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Background: Pervasive usage of alternative promoters leads to the deregulation of gene expression in carcinogenesis and may drive the emergence of new genes in spermatogenesis. However, little is known regarding the mechanisms underpinning the activation of alternative promoters. Results: Here we describe how alternative cancer-testis-specific transcription is activated. We show that intergenic and intronic CTCF binding sites, which are transcriptionally inert in normal somatic cells, could be epigenetically reprogrammed into active de novo promoters in germ and cancer cells. BORIS/CTCFL, the testis-specific paralog of the ubiquitously expressed CTCF, triggers the epigenetic reprogramming of CTCF sites into units of active transcription. BORIS binding initiates the recruitment of the chromatin remodeling factor, SRCAP, followed by the replacement of H2A histone with H2A.Z, resulting in a more relaxed chromatin state in the nucleosomes flanking the CTCF binding sites. The relaxation of chromatin around CTCF binding sites facilitates the recruitment of multiple additional transcription factors, thereby activating transcription from a given binding site. We demonstrate that the epigenetically reprogrammed CTCF binding sites can drive the expression of cancer-testis genes, long noncoding RNAs, retro-pseudogenes, and dormant transposable elements. Conclusions: Thus, BORIS functions as a transcription factor that epigenetically reprograms clustered CTCF binding sites into transcriptional start sites, promoting transcription from alternative promoters in both germ cells and cancer cells.

ABSTRACTCCCTC-binding factor (CTCF) is a conserved, essential regulator of chromatin architecture containing a unique array of 11 zinc fingers (ZFs). Gene duplication and sequence divergence during early amniote evolution generated the CTCF paralog Brother Of the Regulator of Imprinted Sites (BORIS), which has a DNA binding specificity identical to that of CTCF but divergent N- and C-termini. While healthy somatic tissues express only CTCF, CTCF and BORIS are normally co-expressed in meiotic and post-meiotic germ cells, and aberrant activation of BORIS occurs in tumors and some cancer cell lines. This has led to a model in which CTCF and BORIS compete for binding to some but not all genomic target sites; however, regulation of CTCF and BORIS genomic co-occupancy is not well understood. We recently addressed this issue, finding evidence for two major classes of CTCF target sequences, some of which contain single CTCF target sites (1xCTSes) and others containing two adjacent CTCF motifs (2xCTSes). The functional and chromatin structural features of 2xCTSes are distinct from those of 1xCTS-containing regions bound by a CTCF monomer. We suggest that these previously overlooked classes of CTCF binding regions may have different roles in regulating diverse chromatin-based phenomena, and may impact our understanding of heritable epigenetic regulation in cancer cells and normal germ cells.

Epigenetic alterations, particularly in DNA methylation, are ubiquitous in cancer, yet the molecular origins and the consequences of these alterations are poorly understood. CTCF, a DNA-binding protein that regulates higher-order chromatin organization, is frequently altered by hemizygous deletion or mutation in human cancer. To date, a causal role for CTCF in cancer has not been established. Here, we show that Ctcfhemizygous knockout mice are markedly susceptible to spontaneous, radiation-, and chemically induced cancer in a broad range of tissues. Ctcf+/−tumors are characterized by increased aggressiveness, including invasion, metastatic dissemination, and mixed epithelial/mesenchymal differentiation. Molecular analysis of Ctcf+/−tumors indicates that Ctcfis haploinsufficient for tumor suppression. Tissues with hemizygous loss of CTCFexhibit increased variability in CpG methylation genome wide. These findings establish CTCFas a prominent tumor-suppressor gene and point to CTCF-mediated epigenetic stability as a major barrier to neoplastic progression.

Animal models of human diseases are widely used to address questions of tumor development. Selection of a particular animal model depends upon a variety of factors, among them: animal cost, species lifespan, and hardiness; availability of biomolecular and genetic tools for that species; and evolutionary distance from humans. In spite of the growth in genomic data in the past several years, many animal models cannot yet be studied extensively due to gaps in genetic mapping, sequencing and functional analyses. Thus, alternative molecular genetic approaches are needed. We have designed an interspecies comparative genomic hybridization approach to analyze genetic changes in radiation-induced brain tumors in the non-human primate, Macaca mulatta. Using homologies between the primate and human genomes, we adapted widely-available CGH techniques to generate cytogenetic profiles of malignant gliomas in 4 monkey tumors. Losses and gains were projected onto the corresponding homologous chromosomal regions in the human genome, thus directly translating the status of the monkey gliomas into human gene content. This represents a novel method of comparative interspecies cytogenetic mapping that permits simultaneous analysis of genomic imbalance of unknown sequences in disparate species and correlation with potential or known human disease-related genes.

ABSTRACTCTCF is a widely expressed and highly conserved multi-Zn-finger (ZF) nuclear factor. Binding to various CTCF target sites (CTSs) is mediated by combinatorial contributions of different ZFs. Different CTSs mediate distinct CTCF functions in transcriptional regulation, including promoter repression or activation and hormone-responsive gene silencing. In addition, the necessary and sufficient core sequences of diverse enhancer-blocking (insulator) elements, including CpG methylation-sensitive ones, have recently been pinpointed to CTSs. To determine whether a posttranslational modification may modulate CTCF functions, we studied CTCF phosphorylation. We demonstrated that most of the modifications that occur at the carboxy terminus in vivo can be reproduced in vitro with casein kinase II (CKII). Major modification sites map to four serines within the S604KKEDS609S610DS612E motif that is highly conserved in vertebrates. Specific mutations of these serines abrogate phosphorylation of CTCF in vivo and CKII-induced phosphorylation in vitro. In addition, we showed that completely preventing phosphorylation by substituting all serines within this site resulted in markedly enhanced repression of the CTS-bearing vertebrate c-mycpromoters, but did not alter CTCF nuclear localization or in vitro DNA-binding characteristics assayed with c-mycCTSs. Moreover, these substitutions manifested a profound effect on negative cell growth regulation by wild-type CTCF. CKII may thus be responsible for attenuation of CTCF activity, either acting on its own or by providing the signal for phosphorylation by other kinases and for CTCF-interacting protein partners.

CTCF is a widely expressed and highly conserved multi-Zn-finger (ZF) nuclear factor. Binding to various CTCF target sites (CTSs) is mediated by combinatorial contributions of different ZFs. Different CTSs mediate distinct CTCF functions in transcriptional regulation, including promoter repression or activation and hormone-responsive gene silencing. In addition, the necessary and sufficient core sequences of diverse enhancer-blocking (insulator) elements, including CpG methylation-sensitive ones, have recently been pinpointed to CTSs. To determine whether a posttranslational modification may modulate CTCF functions, we studied CTCF phosphorylation. We demonstrated that most of the modifications that occur at the carboxy terminus in vivo can be reproduced in vitro with casein kinase II (CKII). Major modification sites map to four serines within the S604KKEDS609S610DS612E motif that is highly conserved in vertebrates. Specific mutations of these serines abrogate phosphorylation of CTCF in vivo and CKII-induced phosphorylation in vitro. In addition, we showed that completely preventing phosphorylation by substituting all serines within this site resulted in markedly enhanced repression of the CTS-bearing vertebrate c-mycpromoters, but did not alter CTCF nuclear localization or in vitro DNA-binding characteristics assayed with c-mycCTSs. Moreover, these substitutions manifested a profound effect on negative cell growth regulation by wild-type CTCF. CKII may thus be responsible for attenuation of CTCF activity, either acting on its own or by providing the signal for phosphorylation by other kinases and for CTCF-interacting protein partners.

CTCF is a unique, highly conserved, and ubiquitously expressed 11 zinc finger (ZF) transcriptional factor with multiple DNA site specificities. It is able to bind to varying target sequences to perform different regulatory roles, including promoter activation or repression, creating hormone-responsive gene silencing elements, and functional block of enhancer-promoter interactions. Because different sets of ZFs are utilized to recognize different CTCF target DNA sites, each of the diverse DNA·CTCF complexes might engage different essential protein partners to define distinct functional readouts. To identify such proteins, we developed an affinity chromatography method based on matrix-immobilized purified recombinant CTCF. This approach resulted in isolation of several CTCF protein partners. One of these was identified as the multifunctional Y-box DNA/RNA-binding factor, YB-1, known to be involved in transcription, replication, and RNA processing. We examined CTCF/YB-1 interaction by reciprocal immunoprecipitation experiments with anti-CTCF and anti-YB-1 antibodies, and found that CTCF and YB-1 form complexes in vivo. We show that the bacterially expressed ZF domain of CTCF is fully sufficient to retain YB-1 in vitro.To assess possible functional significance of CTCF/YB-1 binding, we employed the very first identified by us, negatively regulated, target for CTCF (c-myconcogene promoter) as a model in co-transfection assays with both CTCF and YB-1 expression vectors. Although expression of YB-1 alone had no effect, co-expression with CTCF resulted in a marked enhancement of CTCF-driven c-myctranscriptional repression. Thus our findings demonstrate, for the first time, the biological relevance of the CTCF/YB-1 interaction.

In mammals, a subset of genes inherit gametic marks that establish parent of origin-dependent expression patterns in the soma ([1]and references therein). The currently most extensively studied examples of this phenomenon, termed genomic imprinting, are the physically linked Igf2(insulin-like growth factor II) and H19genes, which are expressed mono-allelically from opposite parental alleles [1,2]. The repressed status of the maternal Igf2allele is due to ciselements that prevent the H19enhancers [3]from accessing the Igf2promoters on the maternal chromosome [4,5]. A differentially methylated domain (DMD) in the 5′ flank of H19is maintained paternally methylated and maternally unmethylated [6,7]. We show here by gel-shift and chromatin immunopurification analyses that binding of the highly conserved multivalent factor CTCF ([8,9]and references therein) to the H19DMD is methylation-sensitive and parent of origin-dependent. Selectively mutating CTCF-contacting nucleotides, which were identified by methylation interference within the extended binding sites initially revealed by nuclease footprinting, abrogated the H19DMD enhancer-blocking property. These observations suggest that molecular mechanisms of genomic imprinting may use an unusual ability of CTCF to interact with a diverse spectrum of variant target sites, some of which include CpGs that are responsible for methylation-sensitive CTCF binding in vitroand in vivo.

DNA target sites for a “multivalent” 11-zinc-finger CCTC-binding factor (CTCF) are unusually long (∼50 base pairs) and remarkably different. In conjunction with the thyroid receptor (TR), CTCF binding to the lysozyme gene transcriptional silencer mediates the thyroid hormone response element (TRE)-dependent transcriptional repression. We tested whether other TREs, which in addition to the presence of a TR binding site require neighboring sequences for transcriptional function, might also contain a previously unrecognized binding site(s) for CTCF. One such candidate DNA region, previously isolated by Bigler and Eisenman (Bigler, J., and Eisenman, R. N. (1995) EMBO J.14, 5710–5723), is the TRE-containing genomic element 144. We have identified a new CTCF target sequence that is adjacent to the TR binding site within the 144 fragment. Comparison of CTCF recognition nucleotides in the lysozyme silencer and in the 144 sequences revealed both similarities and differences. Several C-terminal CTCF zinc fingers contribute differently to binding each of these sequences. Mutations that eliminate CTCF binding impair 144-mediated negative transcriptional regulation. Thus, the 144 element provides an additional example of a functionally significant composite “TRE plus CTCF binding site” regulatory element suggesting an important role for CTCF in cooperation with the steroid/thyroid superfamily of nuclear receptors to mediate TRE-dependent transcriptional repression.

The transcriptional repressor negative protein 1 (NeP1) binds specifically to the F1 element of the chicken lysozyme gene silencer and mediates synergistic repression by v-ERBA, thyroid hormone receptor, or retinoic acid receptor. Another protein, CCCTC-binding factor (CTCF), specifically binds to 50-bp-long sequences that contain repetitive CCCTC elements in the vicinity of vertebrate c-mycgenes. Previously cloned chicken, mouse, and human CTCF cDNAs encode a highly conserved 11-Zn-finger protein. Here, NeP1 was purified and DNA bases critical for NeP1-F1 interaction were determined. NeP1 is found to bind a 50-bp stretch of nucleotides without any obvious sequence similarity to known CTCF binding sequences. Despite this remarkable difference, these two proteins are identical. They have the same molecular weight, and NeP1 contains peptide sequences which are identical to sequences in CTCF. Moreover, NeP1 and CTCF specifically recognize each other’s binding DNA sequence and induce identical conformational alterations in the F1 DNA. Therefore, we propose to replace the name NeP1 with CTCF. To analyze the puzzling sequence divergence in CTCF binding sites, we studied the DNA binding of 12 CTCF deletions with serially truncated Zn fingers. While fingers 4 to 11 are indispensable for CTCF binding to the human c-mycP2 promoter site A, a completely different combination of fingers, namely, 1 to 8 or 5 to 11, was sufficient to bind the lysozyme silencer site F1. Thus, CTCF is a true multivalent factor with multiple repressive functions and multiple sequence specificities.

CTCFis a multifunctional transcription factor encoded by a novel candidate tumor suppressor gene (Filippova, G. N., Lindblom, A., Meinke, L. J., Klenova, E. M., Neiman, P. E., Collins, S. J., Doggett, N. D., and Lobanenkov, V. V. (1998) Genes Chromosomes Cancer22, 26–36). We characterized genomic organization of the chicken CTCF(chCTCF) gene, and studied the chCTCFpromoter. Genomic locus of chCTCFcontains a GC-rich untranslated exon separated from seven coding exons by a long intron. The 2-kilobase pair region upstream of the major transcription start site contains a CpG island marked by a “Not-knot” that includes sequence motifs characteristic of a TATA-less promoter of housekeeping genes. When fused upstream of a reporter chloramphenicol acetyltransferase gene, it acts as a strong transcriptional promoter in transient transfection experiments. The minimal 180-base pair chCTCFpromoter region that is fully sufficient to confer high level transcriptional activity to the reporter contains high affinity binding element for the transcription factor YY1. This element is strictly conserved in chicken, mouse, and human CTCFgenes. Mutations in the core nucleotides of the YY1 element reduce transcriptional activity of the minimal chCTCFpromoter, indicating that the conserved YY1-binding sequence is critical for transcriptional regulation of vertebrate CTCFgenes. We also noted in thechCTCFpromoter several elements previously characterized in cell cycle-regulated genes, including the “cell cycle-dependent element” and “cell cycle gene homology region” motifs shown to be important for S/G2-specific up-regulation of cdc25C, cdc2, cyclin A, and Plk(polo-like kinase) gene promoters. Presence of the cell cycle-dependent element/cell cycle gene homology region element suggested that chCTCFexpression may be cell cycle-regulated. We show that both levels of the endogenous chCTCFmRNA, and the activity of the stably transfected chCTCFpromoter constructs, increase in S/G2cells.

CTCF belongs to the Zn finger transcription factors family and binds to the promoter region of c-myc. CTCF is highly conserved between species, ubiquitous and localised in nuclei. The endogenous CTCF migrates as a 130 kDa (CTCF-130) protein on SDS-PAGE, however, the open reading frame (ORF) of the CTCF cDNA encodes only a 82 kDa protein (CTCF-82). In the present study we investigate this phenomenon and show with mass-spectra analysis that this occurs due to aberrant mobility of the CTCF protein. Another paradox is that our original cDNA, composed of the ORF and 3′-untranslated region (3′-UTR), produces a protein with the apparent molecular weight of 70 kDa (CTCF-70). This paradox has been found to be an effect of the UTRs and sequences within the coding region of the CTCF gene resulting in C-terminal truncation of CTCF-130. The potential attenuator has been identified and point-mutated. This restored the electrophoretic mobility of the CTCF protein to 130 kDa. CTCF-70, the aberrantly migrating CTCF N-terminus per se, is also detected in some cell types and therefore may have some biological implications. In particular, CTCF-70 interferes with CTCF-130 normal function, enhancing transactivation induced by CTCF-130 in COS6 cells. The mechanism of CTCF-70 action and other possible functions of CTCF-70 are discussed.