Human CTCF Research Articles

The N-terminal dimerization domains of human and Drosophila CTCF have similar functionality

BackgroundCTCF is highly likely to be the ancestor of proteins that contain large clusters of C2H2 zinc finger domains, and its conservation is observed across most bilaterian organisms. In mammals, CTCF is the primary architectural protein involved in organizing chromosome topology and mediating enhancer–promoter interactions over long distances. In Drosophila, CTCF (dCTCF) cooperates with other architectural proteins to establish long-range interactions and chromatin boundaries. CTCFs of various organisms contain an unstructured N-terminal dimerization domain (DD) and clusters comprising eleven zinc-finger domains of the C2H2 type. The Drosophila (dCTCF) and human (hCTCF) CTCFs share sequence homology in only five C2H2 domains that specifically bind to a conserved 15 bp motif.ResultsPreviously, we demonstrated that CTCFs from different organisms carry unstructured N-terminal dimerization domains (DDs) that lack sequence homology. Here we used the CTCFattP(mCh) platform to introduce desired changes in the Drosophila CTCF gene and generated a series of transgenic lines expressing dCTCF with different variants of the N-terminal domain. Our findings revealed that the functionality of dCTCF is significantly affected by the deletion of the N-terminal DD. Additionally, we observed a strong impact on the binding of the dCTCF mutant to chromatin upon deletion of the DD. However, chromatin binding was restored in transgenic flies expressing a chimeric CTCF protein with the DD of hCTCF. Although the chimeric protein exhibited lower expression levels than those of the dCTCF variants, it efficiently bound to chromatin similarly to the wild type (wt) protein.ConclusionsOur findings suggest that one of the evolutionarily conserved functions of the unstructured N-terminal dimerization domain is to recruit dCTCF to its genomic sites in vivo.

CTCF: an R/bioconductor data package of human and mouse CTCF binding sites.

CTCF (CCCTC-binding factor) is an 11-zinc-finger DNA binding protein which regulates much of the eukaryotic genome's 3D structure and function. The diversity of CTCF binding motifs has led to a fragmented landscape of CTCF binding data. We collected position weight matrices of CTCF binding motifs and defined strand-oriented CTCF binding sites in the human and mouse genomes, including the recent Telomere to Telomere and mm39 assemblies. We included selected experimentally determined and predicted CTCF binding sites, such as CTCF-bound cis-regulatory elements from SCREEN ENCODE. We recommend filtering strategies for CTCF binding motifs and demonstrate that liftOver is a viable alternative to convert CTCF coordinates between assemblies. Our comprehensive data resource and usage recommendations can serve to harmonize and strengthen the reproducibility of genomic studies utilizing CTCF binding data. https://bioconductor.org/packages/CTCF. Companion website: https://dozmorovlab.github.io/CTCF/; Code to reproduce the analyses: https://github.com/dozmorovlab/CTCF.dev. Supplementary data are available at Bioinformatics Advances online.

Study of the N-Terminal Domain Homodimerization in Human Proteins with Zinc Finger Clusters

CTCF belongs to a large family of transcription factors with clusters of C2H2-type zinc finger domains (C2H2 proteins) and is a main architectural protein in mammals. Human CTCF has a homodimerizing unstructured domain at the N-terminus which is involved in long-distance interactions. To test the presence of similar N-terminal domains in other human C2H2 proteins, a yeast two-hybrid system was used. In total, the ability of unstructured N-terminal domains to homodimerize was investigated for six human C2H2 proteins with an expression profile similar to CTCF. The data indicate the lack of the homodimerization ability of these domains. On the other hand, three C2H2 proteins containing the structured domain DUF3669 at the N-terminus demonstrated homo- and heterodimerization activity.

Human CTCF Interacts with Drosophila CP190, but not with Kaiso.

Human CTCF (hCTCF) is a major architectural protein in mammals. In Drosophila, the CTCF homologue (dCTCF) interacts with the BTB domain of the CP190 protein, which is involved in the establishment of open chromatin and activity of insulators. Previously, it was shown that the BTB protein Kaiso interacts with hCTCF and regulates its activity. We have carried out a detailed study of the interaction between these proteins in the yeast two-hybrid assay. Surprisingly, Kaiso did not interact with hCTCF and its Drosophila homologue. On the other hand, CP190 interacted with the C-terminus of hCTCF. The results obtained demonstrate that the interaction between CTCF and CP190 proteins is highly conserved. It is likely that humans have other BTB proteins that perform the functions described for the Drosophila CP190.

Abstract 5228: Acute depletion of CTCF disrupts enhancer-promoter regulation ofMYCin B-cell acute lymphoblastic leukemia

Abstract Acute lymphoblastic leukemia (ALL), including B-ALL and T-ALL, is the most common childhood cancer and the leading cause of cancer death in children and young adults. Comprehensively genomic sequencing strategies have successfully sub-classified ALL based on genetic alterations including chromosomal rearrangement and aberrant gene mutations, of which frequent coding mutations of CTCF were discovered. However, the function of CTCF in ALL was not clearly defined yet. In a cohort of 264 human pediatric B-ALL patients, particularly in those carrying CTCF loss-of-function mutations, we identified a positive correlation of mRNA expression between CTCF and MYC. Then we integrated the miniAID-mClover3 cassette to the both alleles of endogenous human CTCF locus, which allows acute auxin-mediated degradation of the CTCF protein in a human pediatric B-ALL cell line. By employing combination strategies including whole transcriptome analysis and Capture-C, we discovered that only small number of genes were differentially expressed in CTCF depleted cells, among which MYC and it's downstream genes, not other B-ALL associated oncogenes, were specifically enriched. Mechanically, chromatin interaction capture assay reveals that acute depletion of CTCF primarily disrupted direct interaction between MYC promoter and its distal enhancer residing ~1.8 Mb downstream, subsequently down-regulating target genes in human B-ALL cancer cells. Thus, CTCF/MYC regulation axis is essential in B-ALL development. Citation Format: Chunliang Li, Yang Zhang. Acute depletion of CTCF disrupts enhancer-promoter regulation ofMYCin B-cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5228.

An alternative CTCF isoform antagonizes canonical CTCF occupancy and changes chromatin architecture to promote apoptosis

CTCF plays key roles in gene regulation, chromatin insulation, imprinting, X chromosome inactivation and organizing the higher-order chromatin architecture of mammalian genomes. Previous studies have mainly focused on the roles of the canonical CTCF isoform. Here, we explore the functions of an alternatively spliced human CTCF isoform in which exons 3 and 4 are skipped, producing a shorter isoform (CTCF-s). Functionally, we find that CTCF-s competes with the genome binding of canonical CTCF and binds a similar DNA sequence. CTCF-s binding disrupts CTCF/cohesin binding, alters CTCF-mediated chromatin looping and promotes the activation of IFI6 that leads to apoptosis. This effect is caused by an abnormal long-range interaction at the IFI6 enhancer and promoter. Taken together, this study reveals a non-canonical function for CTCF-s that antagonizes the genomic binding of canonical CTCF and cohesin, and that modulates chromatin looping and causes apoptosis by stimulating IFI6 expression.

DNA Binding Kinetics of CTCF in vitro

CTCF is a ubiquitously expressed transcription factor whose architectural role as an insulator is tied to its ability to form genomic loops and delimit boundaries of topologically associated domains (TADs). This capacity for binding of a single protein to affect distal regulation over megabases is both remarkable and poorly understood mechanistically. In current models, a cohesin‐associated complex expands a loop by tracking along DNA in both directions, while CTCF defines the loop size and position by halting this complex. However, a recent single molecule imaging study finds the DNA‐association lifetime of CTCF to be ten‐fold shorter than that of cohesin in vivo, suggesting that CTCF and cohesin act independently and dynamically (Hansen et al., 2017). This raises key questions about how CTCF forms and maintains domain boundaries and loops, and highlights the need for a careful study of CTCF‐DNA binding kinetics.Here, we purified human CTCF and characterized its DNA‐binding activity in vitro. Using mobility shift assays, we recapitulated the known high affinity of full length CTCF and the zinc finger domain for CTCF binding sites and its dependence on DNA methylation. We further used surface plasmon resonance to measure real time binding and unbinding kinetics of CTCF to methylated and unmethylated DNA targets. These experiments illuminate the role of DNA methylation in CTCF binding site selection, shed light on in vivo binding kinetics, and represent a first step towards the study of CTCF‐mediated looping in vitro.Support or Funding InformationThis research was supported by NIH Grants GM36659 and AI21144 (to R.D.K.) and a National Defense Science and Engineering Fellowship (to A.L.S.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

CTCF-Induced Circular DNA Complexes Observed by Atomic Force Microscopy

The CTCF protein has emerged as a key architectural protein involved in genome organization. Although hypothesized to initiate DNA looping, direct evidence of CTCF-induced DNA loop formation is still missing. Several studies have shown that the 11 zinc finger (11 ZF) domain of CTCF is actively involved in DNA binding. We here use atomic force microscopy to examine the effect of the 11 ZF domain comprising residues 266–579 (11 ZF CTCF) and the 3 ZF domain comprising residues 402–494 (6–8 ZF CTCF) of human CTCF on the DNA morphology. Our results show that both domains alter the DNA architecture from the relaxed morphology observed in control DNA samples to compact circular complexes, meshes, and networks, offering important insights into the multivalent character of the 11 ZF CTCF domain. Atomic force microscopy images reveal quasi-circular DNA/CTCF complexes, which are destabilized upon replacing the 11 ZF CTCF by the 6–8 ZF CTCF domain, highlighting the role of the 11 ZF motif in loop formation. Intriguingly, the formation of circular DNA/CTCF complexes is dominated by non-specific binding, whereby contour length and height profiles suggest a single DNA molecule twice wrapped around the protein.

Transcription and Remodeling Produce Asymmetrically Unwrapped Nucleosomal Intermediates

Nucleosomes are disrupted during transcription and other active processes, but the structural intermediates during nucleosome disruption invivo are unknown. To identify intermediates, we mapped subnucleosomal protections in Drosophila cells using Micrococcal Nuclease followed by sequencing. At the first nucleosome position downstream ofthetranscription start site, we identified unwrapped intermediates, including hexasomes that lackeitherproximal or distal contacts. Inhibiting topoisomerases or depleting histone chaperones increased unwrapping, whereas inhibiting release of paused RNAPII or reducing RNAPII elongation decreased unwrapping. Our results indicate that positive torsion generated by elongating RNAPII causes transient loss of histone-DNA contacts. Using this mapping approach, we found that nucleosomes flanking human CTCF insulation sites are similarly disrupted. We also identified diagnostic subnucleosomal particle remnants in cell-free human DNA data as a relic of transcribed genes from apoptosing cells. Thus identification of subnucleosomal fragments from nuclease protection datarepresents a general strategy for structural epigenomics.

Molecular mechanism of directional CTCF recognition of a diverse range of genomic sites.

CTCF, a conserved 3D genome architecture protein, determines proper genome-wide chromatin looping interactions through directional binding to specific sequence elements of four modules within numerous CTCF-binding sites (CBSs) by its 11 zinc fingers (ZFs). Here, we report four crystal structures of human CTCF in complex with CBSs of the protocadherin (Pcdh) clusters. We show that directional CTCF binding to cognate CBSs of the Pcdh enhancers and promoters is achieved through inserting its ZF3, ZFs 4-7, and ZFs 9-11 into the major groove along CBSs, resulting in a sequence-specific recognition of module 4, modules 3 and 2, and module 1, respectively; and ZF8 serves as a spacer element for variable distances between modules 1 and 2. In addition, the base contact with the asymmetric "A" in the central position of modules 2-3, is essential for directional recognition of the CBSs with symmetric core sequences but lacking module 1. Furthermore, CTCF tolerates base changes at specific positions within the degenerated CBS sequences, permitting genome-wide CTCF binding to a diverse range of CBSs. Together, these complex structures provide important insights into the molecular mechanisms for the directionality, diversity, flexibility, dynamics, and conservation of multivalent CTCF binding to its cognate sites across the entire human genome.

An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites.

We describe Cleavage Under Targets and Release Using Nuclease (CUT&RUN), a chromatin profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing. Unlike Chromatin Immunoprecipitation (ChIP), which fragments and solubilizes total chromatin, CUT&RUN is performed in situ, allowing for both quantitative high-resolution chromatin mapping and probing of the local chromatin environment. When applied to yeast and human nuclei, CUT&RUN yielded precise transcription factor profiles while avoiding crosslinking and solubilization issues. CUT&RUN is simple to perform and is inherently robust, with extremely low backgrounds requiring only ~1/10th the sequencing depth as ChIP, making CUT&RUN especially cost-effective for transcription factor and chromatin profiling. When used in conjunction with native ChIP-seq and applied to human CTCF, CUT&RUN mapped directional long range contact sites at high resolution. We conclude that in situ mapping of protein-DNA interactions by CUT&RUN is an attractive alternative to ChIP-seq.

MACE: model based analysis of ChIP-exo.

Understanding the role of a given transcription factor (TF) in regulating gene expression requires precise mapping of its binding sites in the genome. Chromatin immunoprecipitation-exo, an emerging technique using λ exonuclease to digest TF unbound DNA after ChIP, is designed to reveal transcription factor binding site (TFBS) boundaries with near-single nucleotide resolution. Although ChIP-exo promises deeper insights into transcription regulation, no dedicated bioinformatics tool exists to leverage its advantages. Most ChIP-seq and ChIP-chip analytic methods are not tailored for ChIP-exo, and thus cannot take full advantage of high-resolution ChIP-exo data. Here we describe a novel analysis framework, termed MACE (model-based analysis of ChIP-exo) dedicated to ChIP-exo data analysis. The MACE workflow consists of four steps: (i) sequencing data normalization and bias correction; (ii) signal consolidation and noise reduction; (iii) single-nucleotide resolution border peak detection using the Chebyshev Inequality and (iv) border matching using the Gale-Shapley stable matching algorithm. When applied to published human CTCF, yeast Reb1 and our own mouse ONECUT1/HNF6 ChIP-exo data, MACE is able to define TFBSs with high sensitivity, specificity and spatial resolution, as evidenced by multiple criteria including motif enrichment, sequence conservation, direct sequence pileup, nucleosome positioning and open chromatin states. In addition, we show that the fundamental advance of MACE is the identification of two boundaries of a TFBS with high resolution, whereas other methods only report a single location of the same event. The two boundaries help elucidate the in vivo binding structure of a given TF, e.g. whether the TF may bind as dimers or in a complex with other co-factors.

Identification and analysis of pig chimeric mRNAs using RNA sequencing data

BackgroundGene fusion is ubiquitous over the course of evolution. It is expected to increase the diversity and complexity of transcriptomes and proteomes through chimeric sequence segments or altered regulation. However, chimeric mRNAs in pigs remain unclear. Here we identified some chimeric mRNAs in pigs and analyzed the expression of them across individuals and breeds using RNA-sequencing data.ResultsThe present study identified 669 putative chimeric mRNAs in pigs, of which 251 chimeric candidates were detected in a set of RNA-sequencing data. The 618 candidates had clear trans-splicing sites, 537 of which obeyed the canonical GU-AG splice rule. Only two putative pig chimera variants whose fusion junction was overlapped with that of a known human chimeric mRNA were found. A set of unique chimeric events were considered middle variances in the expression across individuals and breeds, and revealed non-significant variance between sexes. Furthermore, the genomic region of the 5′ partner gene shares a similar DNA sequence with that of the 3′ partner gene for 458 putative chimeric mRNAs. The 81 of those shared DNA sequences significantly matched the known DNA-binding motifs in the JASPAR CORE database. Four DNA motifs shared in parental genomic regions had significant similarity with known human CTCF binding sites.ConclusionsThe present study provided detailed information on some pig chimeric mRNAs. We proposed a model that trans-acting factors, such as CTCF, induced the spatial organisation of parental genes to the same transcriptional factory so that parental genes were coordinatively transcribed to give birth to chimeric mRNAs.

Changes of the Nucleolus Architecture in Absence of the Nuclear Factor CTCF

CTCF is a multifunctional nuclear factor involved in many cellular processes like gene regulation, chromatin insulation and genomic organization. Recently, CTCF has been shown to be involved in the transcriptional regulation of ribosomal genes and nucleolar organization in Drosophila cells and different murine cell types, including embryonic stem cells. Moreover, it has been suggested that CTCF could be associated to the nucleolus of human erythroleukemic K562 cells. In the present work, we took advantage of efficient small hairpin RNA interference against human CTCF to analyze nucleolar organization in HeLa cells. We have found that key components of the nucleolar architecture are altered. As a consequence of such alterations, an upregulation of ribosomal gene transcription was observed. We propose that CTCF contributes to the structural organization of the nucleolus and, through epigenetic mechanisms, to the regulation of the ribosomal gene expression.

Comprehensive Genome-wide Protein-DNA Interactions Detected at Single-Nucleotide Resolution

Chromatin immunoprecipitation (ChIP-chip and ChIP-seq) assays identify where proteins bind throughout a genome. However, DNA contamination and DNA fragmentation heterogeneity produce false positives (erroneous calls) and imprecision in mapping. Consequently, stringent data filtering produces false negatives (missed calls). Here we describe ChIP-exo, where an exonuclease trims ChIP DNA to a precise distance from the crosslinking site. Bound locations are detectable as peak pairs by deep sequencing. Contaminating DNA is degraded or fails to form complementary peak pairs. With the single bp accuracy provided by ChIP-exo, we show an unprecedented view into genome-wide binding of the yeast transcription factors Reb1, Gal4, Phd1, Rap1, and human CTCF. Each of these factors was chosen to address potential limitations of ChIP-exo. We found that binding sites become unambiguous and reveal diverse tendencies governing in vivo DNA-binding specificity that include sequence variants, functionally distinct motifs, motif clustering, secondary interactions, and combinatorial modules within a compound motif.

Active promoters and insulators are marked by the centrosomal protein 190

For the compact Drosophila genome, several factors mediating insulator function, such as su(Hw) and dCTCF, have been identified. Recent analyses showed that both these insulator-binding factors are functionally dependent on the same cofactor, CP190. Here we analysed genome-wide binding of CP190 and dCTCF. CP190 binding was detected at CTCF, su(Hw) and GAF sites and unexpectedly at the transcriptional start sites of actively transcribed genes. Both insulator and transcription start site CP190-binding elements are strictly marked by a depletion of histone H3 and, therefore, a loss of nucleosome occupancy. In addition, CP190/dCTCF double occupancy was seen at the borders of many H3K27me3 'islands'. As before, these sites were also depleted of H3. Loss of either dCTCF or CP190 causes an increase of H3 and H3K27 trimethylation at these sites. Thus, for both types of cis-regulatory elements, domain borders and promoters, the chromatin structure is dependent on CP190.

Cloning and characterization of zebrafish CTCF: Developmental expression patterns, regulation of the promoter region, and evolutionary aspects of gene organization

CTCF is a nuclear phosphoprotein capable of using different subsets of its 11 Zn fingers (ZF) for sequence-specific binding to many dissimilar DNA CTCF-target sites. Such sites were identified in the genomic DNA of various multicellular organisms, in which the CTCF gene was cloned, including insects, birds, rodents, and primates. CTCF/DNA-complexes formed in vivo with different 50-bp-long sequences mediate diverse functions such as positive and negative regulation of promoters, and organization of all known enhancer-blocking elements (“chromatin insulators”) including constitutive and epigenetically regulated elements. Abnormal functions of certain CTCF sites are implicated in cancer and in epigenetic syndromes such as BWS and skewed X-inactivation. We describe here the cloning and characterization of the CTCF cDNA and promoter region from zebrafish, a valuable vertebrate model organism. The full-length zebrafish CTCF cDNA clone is 4244 bp in length with an open reading frame (ORF) of 2391 bp that encodes 797 amino acids. The zebrafish CTCF amino acid sequence shows high identity (up to 98% in the zinc finger region) with human CTCF, and perfect conservation of exon–intron organization. Southern blot analyses indicated that the zebrafish genome contains a single copy of the CTCF gene. In situ hybridization revealed the presence of zebrafish CTCF transcripts in all early stages of embryogenesis. Transfection assays with luciferase reporter-constructs identified a core promoter region within 146 bp immediately upstream of the transcriptional start site of zebrafish CTCF that is located at a highly conserved YY1/Initiator element.

CTCF Regulates Growth and Erythroid Differentiation of Human Myeloid Leukemia Cells

CTCF is a transcription factor and a candidate tumor suppressor that contains a DNA-binding domain composed of 11 zinc fingers. We reported previously that CTCF is differentially regulated during differentiation of human myeloid leukemia cells. In this study we aimed to investigate the role of CTCF in myeloid cell differentiation. A human cell line, K562, that can be chemically induced to differentiate into various hematopoietic lineages was chosen as a model system for this study. Several K562 cell lines with constitutive and conditional expression of CTCF have been generated. By using these model systems we demonstrated that: (i) ectopic expression of CTCF in K562 cells led to growth retardation and promotion of differentiation into the erythroid lineage; (ii) CTCF knock-down significantly inhibited differentiation of K562 cells into erythroid lineage; (iii) differentiation of K562 into the megakaryocytic lineage was not significantly affected; and (iv) down-regulation of MYC has been identified as one of the mechanisms by which CTCF promotes erythroid differentiation. Taken together our results demonstrate that CTCF is involved in the control of myeloid cell growth and differentiation.

Characterization of the Chicken CTCF Genomic Locus, and Initial Study of the Cell Cycle-regulated Promoter of the Gene

CTCF is 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 Cancer 22, 26-36). We characterized genomic organization of the chicken CTCF (chCTCF) gene, and studied the chCTCF promoter. Genomic locus of chCTCF contains 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 chCTCF promoter 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 CTCF genes. Mutations in the core nucleotides of the YY1 element reduce transcriptional activity of the minimal chCTCF promoter, indicating that the conserved YY1-binding sequence is critical for transcriptional regulation of vertebrate CTCF genes. We also noted in the chCTCF promoter 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 chCTCF expression may be cell cycle-regulated. We show that both levels of the endogenous chCTCF mRNA, and the activity of the stably transfected chCTCF promoter constructs, increase in S/G2 cells.

A widely expressed transcription factor with multiple DNA sequence specificity,CTCF, is localized at chromosome segment 16q22.1 within one of the smallest regions of overlap for common deletions in breast and prostate cancers

The cellular protooncogene MYC encodes a nuclear transcription factor that is involved in regulating important cellular functions, including cell cycle progression, differentiation, and apoptosis. Dysregulated MYC expression appears critical to the development of various types of malignancies, and thus factors involved in regulating MYC expression may also play a key role in the pathogenesis of certain cancers. We have cloned one such MYC regulatory factor, termed CTCF, which is a highly evolutionarily conserved-11-zinc finger transcriptional factor possessing multiple DNA sequence specificity. CTCF binds to a number of important regulatory regions within the 5' noncoding sequence of the human MYC oncogene, and it can regulate its transcription in several experimental systems. CTCF mRNA is expressed in cells of multiple different lineages. Enforced ectopic expression of CTCF inhibits cell growth in culture. Southern blot analyses and fluorescence in situ hybridization (FISH) with normal human metaphase chromosomes showed that the human CTCF is a single-copy gene situated at chromosome locus 16q22. Cytogenetic studies have pointed out that chromosome abnormalities (deletions) at this locus frequently occur in many different human malignancies, suggesting the presence of one or more tumor suppressor genes in the region. To narrow down their localization, several loss of heterozygosity (LOH) studies of chromosome arm 16q in sporadic breast and prostate cancers have been carried out to define the most recurrent and smallest region(s) of overlap (SRO) for commonly deleted chromosome arm 16q material. For CTCF to be considered as a candidate tumor suppressor gene associated with tumorigenesis, it should localize within one of the SROs at 16q. Fine-mapping of CTCF has enabled us to assign the CTCF gene to about a 2 centiMorgan (cM) interval of 16q22.1 between the somatic cell hybrid breakpoints CY130(D) and CY4, which is between markers D16S186 (16AC16-101) and D16S496 (AFM214zg5). This relatively small region, containing the CTCF gene, overlaps the most frequently observed SROs for common chromosomal deletions found in sporadic breast and prostate tumors. In one of four analyzed paired DNA samples from primary breast cancer patients, we have detected a tumor-specific rearrangement of CTCF exons encoding the 11-zinc-finger domain. Therefore, taken together with other CTCF properties, localization of CTCF to a narrow cancer-associated chromosome region suggests that CTCF is a novel candidate tumor suppressor gene at 16q22.1.