CTCF-binding sites flank CTG/CAG repeats and form a methylation-sensitive insulator at the DM1 locus.

An expansion of a CTG repeat at the DM1 locus causes myotonic dystrophy (DM) by altering the expression of the two adjacent genes, DMPK and SIX5, and through a toxic effect of the repeat-containing RNA. Here we identify two CTCF-binding sites that flank the CTG repeat and form an insulator element between DMPK and SIX5. Methylation of these sites prevents binding of CTCF, indicating that the DM1 locus methylation in congenital DM would disrupt insulator function. Furthermore, CTCF-binding sites are associated with CTG/CAG repeats at several other loci. We suggest a general role for CTG/CAG repeats as components of insulator elements at multiple sites in the human genome.

Dual role of DNA methylation inside and outside of CTCF-binding regions in the transcriptional regulation of the telomerase hTERT gene.

Expression of hTERT is the major limiting factor for telomerase activity. We previously showed that methylation of the hTERT promoter is necessary for its transcription and that CTCF can repress hTERT transcription by binding to the first exon. In this study, we used electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) to show that CTCF does not bind the methylated first exon of hTERT. Treatment of telomerase-positive cells with 5-azadC led to a strong demethylation of hTERT 5'-regulatory region, reactivation of CTCF binding and downregulation of hTERT. Although complete hTERT promoter methylation was associated with full transcriptional repression, detailed mapping showed that, in telomerase-positive cells, not all the CpG sites were methylated, especially in the promoter region. Using a methylation cassette assay, selective demethylation of 110 bp within the core promoter significantly increased hTERT transcriptional activity. This study underlines the dual role of DNA methylation in hTERT transcriptional regulation. In our model, hTERT methylation prevents binding of the CTCF repressor, but partial hypomethylation of the core promoter is necessary for hTERT expression.

DNA, but not protein vaccine based on mutated BORIS antigen significantly inhibits tumor growth and prolongs the survival of mice.

The ideal immunological target for cancer vaccine development would meet the criteria of tumor specificity, immunogenicity and vital dependency of the tumor on the functional activities of the antigenic target so as to avoid antigenic loss by mutation. Given that at face value the brother of regulator of imprinted sites (BORIS) transcription factor meets these criteria, we have developed a mutant variant of this molecule (mBORIS) that lacks tumorigenic ability, while retaining immunogenic epitopes that elicits responses against histologically irrelevant tumor cells. Here we compared vaccine strategies employing as an immunogen either mBORIS recombinant protein formulated in a strong Th1-type adjuvant, QuilA or DNA encoding this immunogen along with plasmids expressing interleukin (IL)12/IL18 molecular adjuvants. In both groups of vaccinated mice induction of tumor-specific immunity (antibody response, T-cell proliferation, cytokine production, T-cell cytotoxicity) as well as ability to inhibit growth of the aggressive breast cancer cell line and to prolong survival of vaccinated animals have been tested. We determined that DNA, but not recombinant protein vaccine, induced potent Th1-like T-cell recall responses that significantly inhibited tumor growth and prolongs the survival of vaccinated mice. These studies demonstrate that DNA immunization is superior to recombinant protein strategy and provide a clear guidance for clinical development of a cancer vaccine targeting what appears to be a universal tumor antigen.

Functional association of CTCF with the insulator upstream of the H19 gene is parent of origin-specific and methylation-sensitive.

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 H19 genes, which are expressed mono-allelically from opposite parental alleles [1] [2]. The repressed status of the maternal Igf2 allele is due to cis elements that prevent the H19 enhancers [3] from accessing the Igf2 promoters on the maternal chromosome [4] [5]. A differentially methylated domain (DMD) in the 5' flank of H19 is 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 H19 DMD 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 H19 DMD 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 vitro and in vivo.

CpG methylation regulates the Igf2/H19 insulator.

The differentially methylated 5'-flank of the mouse H19 gene unidirectionally regulates the communication between enhancer elements and gene promoters and presumably represses maternal Igf2 expression in vivo [1-6]. The specific activation of the paternally inherited Igf2 allele has been proposed to involve methylation-mediated inactivation of the H19 insulator function during male germline development [1-4, 6]. Here, we addressed the role of methylation by inserting a methylated fragment of the H19-imprinting control region (ICR) into a nonmethylated episomal H19 minigene construct, followed by the transfection of ligation mixture into Hep3B cells. Individual clones were expanded and analyzed for genotype, methylation status, chromatin conformation, and insulator function. The results show that the methylated status of the H19 ICR could be propagated for several passages without spreading into the episomal vector. Moreover, the nuclease hypersensitive sites, which are typical for the maternally inherited H19 ICR allele [1], were absent on the methylated ICR, underscoring the suggestion that the methylation mark dictates parent of origin-specific chromatin conformations [1] that involve CTCF [2]. Finally, the insulator function was strongly attenuated in stably maintained episomes. Collectively, these results provide the first experimental support that the H19 insulator function is regulated by CpG methylation.

CTCF is a uniquely versatile transcription regulator linked to epigenetics and disease.

CTCF is an evolutionarily conserved zinc finger (ZF) phosphoprotein that binds through combinatorial use of its 11 ZFs to approximately 50 bp target sites that have remarkable sequence variation. Formation of different CTCF-DNA complexes, some of which are methylation-sensitive, results in distinct functions, including gene activation, repression, silencing and chromatin insulation. Disrupting the spectrum of target specificities by ZF mutations or by abnormal selective methylation of targets is associated with cancer. CTCF emerges, therefore, as a central player in networks linking expression domains with epigenetics and cell growth regulation.

CTCF, a conserved nuclear factor required for optimal transcriptional activity of the chicken c-myc gene, is an 11-Zn-finger protein differentially expressed in multiple forms.

A novel sequence-specific DNA-binding protein, CTCF, which interacts with the chicken c-myc gene promoter, has been identified and partially characterized (V. V. Lobanenkov, R. H. Nicolas, V. V. Adler, H. Paterson, E. M. Klenova, A. V. Polotskaja, and G. H. Goodwin, Oncogene 5:1743-1753, 1990). In order to test directly whether binding of CTCF to one specific DNA region of the c-myc promoter is important for chicken c-myc transcription, we have determined which nucleotides within this GC-rich region are responsible for recognition of overlapping sites by CTCF and Sp1-like proteins. Using missing-contact analysis of all four nucleotides in both DNA strands and homogeneous CTCF protein purified by sequence-specific chromatography, we have identified three sets of nucleotides which contact either CTCF or two Sp1-like proteins binding within the same DNA region. Specific mutations of 3 of 15 purines required for CTCF binding were designed to eliminate binding of CTCF without altering the binding of other proteins. Electrophoretic mobility shift assay of nuclear extracts showed that the mutant DNA sequence did not bind CTCF but did bind two Sp1-like proteins. When introduced into a 3.3-kbp-long 5'-flanking noncoding c-myc sequence fused to a reporter CAT gene, the same mutation of the CTCF binding site resulted in 10- and 3-fold reductions, respectively, of transcription in two different (erythroid and myeloid) stably transfected chicken cell lines. Isolation and analysis of the CTCF cDNA encoding an 82-kDa form of CTCF protein shows that DNA-binding domain of CTCF is composed of 11 Zn fingers: 10 are of C2H2 class, and 1 is of C2HC class. CTCF was found to be abundant and conserved in cells of vertebrate species. We detected six major nuclear forms of CTCF protein differentially expressed in different chicken cell lines and tissues. We conclude that isoforms of 11-Zn-finger factor CTCF which are present in chicken hematopoietic HD3 and BM2 cells can act as a positive regulator of the chicken c-myc gene transcription. Possible functions of other CTCF forms are discussed.

Negative protein 1, which is required for function of the chicken lysozyme gene silencer in conjunction with hormone receptors, is identical to the multivalent zinc finger repressor CTCF.

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-myc genes. 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-myc P2 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.

An exceptionally conserved transcriptional repressor, CTCF, employs different combinations of zinc fingers to bind diverged promoter sequences of avian and mammalian c-myc oncogenes.

We have isolated and analyzed human CTCF cDNA clones and show here that the ubiquitously expressed 11-zinc-finger factor CTCF is an exceptionally highly conserved protein displaying 93% identity between avian and human amino acid sequences. It binds specifically to regulatory sequences in the promoter-proximal regions of chicken, mouse, and human c-myc oncogenes. CTCF contains two transcription repressor domains transferable to a heterologous DNA binding domain. One CTCF binding site, conserved in mouse and human c-myc genes, is found immediately downstream of the major P2 promoter at a sequence which maps precisely within the region of RNA polymerase II pausing and release. Gel shift assays of nuclear extracts from mouse and human cells show that CTCF is the predominant factor binding to this sequence. Mutational analysis of the P2-proximal CTCF binding site and transient-cotransfection experiments demonstrate that CTCF is a transcriptional repressor of the human c-myc gene. Although there is 100% sequence identity in the DNA binding domains of the avian and human CTCF proteins, the regulatory sequences recognized by CTCF in chicken and human c-myc promoters are clearly diverged. Mutating the contact nucleotides confirms that CTCF binding to the human c-myc P2 promoter requires a number of unique contact DNA bases that are absent in the chicken c-myc CTCF binding site. Moreover, proteolytic-protection assays indicate that several more CTCF Zn fingers are involved in contacting the human CTCF binding site than the chicken site. Gel shift assays utilizing successively deleted Zn finger domains indicate that CTCF Zn fingers 2 to 7 are involved in binding to the chicken c-myc promoter, while fingers 3 to 11 mediate CTCF binding to the human promoter. This flexibility in Zn finger usage reveals CTCF to be a unique "multivalent" transcriptional factor and provides the first feasible explanation of how certain homologous genes (i.e., c-myc) of different vertebrate species are regulated by the same factor and maintain similar expression patterns despite significant promoter sequence divergence.

Functional phosphorylation sites in the C-terminal region of the multivalent multifunctional transcriptional factor CTCF.

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 S(604)KKEDS(609)S(610)DS(612)E 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-myc promoters, but did not alter CTCF nuclear localization or in vitro DNA-binding characteristics assayed with c-myc CTSs. 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.

Transcriptional repression by the insulator protein CTCF involves histone deacetylases.

The highly conserved zinc-finger protein, CTCF, is a candidate tumor suppressor protein that binds to highly divergent DNA sequences. CTCF has been connected to multiple functions in chromatin organization and gene regulation including chromatin insulator activity and transcriptional enhancement and silencing. Here we show that CTCF harbors several autonomous repression domains. One of these domains, the zinc-finger cluster, silences transcription in all cell types tested and binds directly to the co-repressor SIN3A. Two distinct regions of SIN3A, the PAH3 domain and the extreme C-terminal region, bind independently to this zinc-finger cluster. Analysis of nuclear extract from HeLa cells revealed that CTCF is also capable of retaining functional histone deacetylase activity. Furthermore, the ability of regions of CTCF to retain deacetylase activity correlates with the ability to bind to SIN3A and to repress gene activity. We suggest that CTCF driven repression is mediated in part by the recruitment of histone deacetylase activity by SIN3A.

Loss of imprinting of insulin-like growth factor-II in Wilms' tumor commonly involves altered methylation but not mutations of CTCF or its binding site.

Loss of imprinting (LOI) is the most common molecular abnormality in Wilms' tumor (WT), other embryonal cancers, and most other tumor types. LOI in WT involves activation of the normally silent maternal allele of the insulin-like growth factor-II (IGF2) gene, silencing of the normally active maternal allele of the H19 gene, and aberrant methylation of a differentially methylated region (DMR) upstream of the maternal copy of H19. Recently, the transcription factor CTCF, which binds to the H19 DMR, has been implicated in the maintenance of H19 and IGF2 imprinting. Here, we show that mutations in the CTCF gene or in the H19 DMR do not occur at significant frequency in WT, nor is there transcriptional silencing of CTCF. We also confirm that methylation of the H19 DMR in WT with LOI includes the CTCF core consensus site. However, some WTs with normal imprinting of IGF2 also show aberrant methylation of CTCF binding sites, indicating that methylation of these sites is necessary but not sufficient for LOI in WT.

Cell growth inhibition by the multifunctional multivalent zinc-finger factor CTCF.

The 11-zinc finger protein CCTC-binding factor (CTCF) employs different sets of zinc fingers to form distinct complexes with varying CTCF- target sequences (CTSs) that mediate the repression or activation of gene expression and the creation of hormone-responsive gene silencers and of diverse vertebrate enhancer-blocking elements (chromatin insulators). To determine how these varying effects would integrate in vivo, we engineered a variety of expression systems to study effects of CTCF on cell growth. Here we show that ectopic expression of CTCF in many cell types inhibits cell clonogenicity by causing profound growth retardation without apoptosis. In asynchronous cultures, the cell-cycle profile of CTCF-expressing cells remained unaltered, which suggested that progression through the cycle was slowed at multiple points. Although conditionally induced CTCF caused the S-phase block, CTCF can also arrest cell division. Viable CTCF-expressing cells could be maintained without dividing for several days. While MYC is the well-characterized CTCF target, the inhibitory effects of CTCF on cell growth could not be ascribed solely to repression of MYC, suggesting that additional CTS-driven genes involved in growth-regulatory circuits, such as p19ARF, are likely to contribute to CTCF-induced growth arrest. These findings indicate that CTCF may regulate cell-cycle progression at multiple steps within the cycle, and add to the growing evidence for the function of CTCF as a tumor suppressor gene.

A novel sequence-specific DNA binding protein which interacts with three regularly spaced direct repeats of the CCCTC-motif in the 5'-flanking sequence of the chicken c-myc gene.

The chicken c-myc 5'-flanking sequence has previously been shown to bind multiple proteins present in undifferentiated and differentiated red blood cells. In this report the protein binding to one specific region within a hypersensitive site approximately 200 base pairs upstream of the start of transcription has been analysed in detail. Using a combination of a modified agarose gel retardation assay with O-phenanthroline-copper footprinting in situ, missing contact point and methylation interference techniques, two proteins were found to bind to overlapping sequences within 180-230 bp upstream of the start of transcription. One protein resembles the transcription factor Sp1, the other is a protein which binds to three regularly spaced repeats of the core sequence CCCTC. This CCCTC-binding factor was termed CTCF. It requires additional sequences outside the three recognition motifs for tight binding. CTCF was purified to near homogeneity by sequence-specific DNA chromatography. The approximate molecular weight of the CTCF was estimated to be 130,000. Removal of 110 bp sequence binding both CTCF and Sp1-like proteins leads to a 4 to 8-fold increase in transcription of stably transfected c-myc fusion constructs in chicken embryonic fibroblasts, suggesting that the CTCF is likely to be one of multiple nuclear factors involved in the transcriptional regulation of the chicken c-myc gene.

A liver-specific nuclear protein that binds to the distal promoter element of the rat tyrosine aminotransferase gene.

Using a gel retardation assay and exonuclease III footprinting, we have analyzed sequence-specific DNA-binding nuclear factors which interact with the distal promoter element of the rat tyrosine aminotransferase gene. A factor called LspA1, binding to a sequence that resembles the consensus binding site for the transcription factor Ap-1, was shown to be present in adult rat-liver nuclear protein extracts but not in the extracts from embryonic liver or spleen nuclei.

[Chromatin proteins specifically interacting with certain DNA sequences. Nuclear factor I]. / Belki khromatina, spetsifichno vzaimodeistvuiushchie s opredelennymi posledovatel'nostiami DNK. Iadernyi faktor I.

A comprehensive survey of the data on the nuclear factor I (NF-I) published by the end of 1987 is given. Among all the known DNA-binding chromosomal protein factors which are capable of specific interaction with certain nucleotide sequences, NF-I compels a particular attention as the first example of a conservative multifunctional factor being involved in the assembly of nuclease-hypersensitive chromatin structure within regulatory regions of active genes, in the trans-activation of RNA polymerase II transcription, and also in the replication of the adenoviral genome and presumably in the replication of some other DNAs including the cellular one.

[Occurrence of DNA structures which differ from the canonic B-form in sites of highly specific interaction with chromatin proteins]. / Vstrechaemost' struktur DNK, otlichnykh ot kanonicheskoi B-formy, v uchastkakh vysokospetsifichnogo vzaimodeistviia s belkami khromatina.

According to the three-dimensional structure of DNase I and the mechanism of its action on linear double-stranded DNA, helix regions in conformations considerably different from the canonical B-form should be resistant to endonucleolysis. A number of DNA sequences specifically bound by nonhistone factors within 5'-flanking regions of the chicken beta A-globin, beta H-globin and c-myc genes are shown to contain short DNase I-resistant DNA domains. Several examples of the occurrence of such DNase I-resistant domains within the sites for high-specific recognition by different proteins are given. The role of the DNA structural polymorphism in site-specific interaction with protein factors is discussed.

[Regulatory protein factor CTCF interacts with a segment of the chicken c-myc oncogene promotor, capable of changing to a noncanonical conformation]. / Reguliatornyi belkovyi faktor CTCF vzaimodeistvuet s uchastkom promotora onkogena c-myc kur, sposobnym k perekhody v nekanonicheskuiu konformatsiiu.

To identify the regions in the chicken c-myc promoter that are necessary for the binding of a nuclear trans-acting factor CTCF--the potential oncogene activator--we used a synthetic analog of the natural binding site that contains three correctly spaced CCCTC-repeats that are known to be involved in CTCF-binding. Gel retardation experiments failed to detect any CTCF-binding activity with this synthetic site. We conclude that GC-transversions made in the regions presumed to be invalid, do in fact interfere with the protein binding. The secondary structure analysis with S1-nuclease shows the presence of an unusual DNA conformation of the CTCF-binding site in the supercoiled plasmids, that can not be detected with the artificial construction. The precise mapping of S1 nuclease cleavage reveals several hypersensitive sites in the CCCTC-zone. Thus, an altered secondary structure may be functionally important for the protein recognition in vivo.

[Specific interaction of a nuclear protein factor from the CTF/NF-I family with 2 different promoter regions of the rat tyrosine aminotransferase gene]. / Spetsificheskoe vzaimodeistvie iadernogo belkovogo faktora semeistva CTF/NF-I s dvumia raznymi uchastkami promotora gena tirozinaminotransferazy krysy.

Using gel-retardation and DNase I footprinting assays, we have analysed sequence-specific DNA-protein interactions within proximal promoter fragment (from -2 to -210 bp relative to the transcription start) of the rat tyrosine aminotransferase (TAT) gene. Two distinct DNase I protection regions flanked at either boundary by sites of DNase I hypersensitivity were observed with the rat-liver nuclear extracts. The internal sequence of the region I non-coding strand, (-155)TGGGCCACCTTCCAAT(-170), is highly homologous to the NF-I consensus sequence TGG(N)6-7TGCCAA and also shares a CCAAT-box; the region II noncoding strand sequence includes asymmetrically positioned (-37)AGCCAAT(-43) recognition motif. Since there have been a number of reports about multiple DNA-binding factors that recognize CCAAT homologies, both regions were likely to interact with either a single or distinct factors. Here we show that both region I and II of the TAT gene promoter are binding to the same factor related to the human CTF/NF-I. The evidence for that is based on competition experiments using the DNA fragment containing a synthetic consensus sequence for the NF-I recognition site and on the indistinguishable chromatographic properties of the activity specifically binding to each of three DNA fragments containing NF-I consensus, region I and region II sequences.

[Nuclear factor A5 from the rat liver that requires a metal for specific interaction in vitro with distal element of the tyrosine aminotransferase gene promoter]. / Iadernyi belkovyi faktor A5 iz pecheni krysy, kotoromu neobkhodim metall dlia spetsificheskogo vzaimodeistviia in vitro s distal'nym élementom promotora gena tirozinaminotransferazy.

To detect nuclear protein factors which might account for a tissue-specific and inducible expression of the rat tyrosine aminotransferase (TAT) gene promoter, extracts from rat liver and spleen nuclei have been fractionated by heparin-sepharose chromatography and the fractions assayed for sequence-specific binding to the distal TAT gene promoter element (sequence between -313 and -210). Gel retardation experiments carried out in the presence or absence of Mg2+, Ca2+, or Zn2+ ions showed that there are at least two nuclear factors (A3 and A4) binding to the distal promoter element only in the presence of the chelator (20 mM EDTA). Incubation of the protein fractions with Zn2+ or Ca2+ instead of commonly used Mg2+allowed: (i) to avoid 3 2P-DNA-probe degradation by "contaminating" endogenous nucleases; and (ii) to detect another sequence-specific nuclear factor, A5. No other specific binding activities were found in the rat-liver nuclear fractions tested under these conditions. As the metal ions became inaccessible to chelation in excess of EDTA and EGTA when protein factor A5 was complexed to DNA we assumed that factor A5 is metalloprotein which requires Zn or Ca to maintain a structure of its DNA-binding domain. To identify the polypeptide possessing this domain, a protein gel blotting procedure was employed. By incubating gel blots with the 3 2P-DNA-probe in the buffer containing Zn2+, specific binding to the only polypeptide with approximate Mr 30 kDa was clearly revealed. Both gel retardation and gel blotting assays consistently showed that nuclear factor A5 is present in the liver, but not in the spleen extracts.