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1.
Genome Biol ; 16: 161, 2015 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-26268681

RESUMO

BACKGROUND: CTCF and BORIS (CTCFL), two paralogous mammalian proteins sharing nearly identical DNA binding domains, are thought to function in a mutually exclusive manner in DNA binding and transcriptional regulation. RESULTS: Here we show that these two proteins co-occupy a specific subset of regulatory elements consisting of clustered CTCF binding motifs (termed 2xCTSes). BORIS occupancy at 2xCTSes is largely invariant in BORIS-positive cancer cells, with the genomic pattern recapitulating the germline-specific BORIS binding to chromatin. In contrast to the single-motif CTCF target sites (1xCTSes), the 2xCTS elements are preferentially found at active promoters and enhancers, both in cancer and germ cells. 2xCTSes are also enriched in genomic regions that escape histone to protamine replacement in human and mouse sperm. Depletion of the BORIS gene leads to altered transcription of a large number of genes and the differentiation of K562 cells, while the ectopic expression of this CTCF paralog leads to specific changes in transcription in MCF7 cells. CONCLUSIONS: We discover two functionally and structurally different classes of CTCF binding regions, 2xCTSes and 1xCTSes, revealed by their predisposition to bind BORIS. We propose that 2xCTSes play key roles in the transcriptional program of cancer and germ cells.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Animais , Sítios de Ligação , Fator de Ligação a CCCTC , Linhagem Celular , Cromatina/química , DNA/química , DNA/metabolismo , Genoma , Humanos , Células K562 , Masculino , Camundongos , Neoplasias/genética , Motivos de Nucleotídeos , Ligação Proteica , Espermátides/metabolismo , Espermatozoides/metabolismo , Transcrição Gênica
2.
J Biol Chem ; 286(31): 27378-88, 2011 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-21659515

RESUMO

Cancer-testis antigens (CTAs) are normally expressed in testis but are aberrantly expressed in a variety of cancers with varying frequency. More than 100 proteins have been identified as CTA including testes-specific protease 50 (TSP50) and the testis-specific paralogue of CCCTC-binding factor, BORIS (brother of the regulator of imprinted sites). Because many CTAs are considered as excellent targets for tumor immunotherapy, understanding the regulatory mechanisms governing their expression is important. In this study we demonstrate that BORIS is directly responsible for the transcriptional activation of TSP50. We found two BORIS binding sites in the TSP50 promoter that are highly conserved between mouse and human. Mutations of the binding sites resulted in loss of BORIS binding and the ability of BORIS to activate the promoter. However, although expression of BORIS was essential, it was not sufficient for high expression of TSP50 in cancer cells. Further studies showed that binding of BORIS to the target sites was methylation-independent but was diminished by nucleosomal occupancy consistent with the findings that high expression of TSP50 was associated with increased DNase I sensitivity and high BORIS occupancy of the promoter. These findings indicate that BORIS-induced expression of TSP50 is governed by accessibility and binding of BORIS to the promoter. To our knowledge this is the first report of regulated expression of one CTA by another to be validated in a physiological context.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Regiões Promotoras Genéticas , Serina Endopeptidases/genética , Animais , Sequência de Bases , Sítios de Ligação , Primers do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Camundongos , Camundongos Knockout , Células NIH 3T3 , Reação em Cadeia da Polimerase , Ligação Proteica , Homologia de Sequência do Ácido Nucleico , Serina Endopeptidases/metabolismo
3.
PLoS One ; 5(11): e13872, 2010 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-21079786

RESUMO

BACKGROUND: BORIS/CTCFL is a paralogue of CTCF, the major epigenetic regulator of vertebrate genomes. BORIS is normally expressed only in germ cells but is aberrantly activated in numerous cancers. While recent studies demonstrated that BORIS is a transcriptional activator of testis-specific genes, little is generally known about its biological and molecular functions. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that BORIS is expressed as 23 isoforms in germline and cancer cells. The isoforms are comprised of alternative N- and C-termini combined with varying numbers of zinc fingers (ZF) in the DNA binding domain. The patterns of BORIS isoform expression are distinct in germ and cancer cells. Isoform expression is activated by downregulation of CTCF, upregulated by reduction in CpG methylation caused by inactivation of DNMT1 or DNMT3b, and repressed by activation of p53. Studies of ectopically expressed isoforms showed that all are translated and localized to the nucleus. Using the testis-specific cerebroside sulfotransferase (CST) promoter and the IGF2/H19 imprinting control region (ICR), it was shown that binding of BORIS isoforms to DNA targets in vitro is methylation-sensitive and depends on the number and specific composition of ZF. The ability to bind target DNA and the presence of a specific long amino terminus (N258) in different isoforms are necessary and sufficient to activate CST transcription. Comparative sequence analyses revealed an evolutionary burst in mammals with strong conservation of BORIS isoproteins among primates. CONCLUSIONS: The extensive repertoire of spliced BORIS variants in humans that confer distinct DNA binding and transcriptional activation properties, and their differential patterns of expression among germ cells and neoplastic cells suggest that the gene is involved in a range of functionally important aspects of both normal gametogenesis and cancer development. In addition, a burst in isoform diversification may be evolutionarily tied to unique aspects of primate speciation.


Assuntos
Processamento Alternativo , Proteínas de Ligação a DNA/genética , Gametogênese/genética , Regiões Promotoras Genéticas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Fator de Ligação a CCCTC , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Feminino , Regulação da Expressão Gênica , Células HCT116 , Células HEK293 , Humanos , Células K562 , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Microscopia de Fluorescência , Dados de Sequência Molecular , Neoplasias/genética , Neoplasias/patologia , Isoformas de Proteínas/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Testículo/citologia , Testículo/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Dedos de Zinco
4.
Mol Cell Biol ; 30(10): 2473-84, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20231363

RESUMO

Previously, it was shown that the CTCF paralogous gene, BORIS (brother of the regulator of imprinted sites) is expressed in male germ cells, but its function in spermatogenesis has not been defined. To develop an understanding of the functional activities of BORIS, we generated BORIS knockout (KO) mice. Mice homozygous for the null allele had a defect in spermatogenesis that resulted in small testes associated with increased cell death. The defect was evident as early as postnatal day 21 and was manifested by delayed production of haploid cells. By gene expression profiling, we found that transcript levels for Gal3st1 (also known as cerebroside sulfotransferase [CST]), known to play a crucial role in meiosis, were dramatically reduced in BORIS KO testes. We found that CST is expressed in testis as a novel testis-specific isoform, CST form F(TS), that has a short exon 1f. We showed that BORIS bound to and activated the promoter of CST form F(TS). Mutation of the BORIS binding site in the promoter reduced the ability of BORIS to activate the promoter. These findings define transcriptional regulation of CST expression as a critical role for BORIS in spermatogenesis.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Isoenzimas/metabolismo , Espermatogênese , Sulfotransferases/metabolismo , Testículo/enzimologia , Animais , Sequência de Bases , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Marcação de Genes , Humanos , Isoenzimas/genética , Masculino , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Espermatogênese/fisiologia , Sulfotransferases/genética
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