signal (NES) located in the coiled-coil region of RFP. Mutation of this NES or treatment with leptomycin B abrogated the nuclear export of RFP in NIH3T3 cells. In addition, fusion of this NES to other nuclear proteins, such as yeast transcription factor Gal4, resulted in their release into the cytoplasm of NIH3T3 cells. Although the NES function of RFP in HepG2 cells is masked by another domain in RFP or by another protein, 12-O-tetradecanoylphorbol-13-acetate treatment or overexpression of constitutively active protein kinase Calpha (PKCalpha) abrogated masking, leading to the cytoplasmic localization of RFP. Furthermore, treatment of NIH3T3 cells with PKC inhibitors blocked the function of NES, resulting in nuclear localization of RFP. Thus, the nuclear export of RFP is regulated positively by PKC activation. However, RFP was not a direct substrate of PKC, and additional signaling pathways may be involved in the regulation of nuclear export of RFP.

Active Transport, Cell Nucleus↗

PubMed2002

Maintenance of integrated proviral gene expression requires Brm, a catalytic subunit of SWI/SNF complex.

We show here that murine leukemia virus-based retrovirus vector transgene expression is rapidly silenced in human tumor cell lines lacking expression of Brm, a catalytic subunit of the SWI/SNF chromatin remodeling complex, even though these vectors can successfully enter, integrate, and initiate transcription. We detected this gene silencing as a reduction in the ratio of cells expressing the exogenous gene rather than a reduction in the average expression levels, indicating that down-regulation occurs in an all-or-none manner. Retroviral gene expression was protected from silencing and maintained in Brm-deficient host cells by exogenous expression of Brm but not BRG1, an alternative ATPase subunit in the SWI/SNF complex. Introduction of exogenous Brm to these cells suppressed recruitment of protein complexes containing YY1 and histone deacetylase (HDAC) 1 and 2 to the 5'-long terminal repeat region of the integrated provirus, leading to the enhancement of acetylation of specific lysine residues in histone H4 located in this region. Consistent with these observations, treatment of Brm-deficient cells with HDAC inhibitors but not DNA methylation inhibitors suppressed retroviral gene silencing. These results suggest that the Brm-containing SWI/SNF complex subfamily (trithorax-G) and a complex including YY1 and HDACs (Polycomb-G) counteract each other to maintain transcription of exogenously introduced genes.

Adrenal Cortex Neoplasms↗

PubMed2003

SuUR protein binds to the boundary regions separating forum domains in Drosophila melanogaster.

Forum domains are 50-150 kb DNA fragments that are released during spontaneous fragmentation of chromosomes. They are separated by islands of putative heterochromatin boundary regions. The SuUR protein, which is involved in the control of chromosome organization, is localized exclusively in heterochromatin and often colocalizes on chromosomes with Polycomb group proteins. To test whether the SuUR protein is associated with boundary regions, we used gel retardation assays and found that the SuUR protein binds specifically to boundary regions and that boundary regions are under-replicated. These results suggest that the regular distribution of boundary regions in chromosomes may represent the dispersion of sites designed for chromosomal silencing.

Amino Acid Sequence↗

PubMed2005

Silencing of the meiotic genes SMC1beta and STAG3 in somatic cells by E2F6.

E2F6, a member of the E2F-family of transcription factors, is a retinoblastoma protein-independent transcriptional repressor. E2F6 associates with polycomb group (Pc-G) multiprotein complexes that contain histone H3 methyltransferases, suggesting that E2F6 represses genes by covalent histone modification. However, genes that are repressed by E2F6 via a mechanism that involves histone H3 methylation have not been identified. Using cDNA microarray experiments comparing wild-type and E2f6-/- mouse embryonic fibroblasts, we now found that E2F6 is required to silence the meiosis-specific genes SMC1beta and STAG3 in somatic cells. Re-expression of E2F6 in E2f6-/- cells was sufficient to restore their repression. E2F6 binds in vivo to the promoters of these genes through a conserved binding site. Transcriptional repression of SMC1beta and STAG3 by E2F6 involves multiple mechanisms, including methylation of histone H3 on lysine 9 and lysine 27. Our findings suggest a molecular mechanism for the stable transcriptional silencing of meiotic genes in somatic cells by E2F6.

Acetylation↗

PubMed2006

Enhancer of polycomb1, a novel homeodomain only protein-binding partner, induces skeletal muscle differentiation.

Homeodomain only protein, Hop, is an unusual small protein that modulates target gene transcription without direct binding to DNA. Here we show that Hop interacts with Enhancer of Polycomb1 (Epc1), a homolog of a Drosophila polycomb group gene product that regulates transcription, to induce the skeletal muscle differentiation. Yeast two-hybrid assay with the human adult heart cDNA library revealed that Hop can associate with Epc1. The amino-terminal domain of Epc1 as well as full Epc1 physically interacted with Hop in mammalian cells and in yeast. Epc1 is highly expressed in the embryonic heart and adult skeletal muscles. Serum deprivation induced differentiation of H9c2, a myoblast cell line, into skeletal myocytes, and Epc1 was up-regulated. Differentiation of H9c2 was induced by Epc1 overexpression, although it was severely impaired in Epc1-knockdown cells. Co-transfection of Hop potentiated Epc1-induced transactivation of myogenin and myotube formation. Hop knock-out mice elicited a decrease in myosin heavy chain and myogenin expressions in skeletal muscle and showed delay in hamstring muscle healing after injury. Differentiation was impaired in skeletal myoblasts from Hop knock-out mice. These results suggest that Epc1 plays a role in the initiation of skeletal muscle differentiation, and its interaction with Hop is required for the full activity.

Animals↗

PubMed2005

Biomarker discovery in urogenital cancer.

Novel approaches for the early detection of urogenital cancers are urgently needed. Metastatic renal cell carcinoma (RCC) has a poor prognosis and unpredictable course and to date there are no molecular markers that reliably protect RCC outcome. A novel kidney cancer marker, carbonic anhydrase IX (CA IX), was investigated as an independent prognostic factor for survival for patients with metastatic RCC. In patients with non-metastatic RCC low CAIX predicted a worse outcome similar to patients with metastatic disease and overall CAIX expression decreased with development of metastasis. CAIX reflects significant changes in tumour biology, which may be used to predict clinical outcome and identify high-risk patients for adjuvant-targeted therapies. With regard to prostate cancer there are a number of putative biomarkers, although there are limited studies providing clinical correlations in humans. Potential biomarkers include caveolin-1, p-Akt, p27, the met oncogene, Ki67 (MIB-1), 8q24 over-expression, polycomb protein EZH2, plasma TGF-B1 and IL-6 among others. The laboratory has concentrated on the Prostate Stem Cell Antigen (PSCA) which is increased in patients with more aggressive features, that is higher Gleason grade and higher stage. Highest expression is seen in metastatic lesions to bone and staining for PSCA may predict for disease progression or recurrence. Also promising is the finding reported by the group that expression of p27 in radical prostatectomy specimens correlates with biochemical recurrence. Loss of p27 (defined as absent expression in more than 70% of the specimen) is an independent predictor of recurrence among all patients and among the sub-set with organ confined and extra-capsular disease. The data also shows that p27 can predict outcome among patients with positive surgical resection margins. As with other biomarkers, major questions still to be addressed is the requirement for universal application with uniform scoring and the need for prospective studies in randomized clinical trials.

Antigens, Neoplasm↗

PubMed1995

The Drosophila snr1 and brm proteins are related to yeast SWI/SNF proteins and are components of a large protein complex.

During most of Drosophila development the regulation of homeotic gene transcription is controlled by two groups of regulatory genes, the trithorax group of activators and the Polycomb group of repressors. brahma (brm), a member of the trithorax group, encodes a protein related to the yeast SWI2/SNF2 protein, a subunit of a protein complex that assists sequence-specific activator proteins by alleviating the repressive effects of chromatin. To learn more about the molecular mechanisms underlying the regulation of homeotic gene transcription, we have investigated whether a similar complex exists in flies. We identified the Drosophila snr1 gene, a potential homologue of the yeast SNF5 gene that encodes a subunit of the yeast SWI/SNF complex. The snr1 gene is essential and genetically interacts with brm and trithorax (trx), suggesting cooperation in regulating homeotic gene transcription. The spatial and temporal patterns of expression of snr1 are similar to those of brm. The snr1 and brm proteins are present in a large (> 2 x 10(6) Da) complex, and they co-immunoprecipitate from Drosophila extracts. These findings provide direct evidence for conservation of the SWI/SNF complex in higher eucaryotes and suggest that the Drosophila brm/snr1 complex plays an important role in maintaining homeotic gene transcription during development by counteracting the repressive effects of chromatin.

Amino Acid Sequence↗

PubMed2006

The Mcp element mediates stable long-range chromosome-chromosome interactions in Drosophila.

Chromosome organization inside the nucleus is not random but rather is determined by a variety of factors, including interactions between chromosomes and nuclear components such as the nuclear envelope or nuclear matrix. Such interactions may be critical for proper nuclear organization, chromosome partitioning during cell division, and gene regulation. An important, but poorly documented subset, includes interactions between specific chromosomal regions. Interactions of this type are thought to be involved in long-range promoter regulation by distant enhancers or locus control regions and may underlie phenomena such as transvection. Here, we used an in vivo microscopy assay based on Lac Repressor/operator recognition to show that Mcp, a polycomb response element from the Drosophila bithorax complex, is able to mediate physical interaction between remote chromosomal regions. These interactions are tissue specific, can take place between multiple Mcp elements, and seem to be stable once established. We speculate that this ability to interact may be part of the mechanism through which Mcp mediates its regulatory function in the bithorax complex.

ATP-Binding Cassette Transporters↗

PubMed2000

The GAGA factor of Drosophila interacts with SAP18, a Sin3-associated polypeptide.

SAP18, a polypeptide associated with the Sin3-HDAC co-repressor complex, was identified in a yeast two-hybrid screen as capable of interacting with the Drosophila GAGA factor. The interaction was confirmed in vitro by glutathione S-transferase pull-down assays using recombinant proteins and crude SL2 nuclear extracts. The first 245 residues of GAGA, including the POZ domain, are necessary and sufficient to bind dSAP18. In polytene chromosomes, dSAP18 and GAGA co-localize at a few discrete sites and, in particular, at the bithorax complex where GAGA binds some silenced polycomb response elements. When the dSAP18 dose is reduced, flies heterozygous for the GAGA mutation Trl67 show the homeotic transformation of segment A6 into A5, indicating that GAGA-dSAP18 interaction contributes to the functional regulation of the iab-6 element of the bithorax complex. These results suggest that, through recruitment of the Sin3-HDAC complex, GAGA might contribute to the regulation of homeotic gene expression.

Abdomen↗

PubMed2002

The Drosophila BRM complex facilitates global transcription by RNA polymerase II.

Drosophila brahma (brm) encodes the ATPase subunit of a 2 MDa complex that is related to yeast SWI/SNF and other chromatin-remodeling complexes. BRM was identified as a transcriptional activator of Hox genes required for the specification of body segment identities. To clarify the role of the BRM complex in the transcription of other genes, we examined its distribution on larval salivary gland polytene chromosomes. The BRM complex is associated with nearly all transcriptionally active chromatin in a pattern that is generally non-overlapping with that of Polycomb, a repressor of Hox gene transcription. Reduction of BRM function dramatically reduces the association of RNA polymerase II with salivary gland chromosomes. A few genes, such as induced heat shock loci, are not associated with the BRM complex; transcription of these genes is not compromised by loss of BRM function. The distribution of the BRM complex thus correlates with a dependence on BRM for gene activity. These data suggest that the chromatin remodeling activity of the BRM complex plays a general role in facilitating transcription by RNA polymerase II.

Animals↗

PubMed1982

Polycomblike: a gene that appears to be required for the normal expression of the bithorax and antennapedia gene complexes of Drosophila melanogaster.

A newly identified gene is described that is required for the maintenance of normal identities in many of the body segments of the fly. The effects of mutants in this gene, which is called Polycomblike (Pcl), suggest that its wild-type allele functions in the regulation of the bithorax gene complex (BX-C) and the Antennapedia gene complex (ANT-C). Evidence in favor of this idea derives from (1) the close correspondence between segmental transformations caused by Pcl mutants and those caused by dominant gain-of-function mutants in the BX-C and ANT-C, (2) the interactions observed between Pcl mutants and mutants in these complexes, and (3) the dependence upon BX-C and ANT-C dosage of the severity of at least one of the transformations caused by Pcl mutants. Arguments are presented that the control of the BX-C and ANT-C by Pcl+ is negative in nature. The results of clonal analysis experiments indicate that, at least for the BX-C, Pcl+ exerts this control until late in development. Since the wild-type allele of another gene, called Polycomb (Pc), has previously been shown to have many of the same properties as Pcl+, it appears that the BX-C and perhaps also the ANT-C are continuously regulated during development by at least two and probably several other genes.

Animals↗

PubMed1990

Dosage requirements of Ultrabithorax and bithoraxoid in the determination of segment identity in Drosophila melanogaster.

The wild-type Ultrabithorax (Ubx) and bithoraxoid (bxd) functions are primarily responsible for establishing the identity of parasegment 6 (PS6) in the Drosophila embryo and thus the identity of the posterior compartment of the third thoracic segment (pT3) and the anterior compartment of the first abdominal segment (aA1) in the adult. The experiments described were designed to test the ability of an increased dosage of Ubx+ and bxd+ to affect the transformation of PS5 toward PS6. The results are consistent with the ideas that (1) multiple copies of Ubx+ and bxd+ cause some cells within PS5 to take on the characteristics of PS6 cells but do not cause an overall parasegmental transformation of PS5 toward PS6, (2) cellular identity depends not only on the activity of Ubx+ but on its concentration as well, and (3) that an interaction between Ubx+ and the wild-type Antennapedia (Antp) gene establishes segmental identity in pT2. In the first instar larvae carrying eight copies of Ubx+ and bxd+ the fine hairs of the T3 setal belt are transformed toward the hook-like structures of the A1 setal belt. Other structures within this segment are unaffected. In the adult, the haltere is reduced in size. The transformation of pT2 cells (wing) toward pT3 cells (haltere) is seen in adults carrying eight doses of wild type Ubx and bxd by decreasing the amount of the bithorax complex (BX-C) regulator Polycomb (Pc). However, the transformation of the T3 setal belt is not enhanced in the larvae of these animals. The interaction between the genes of the Antennapedia complex (ANT-C) and the Ubx+ and bxd+ functions in pT2 is dosage sensitive only when the animals carry one copy of Pc. In these animals, the transformation of wing toward haltere is significantly enhanced.

Animals↗

PubMed1990

Transvection in the Drosophila Ultrabithorax gene: a Cbx1 mutant allele induces ectopic expression of a normal allele in trans.

In wild-type Drosophila melanogaster larvae, the Ultrabithorax (Ubx) gene is expressed in the haltere imaginal discs but not in the majority of cells of the wing imaginal discs. Ectopic expression of the Ubx gene in wing discs can be elicited by the presence of Contrabithorax (Cbx) gain-of-function alleles of the Ubx gene or by loss-of-function mutations in Polycomb (Pc) or in other trans-regulatory genes which behave as repressors of Ubx gene activity. Several Ubx loss-of-function alleles cause the absence of detectable Ubx proteins (UBX) or the presence of truncated UBX lacking the homeodomain. We have compared adult wing phenotypes with larval wing disc UBX patterns in genotypes involving double mutant chromosomes carrying in cis one of those Ubx mutations and the Cbx1 mutation. We show that such double mutant genes are (1) active in the same cells in which the single mutant Cbx1 is expressed, although they are unable to yield functional proteins, and (2) able to induce ectopic expression of a normal homologous Ubx allele in a part of the cells in which the single mutant Cbx1 is active. That induction is conditional upon pairing of the homologous chromosomes (the phenomenon known as transvection), and it is not mediated by UBX. Depletion of Pc gene products by Pc3 mutation strongly enhances the induction phenomenon, as shown by (1) the increase of the number of wing disc cells in which induction of the homologous allele is detectable, and (2) the induction of not only a paired normal allele but also an unpaired one.

Alleles↗

PubMed1991

Molecular genetics of the Posterior sex combs/Suppressor 2 of zeste region of Drosophila: aberrant expression of the Suppressor 2 of zeste gene results in abnormal bristle development.

We report the molecular characterization of the Posterior sex combs-Suppressor 2 of zeste region of Drosophila melanogaster. The distal breakpoint of the Aristapedioid inversion divides the region into two parts. We have molecularly mapped the lesions associated with several loss of function mutations in the Polycomb group gene Posterior sex combs (Psc) proximal to this breakpoint. In addition, we have found that lesions associated with several loss of function mutations in the Suppressor 2 of zeste [Su(z)2] gene lie distal to this breakpoint. Since the breakpoint does not cause a loss of function in either gene, no essential sequences are shared by these two neighboring genes. There are three dominant gain of function mutations in the region that result in abnormal bristle development. We find that all three juxtapose foreign DNA sequences upstream of the Su(z)2 gene, and that at least two of these mutations (Arp1 and vgD) behave genetically as gain of function mutations in Su(z)2. Northern and in situ hybridization analyses show that the mutations result in increased accumulation of the Su(z)2 mRNA, which we argue is responsible for the bristle loss phenotype.

Animals↗

PubMed1992

Genetic analysis of the additional sex combs locus of Drosophila melanogaster.

Additional sex combs (Asx) is a member of the Polycomb group of genes, which are thought to be required for maintenance of chromatin structure. To better understand the function of Asx, we have isolated nine new alleles, each of which acts like a gain of function mutation. Asx is required for normal determination of segment identity. AsxP1 shows an unusual phenotype in that anterior and posterior homeotic transformations are seen in the same individuals, suggesting that AsxP1 might upset chromatin structure in a way that makes both activation and repression of homeotic genes more difficult. Analysis of embryonic and adult phenotypes of Asx alleles suggests that Asx is required zygotically for determination of segment number and polarity. The expression pattern of even-skipped is altered in Asx mutant embryos, suggesting that Asx is required for normal expression of this gene. We have transposon-tagged the Asx gene, and can thus begin molecular analysis of its function.

Alleles↗

PubMed1996

The Drosophila ash1 gene product, which is localized at specific sites on polytene chromosomes, contains a SET domain and a PHD finger.

The determined state of Drosophila imaginal discs depends on stable patterns of homeotic gene expression. The stability of these patterns requires the function of the ash1 gene, a member of the trithorax group. The primary translation product of the 7.5-kb ash1 transcript is predicted to be a basic protein of 2144 amino acids. The ASH1 protein contains a SET domain and a PHD finger. Both of these motifs are found in the products of some trithorax group and Polycomb group genes. We have determined the nucleotide sequence alterations in 10 ash1 mutant alleles and have examined their mutant phenotype. The best candidate for a null allele is ash1. The truncated protein product of this mutant allele is predicted to contain only 47 amino acids. The ASH1 protein is localized on polytene chromosomes of larval salivary glands at > 100 sites. The chromosomal localization of ASH1 implies that it functions at the transcriptional level to maintain the expression pattern of homeotic selector genes.

Amino Acid Sequence↗

PubMed1996

Molecular genetic analysis of Drosophila ash2, a member of the trithorax group required for imaginal disc pattern formation.

The ash2 gene is a member of the trithorax group of genes whose products function to maintain active transcription of homeotic selector genes. Mutations in ash2 cause the homeotic transformations expected for a gene in this group but, in addition, cause a variety of pattern formation defects that are not necessarily expected. The ash2 gene is located in cytogenetic region 96A17-19 flanked by slowpoke and tolloid and is included in a cosmid that contains part of slowpoke. The ash2 transcript is 2.0 kb and is present throughout development. The ASH2 protein predicted from the nucleotide sequence of the open reading frame has a putative double zinc-finger domain, called a PHD finger, that is present not only in the products of other trithorax group genes such as TRX and ASH1, but also in the product of a Polycomb group gene, PCL. Polyclonal antibodies directed against ASH2 detect the protein in imaginal discs and in the nuclei of salivary gland and fat body cells. On immunoblots these affinity-purified antibodies detect a 70-kDa protein in larvae and a 53-kDa protein in pupae.

Amino Acid Sequence↗