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.

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CTCF function is modulated by neighboring DNA binding factors

Biochemistry and Cell Biology ◽

10.1139/o11-033 ◽

2011 ◽

Vol 89 (5) ◽

pp. 459-468 ◽

Cited By ~ 42

Author(s):

Oliver Weth ◽

Rainer Renkawitz

Keyword(s):

Dna Binding ◽

Binding Site ◽

Zinc Finger ◽

Binding Sites ◽

Zinc Finger Protein ◽

Gene Activation ◽

Ctcf Binding ◽

Paternal Allele ◽

Ctcf Binding Site ◽

Enhancer Blocking

The zinc-finger protein CTCF was originally identified in the context of gene silencing and gene repression (Baniahmad et al. 1990; Lobanenkov et al. 1990). CTCF was later shown to be involved in several transcriptional mechanisms such as gene activation (Vostrov et al. 2002) and enhancer blocking (Filippova et al. 2001; Hark et al. 2000; Kanduri et al. 2000; Lutz et al. 2003; Szabó et al. 2000; Tanimoto et al. 2003; Phillips and Corces 2009; Bell et al. 1999; Zlatanova and Caiafa 2009a, 2009b). Insulators block the action of enhancers when positioned between enhancer and promoter. CTCF was found to be required in almost all cases of enhancer blocking tested in vertebrates. This CTCF-mediated enhancer blocking is in many instances conferred by constitutive CTCF action. For some examples however, a modulation of the enhancer blocking activity was documented (Lutz et al. 2003; Weth et al. 2010). One mechanism is achieved by regulation of binding to DNA. It was shown that CTCF is not able to bind to those binding-sites containing methylated CpG sequences. At the imprinting control region (ICR) of the Igf2/H19 locus the binding-site for CTCF on the paternal allele is methylated. This prevents DNA-binding of CTCF, resulting in the loss of enhancer blocking (Bell and Felsenfeld 2000; Chao et al. 2002; Filippova et al. 2001; Hark et al. 2000; Kanduri et al. 2000, 2002; Szabó et al. 2000; Takai et al. 2001). Not only can DNA methylation interfere with CTCF binding to DNA, it was also shown in one report that RNA transcription through the CTCF binding site results in CTCF eviction (Lefevre et al. 2008). In contrast to these cases most of the DNA sites are not differentially bound by CTCF. Even CTCF interaction with its cofactor cohesin does not seem to differ in different cell types (Schmidt et al. 2010). These results indicate that regulation of CTCF activity might be achieved by neighboring factors bound to DNA. In fact, whole genome analyses of CTCF binding sites identified several classes of neighboring sequences (Dickson et al. 2010; Boyle et al. 2010; Essien et al. 2009). Therefore, in this review we will summarize those results for which a combined action of CTCF with factors bound adjacently was found. These neighboring factors include the RNA polymerases I, II and III, another zinc finger factor VEZF1 and the factors YY1, SMAD, TR and Oct4. Each of these seems to influence, modulate or determine the function of CTCF. Thereby, at least some of the pleiotropic effects of CTCF can be explained.

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3'HS1 CTCF binding site in human β-globin locus regulates fetal hemoglobin expression

10.1101/2021.05.18.444713 ◽

2021 ◽

Author(s):

Pamela Himadewi ◽

Xue Qing David Wang ◽

Fan Feng ◽

Haley Gore ◽

Yushuai Liu ◽

...

Keyword(s):

Binding Site ◽

Progenitor Cells ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Ctcf Binding ◽

Ctcf Binding Site ◽

Distal Enhancer ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Ctcf Site

Mutations in the adult β-globin gene can lead to a variety of hemoglobinopathies, including sickle cell disease and β-thalassemia. An increase in fetal hemoglobin expression throughout adulthood, a condition named Hereditary Persistence of Fetal Hemoglobin (HPFH), has been found to ameliorate hemoglobinopathies. Deletional HPFH occurs through the excision of a significant portion of the 3 prime end of the β-globin locus, including a CTCF binding site termed 3'HS1. Here, we show that the deletion of this CTCF site alone induces fetal hemoglobin expression in both adult CD34+ hematopoietic stem and progenitor cells and HUDEP-2 erythroid progenitor cells. This induction is driven by the ectopic access of a previously postulated distal enhancer located in the OR52A1 gene downstream of the locus, which can also be insulated by the inversion of the 3'HS1 CTCF site. This suggests that genetic editing of this binding site can have therapeutic implications to treat hemoglobinopathies.

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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

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7612-7624.1993 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7612-7624

Author(s):

E M Klenova ◽

R H Nicolas ◽

H F Paterson ◽

A F Carne ◽

C M Heath ◽

...

Keyword(s):

Dna Binding ◽

Cell Lines ◽

Gene Promoter ◽

Differentially Expressed ◽

Ctcf Binding ◽

Ctcf Binding Site ◽

Mobility Shift ◽

Chicken Cell ◽

Nuclear Extracts ◽

Myc Gene

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.

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Allele-specific methylation of a functional CTCF binding site upstream of MEG3 in the human imprinted domain of 14q32

Chromosome Research ◽

10.1007/s10577-005-1015-4 ◽

2005 ◽

Vol 13 (8) ◽

pp. 809-818 ◽

Cited By ~ 29

Author(s):

Alberto L. Rosa ◽

Yuan-Qing Wu ◽

Bernard Kwabi-Addo ◽

Karen J. Coveler ◽

V. Reid Sutton ◽

...

Keyword(s):

Binding Site ◽

Ctcf Binding ◽

Ctcf Binding Site ◽

Allele Specific ◽

Allele Specific Methylation

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Functional Differences in GATA-1 Affinity for Double Versus Single GATA-1 Binding Sites Dictate Synergistic Versus Antagonistic Interactions of PU.1 and GATA-1 in Myeloid Gene Transcription.

Blood ◽

10.1182/blood.v104.11.1608.1608 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1608-1608

Author(s):

Jian Du ◽

Dharmesh Vyas ◽

Qing Xi ◽

Steven J. Ackerman

Keyword(s):

Dna Binding ◽

Binding Site ◽

Gene Transcription ◽

Binding Sites ◽

Zinc Fingers ◽

Specific Expression ◽

High Affinity ◽

Dual Versus ◽

P2 Promoter ◽

Dual Site

Abstract Instructive roles for both GATA-1 and PU.1 have been demonstrated in hematopoiesis, and recent studies have identified both antagonistic and synergistic interactions between them in myeloid gene transcription and lineage development. In prior studies, we reported that PU.1 synergizes with rather than antagonizes GATA-1 for transactivation of a hallmark eosinophil gene, the major basic protein P2 promoter (MBP-P2), which possesses a novel dual (double) GATA-binding site, similar to the palindromic double site in the murine GATA-1 control locus that may specify eosinophil lineage-specific expression of GATA-1 and eosinophil development. To address the transcriptional mechanism for PU.1-GATA-1 synergy through the MBP-P2 dual GATA site, we investigated GATA-1 and PU.1 physical and functonal interactions via their binding sites in the MBP-P2 promoter. DNA binding affinities of GATA-1 and its C- versus N-terminal zinc fingers were assessed for single versus double GATA sites in the presence or absence of PU.1. Our results show that the dual GATA site strongly binds full length GATA-1 with higher affinity than either of the single sites, using both zinc fingers, but that mutant GATA-1 proteins with C-finger or N-finger deletions retain their ability to bind, albeit at lower affinity, to the dual site. DNA binding activities of the two zinc fingers with the dual GATA site were confirmed using peptides containing only the C-finger or N-finger region. Of note, formation of GATA-1 complexes with the dual GATA site was not inhibited by the addition of PU.1, whereas formation of binding complexes for mutants of GATA-1 containing only the C- or N-finger region could be completely inhibited in a dose-response fashion by PU.1. These unique features of PU.1/GATA-1 interactions on a dual versus single GATA-1 site were confirmed using peptides containing only the C- or N-finger regions of GATA-1. Our findings indicate that both zinc fingers of GATA-1 are involved in formation of the high-affinity GATA-1 complex with the dual site. Importantly, we show that the higher affinity dual GATA-1 site complex is not affected by the addition of PU.1, whereas formation of the binding complex with a single GATA-1 site is eliminated by PU.1, emphasizing the different mechanisms of GATA-1/PU.1 interactions on dual versus single GATA binding sites. Functional analyses by transactivation confirmed that synergistic activation of the MBP-P2 promoter by GATA-1 and PU.1 is mediated by their protein-protein interactions through this unique high affinity dual GATA-1 binding site. We suggest two possible mechanisms for PU.1/GATA-1 synergy on dual GATA sites: (1) PU.1 may change GATA-1 conformation and its high affinity for the dual site, enhancing its availability for interaction with the basal transcriptional machinery. Alternatively, (2) PU.1 could impede interactions of GATA-1 with a co-repressor, e.g. FOG-1, which we and others have shown represses GATA-1 function in the eosinophil lineage.

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CTCFBSDB: a CTCF-binding site database for characterization of vertebrate genomic insulators

Nucleic Acids Research ◽

10.1093/nar/gkm875 ◽

2007 ◽

Vol 36 (Database) ◽

pp. D83-D87 ◽

Cited By ~ 66

Author(s):

L. Bao ◽

M. Zhou ◽

Y. Cui

Keyword(s):

Binding Site ◽

Ctcf Binding ◽

Ctcf Binding Site

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cDNA cloning and expression of rat homeobox gene, Hex, and functional characterization of the protein

Biochemical Journal ◽

10.1042/bj3390111 ◽

1999 ◽

Vol 339 (1) ◽

pp. 111-117 ◽

Cited By ~ 31

Author(s):

Takashi TANAKA ◽

Tetsuya INAZU ◽

Kazuya YAMADA ◽

Zaw MYINT ◽

Vincent W. KENG ◽

...

Keyword(s):

Dna Binding ◽

Binding Site ◽

Rat Liver ◽

Functional Characterization ◽

Homeobox Gene ◽

Hepatoma Cells ◽

Cloning And Expression ◽

Expression Vectors ◽

Luciferase Reporter ◽

Cdna Clones

We isolated two cDNA clones of rat Hex, a homeobox protein, studied its expression in rat liver and various cells, and characterized the protein. The levels of Hex mRNA were only slightly increased in liver of rats refed with a high-carbohydrate diet or after partial hepatectomy. Whereas the expression of Hex mRNA was detected in hepatocytes isolated from adult rat liver and also in highly differentiated hepatoma cells, no Hex mRNA was detected in poorly differentiated hepatoma cells. Hex mRNA was also detected in liver from embryo aged 15 days. Expression of Hex was increased in F9 cells during differentiation into visceral endoderm cells by treatment with retinoic acid. This stimulation occurred prior to an increase in the level of α-fetoprotein mRNA. When fusion-protein expression vectors of GAL4 DNA-binding domain and Hex were co-transfected with luciferase reporter plasmid, with or without five copies of the GAL4-binding site, into HepG2 cells, the luciferase activities were decreased in concentration- and GAL4-binding site-dependent manners. This repression did not require the presence of the homeodomain, which is located between the amino acid residues 137 and 196. Its repression domain was mapped between the residues 45 and 136 in the proline-rich N-terminal region. In addition, the homeodomain was responsible for DNA-binding of Hex. These results indicate that Hex functions as a transcriptional repressor and may be involved in the differentiation and/or maintenance of the differentiated state in hepatocytes.

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