African-American TOMM40'523-APOE haplotypes are admixture of West African and Caucasian alleles.

BACKGROUND: Several studies have demonstrated a lower apolipoprotein E4 (APOE ε4) allele frequency in African-Americans, but yet an increased age-related prevalence of AD. An algorithm for prevention clinical trials incorporating TOMM40'523 (Translocase of Outer Mitochondria Membrane) and APOE depends on accurate TOMM40'523-APOE haplotypes. METHODS: We have compared the APOE and TOMM40'523 phased haplotype frequencies of a 9.5 kb TOMM40/APOE genomic region in West African, Caucasian, and African-American cohorts. RESULTS: African-American haplotype frequency scans of poly-T lengths connected in phase with either APOE ε4 or APOE ε3 differ from both West Africans and Caucasians and represent admixture of several distinct West African and Caucasian haplotypes. A new West African TOMM40'523 haplotype, with APOE ε4 connected to a short TOMM40'523 allele, is observed in African-Americans but not Caucasians. CONCLUSION: These data have therapeutic implications for the age of onset risk algorithm estimates and the design of a prevention trial for African-Americans or other mixed ethnic populations.

BORIS/CTCFL-mediated transcriptional regulation of the hTERT telomerase gene in testicular and ovarian tumor cells.

Telomerase activity, not detectable in somatic cells but frequently activated during carcinogenesis, confers immortality to tumors. Mechanisms governing expression of the catalytic subunit hTERT, the limiting factor for telomerase activity, still remain unclear. We previously proposed a model in which the binding of the transcription factor CTCF to the two first exons of hTERT results in transcriptional inhibition in normal cells. This inhibition is abrogated, however, by methylation of CTCF binding sites in 85% of tumors. Here, we showed that hTERT was unmethylated in testicular and ovarian tumors and in derivative cell lines. We demonstrated that CTCF and its paralogue, BORIS/CTCFL, were both present in the nucleus of the same cancer cells and bound to the first exon of hTERT in vivo. Moreover, exogenous BORIS expression in normal BORIS-negative cells was sufficient to activate hTERT transcription with an increasing number of cell passages. Thus, expression of BORIS was sufficient to allow hTERT transcription in normal cells and to counteract the inhibitory effect of CTCF in testicular and ovarian tumor cells. These results define an important contribution of BORIS to immortalization during tumorigenesis.

Transcription factor BORIS (Brother of the Regulator of Imprinted Sites) directly induces expression of a cancer-testis antigen, TSP50, through regulated binding of BORIS to the promoter.

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.

Multiple nucleosome positioning sites regulate the CTCF-mediated insulator function of the H19 imprinting control region.

The 5' region of the H19 gene harbors a methylation-sensitive chromatin insulator within an imprinting control region (ICR). Insertional mutagenesis in combination with episomal assays identified nucleosome positioning sequences (NPSs) that set the stage for the remarkably precise distribution of the four target sites for the chromatin insulator protein CTCF to nucleosome linker sequences in the H19 ICR. Changing positions of the NPSs resulted in loss of both CTCF target site occupancy and insulator function, suggesting that the NPSs optimize the fidelity of the insulator function. We propose that the NPSs ensure the fidelity of the repressed status of the maternal Igf2 allele during development by constitutively maintaining availability of the CTCF target sites.

A novel mechanism for CTCF in the epigenetic regulation of Bax in breast cancer cells.

We previously reported the association of elevated levels of the multifunctional transcription factor, CCCTC binding factor (CTCF), in breast cancer cells with the specific anti-apoptotic function of CTCF. To understand the molecular mechanisms of this phenomenon, we investigated regulation of the human Bax gene by CTCF in breast and non-breast cells. Two CTCF binding sites (CTSs) within the Bax promoter were identified. In all cells, breast and non-breast, active histone modifications were present at these CTSs, DNA harboring this region was unmethylated, and levels of Bax mRNA and protein were similar. Nevertheless, up-regulation of Bax mRNA and protein and apoptotic cell death were observed only in breast cancer cells depleted of CTCF. We proposed that increased CTCF binding to the Bax promoter in breast cancer cells, by comparison with non-breast cells, may be mechanistically linked to the specific apoptotic phenotype in CTCF-depleted breast cancer cells. In this study, we show that CTCF binding was enriched at the Bax CTSs in breast cancer cells and tumors; in contrast, binding of other transcription factors (SP1, WT1, EGR1, and c-Myc) was generally increased in non-breast cells and normal breast tissues. Our findings suggest a novel mechanism for CTCF in the epigenetic regulation of Bax in breast cancer cells, whereby elevated levels of CTCF support preferential binding of CTCF to the Bax CTSs. In this context, CTCF functions as a transcriptional repressor counteracting influences of positive regulatory factors; depletion of breast cancer cells from CTCF therefore results in the activation of Bax and apoptosis.

A nuclear factor-binding domain in the 5'-untranslated region of the amyloid precursor protein promoter: implications for the regulation of gene expression.

BACKGROUND: The extracellular deposition of aggregated amyloid beta-protein is a neuropathological manifestation of Alzheimer disease and Down syndrome. The Amyloid beta-protein is derived from a group of larger differentially spliced proteins, the amyloid protein precursors (APP). Data suggests that the level of APP gene expression could contribute to the pathological processes leading to amyloid depositions. FINDINGS: The 5' untranslated region (UTR) of the APP gene, encompassing 147 base pairs between the transcriptional (+1) and the translational start site, was examined for its role in APP expression. Deletions close to the transcriptional start site reduced expression from the APP promoter in part by transcriptional mechanisms. However, deletions between position +50 and +104 had no effect on transcriptional activity while significantly reducing overall expression from the promoter. A nuclear factor-binding domain designated as DAPB was identified between position +72 and +115 of the 5'-APP-UTR. The binding-recognition sequence was localized between position +96 and +105. The same mutations that eliminated factor-binding also reduced expression from the APP promoter while having no effect on in vitro transcription or the RNA levels transcribed from transfected constructs. CONCLUSIONS: A nuclear factor-binding domain designated as DAPB was identified in the 5'-UTR of the APP gene. Elimination of factor-binding correlated with an overall decline in expression from the APP promoter while in vitro transcription and the total amount of in vivo transcribed RNA remained unaffected. This suggests that the binding-factor may have a function in post-transcriptional regulation, including nuclear export of mRNA.

The structural complexity of the human BORIS gene in gametogenesis and cancer.

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.

Two adjacent nuclear factor-binding domains activate expression from the human PRNP promoter.

BACKGROUND: The transmissible spongiform encephalopathies (TSEs) comprise a group of fatal degenerative neurological diseases in humans and other mammals. After infection, the cellular prion protein isoform PrPC is converted to the pathological PrPSC scrapie isoform. The continued conversion of PrPC to PrPSC requires de novo endogenous PrP synthesis for disease progression. The human prion protein gene (PRNP) promoter was therefore investigated to identify regulatory elements that could serve as targets for therapeutic intervention. FINDINGS: The human prion protein gene (PRNP) promoter from position -1593 to +134 relative to the putative transcriptional start site (+1) was analyzed by transient transfection in HeLa cells. Deletions from the 5' end between positions -1593 and -232 yielded little change in activity. A further 5' deletion at position -90 resulted in a decline in activity to a level of about 30% of the full-length value. DNase I footprinting of the region between positions -259 and +2 identified two adjacent protected domains designated as prpA (-116 to -143) and prpB (-147 to -186). Internal deletions combined with mobility shift electrophoresis and methylation interference assays indicated the presence of sequence specific nuclear factor complexes that bind to the prpA and prpB domains and activate expression from the human PRNP promoter in an additive fashion. CONCLUSION: Results from transient transfection, DNase I footprinting, mobility shift electrophoresis, and methylation interference experiments suggest that two DNase I protected domains designated as prpA and prpB are binding sites for as yet unidentified regulatory factors that independently activate expression from the PRNP promoter.

Coordinated activation of candidate proto-oncogenes and cancer testes antigens via promoter demethylation in head and neck cancer and lung cancer.

BACKGROUND: Epigenetic alterations have been implicated in the pathogenesis of solid tumors, however, proto-oncogenes activated by promoter demethylation have been sporadically reported. We used an integrative method to analyze expression in primary head and neck squamous cell carcinoma (HNSCC) and pharmacologically demethylated cell lines to identify aberrantly demethylated and expressed candidate proto-oncogenes and cancer testes antigens in HNSCC. METHODOLOGY/PRINCIPAL FINDINGS: We noted coordinated promoter demethylation and simultaneous transcriptional upregulation of proto-oncogene candidates with promoter homology, and phylogenetic footprinting of these promoters demonstrated potential recognition sites for the transcription factor BORIS. Aberrant BORIS expression correlated with upregulation of candidate proto-oncogenes in multiple human malignancies including primary non-small cell lung cancers and HNSCC, induced coordinated proto-oncogene specific promoter demethylation and expression in non-tumorigenic cells, and transformed NIH3T3 cells. CONCLUSIONS/SIGNIFICANCE: Coordinated, epigenetic unmasking of multiple genes with growth promoting activity occurs in aerodigestive cancers, and BORIS is implicated in the coordinated promoter demethylation and reactivation of epigenetically silenced genes in human cancers.

Familial cases of point mutations in the XIST promoter reveal a correlation between CTCF binding and pre-emptive choices of X chromosome inactivation.

The choice mechanisms that determine the future inactive X chromosome in somatic cells of female mammals involve the regulated expression of the XIST gene. A familial C(-43)G mutation in the XIST promoter results in skewing of X chromosome inactivation (XCI) towards the inactive X chromosome of heterozygous females, whereas a C(-43)A mutation found primarily in the active X chromosome results in the opposite skewing pattern. Both mutations point to the existence of a factor that might be responsible for the skewed patterns. Here we identify this factor as CTCF, a conserved protein with a 11 Zn-finger (ZF) domain that can mediate multiple sequence-specificity and interactions between DNA-bound CTCF molecules. We show that mouse and human Xist/XIST promoters contain one homologous CTCF-binding sequence with the matching dG-contacts, which in the human XIST include the -43 position within the DNase I footprint of CTCF. While the C(-43)A mutation abrogates CTCF binding, the C(-43)G mutation results in a dramatic increase in CTCF-binding efficiency by altering ZF-usage mode required for recognition of the altered dG-contacts of the mutant site. Thus, the skewing effect of the two -43C mutations correlates with their effects on CTCF binding. Finally, CTCF interacts with the XIST/Xist promoter only in female human and mouse cells. The interpretation that this reflected a preferential interaction with the promoter of the active Xist allele was confirmed in mouse fetal placenta. These observations are in keeping with the possibility that the choice of X chromosome inactivation reflects stabilization of a higher order chromatin conformation impinging on the CTCF-XIST promoter complex.

A region to the N-terminal side of the CTCF zinc finger domain is essential for activating transcription from the amyloid precursor protein promoter.

Transcription from the amyloid precursor protein (APP) promoter is largely dependent on a nuclear factor binding site designated as APBbeta. The protein that binds to this site is the multifunctional transcription factor CTCF, which consists of 727 amino acids and contains a domain of 11 zinc finger motifs that is flanked by 267 amino acids on the N-terminal side and 150 amino acids on the C-terminal side. Depleting HeLa cell nuclear extract of endogenous CTCF specifically reduced transcriptional activity from the APP promoter. However, transcriptional activity was restored by replenishing the depleted extract with recombinant CTCF. Deleting 201 amino acids from the C-terminal end of CTCF had no detrimental effect on transcriptional activation, whereas deleting either 248 or 284 amino acids from the N-terminal end abolished transcriptional activation. Competing endogenous CTCF in vivo was accomplished by cotransfecting COS-1 cells with a plasmid overexpressing CTCF constructs and a reporter plasmid containing the APP promoter. Under these conditions, an N-terminal deletion of CTCF reduced expression from the APP promoter, whereas the C-terminal deletion had no effect. These results demonstrate that CTCF activates transcription from the APP promoter and that the activation domain is located on the N-terminal side of the zinc finger domain.

The binding sites for the chromatin insulator protein CTCF map to DNA methylation-free domains genome-wide.

All known vertebrate chromatin insulators interact with the highly conserved, multivalent 11-zinc finger nuclear factor CTCF to demarcate expression domains by blocking enhancer or silencer signals in a position-dependent manner. Recent observations document that the properties of CTCF include reading and propagating the epigenetic state of the differentially methylated H19 imprinting control region. To assess whether these findings may reflect a universal role for CTCF targets, we identified more than 200 new CTCF target sites by generating DNA microarrays of clones derived from chromatin-immunopurified (ChIP) DNA followed by ChIP-on-chip hybridization analysis. Target sites include not only known loci involved in multiple cellular functions, such as metabolism, neurogenesis, growth, apoptosis, and signalling, but potentially also heterochromatic sequences. Using a novel insulator trapping assay, we also show that the majority of these targets manifest insulator functions with a continuous distribution of stringency. As these targets are generally DNA methylation-free as determined by antibodies against 5-methylcytidine and a methyl-binding protein (MBD2), a CTCF-based network correlates with genome-wide epigenetic states.

BORIS, a novel male germ-line-specific protein associated with epigenetic reprogramming events, shares the same 11-zinc-finger domain with CTCF, the insulator protein involved in reading imprinting marks in the soma.

CTCF, a conserved, ubiquitous, and highly versatile 11-zinc-finger factor involved in various aspects of gene regulation, forms methylation-sensitive insulators that regulate X chromosome inactivation and expression of imprinted genes. We document here the existence of a paralogous gene with the same exons encoding the 11-zinc-finger domain as mammalian CTCF genes and thus the same DNA-binding potential, but with distinct amino and carboxy termini. We named this gene BORIS for Brother of the Regulator of Imprinted Sites. BORIS is present only in the testis, and expressed in a mutually exclusive manner with CTCF during male germ cell development. We show here that erasure of methylation marks during male germ-line development is associated with dramatic up-regulation of BORIS and down-regulation of CTCF expression. Because BORIS bears the same DNA-binding domain that CTCF employs for recognition of methylation marks in soma, BORIS is a candidate protein for the elusive epigenetic reprogramming factor acting in the male germ line.