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-zincfinger domain as mammalian CTCF genes and thus the same DNAbinding 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.

The promoter of the amyloid ␤-protein precursor (APP) gene directs high levels of cell type-specific transcription with 94 base pairs 5 to the main transcriptional start site. An essential activator domain in this proximal APP promoter is a nuclear factor binding site designated as APB␤. The recognition domain for the APB␤ binding factor is located between position ؊93 and ؊82 relative to the main transcriptional start site.The nuclear factor that binds to the APB␤ site was partially purified by multiple steps of ion exchange and hydroxyapatite chromatography. Based on UV crosslinking results, a protein with an apparent molecular mass of 140 kDa was selected as the putative APB␤ binding protein. After the final purification step consisting of preparative SDS-polyacrylamide gel electrophoresis, partial peptide sequences were obtained that completely matched the transcriptional factor CTCF. This protein is a known regulator of c-myc and lysozyme gene expression, and it binds to a variety of diverse DNA sequences.The binding of CTCF to the APB␤ domain was further established by competition with CTCF binding oligonucleotides in mobility shift electrophoresis. The identity was also confirmed by the observation that the APB␤ binding factor is recognized by antibodies against Cand N-terminal sequences of CTCF. In addition, oligonucleotide competition during in vitro transcription affirmed that CTCF acts as a transcriptional activator in the APP gene promoter.

BackgroundEpigenetic 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 FindingsWe 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/SignificanceCoordinated, 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.

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.