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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
Mol Biol (Mosk)
PMID:[Regulatory protein factor CTCF interacts with a segment of the chicken c-myc oncogene promotor, capable of changing to a noncanonical conformation]. 179 97

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.
Mol Cell Biol 1993 Dec
PMID: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. 824 78

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.
Mol Cell Biol 1996 Jun
PMID:An exceptionally conserved transcriptional repressor, CTCF, employs different combinations of zinc fingers to bind diverged promoter sequences of avian and mammalian c-myc oncogenes. 864 89

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.
Mol Cell Biol 2001 Mar
PMID:Functional phosphorylation sites in the C-terminal region of the multivalent multifunctional transcriptional factor CTCF. 1123 55

The methylation status of binding sites of the insulator protein, CTCF, in the H19 promoter has been suggested as being critical to the regulation of imprinting of the H19/IGF2 locus located in chromosome 11p15. In this study, we have analyzed the methylation of all of seven potential CTCF-binding sites in the human H19 promoter since the methylation status of these sites has not been reported. We found that all the binding sites except the sixth were hypermethylated whereas only the sixth binding site showed allele-specific methylation in normal human embryonic ureteral tissue. We also analyzed the methylation status of these sites in human-mouse somatic-cell-hybrid clones containing a single copy of human chromosome 11 and which were treated with 5-aza-2'-deoxycytidine (5-aza-CdR) to yield clones which expressed human IGF2 and H19 mutually exclusively of each other. In most of the clones, a correlation between methylation of the sixth CTCF-binding site and expression of IGF2 was observed. Therefore, we analyzed the methylation status of this site in human bladder cancer and found hypomethylation of the paternal allele in two of six informative cases. These results demonstrate that only the sixth CTCF-binding site acts as a key regulatory domain for switching between H19 or IGF2 expression, whereas the other sites are not subject to allele-specific methylation. Loss of methylation imprinting of H19 is linked to hypomethylation of the paternal allele in human bladder cancer, unlike the situation in Wilms' tumor and colon cancer where the maternal allele becomes hypermethylated.
Hum Mol Genet 2001 Nov 01
PMID:Large scale mapping of methylcytosines in CTCF-binding sites in the human H19 promoter and aberrant hypomethylation in human bladder cancer. 1172 48

Dlk1 and Gtl2 are reciprocally imprinted genes located 80 kb apart on mouse chromosome 12. Similarities between this domain and that of the well characterized Igf2-H19 locus have been previously noted. Comparative genomic and epigenetic analysis of these two domains might help identify allele-specific epigenetic regulatory elements and common features involved in aspects of imprinting control. Here we describe a detailed methylation analysis of the Dlk1-Gtl2 domain on both parental alleles in the mouse. Like the Igf2-H19 domain, areas of differential methylation are hypermethylated on the paternal allele and hypomethylated on the maternal allele. Three differentially methylated regions (DMRs), each with different epigenetic characteristics, have been identified. One DMR is intergenic, contains tandem repeats and is the only region that inherits a paternal methylation mark from the germline. An intronic DMR contains a conserved putative CTCF-binding domain. All three DMRs have both unique and common features compared to those identified in the Igf2-H19 domain.
Hum Mol Genet 2002 Jan 01
PMID:Epigenetic analysis of the Dlk1-Gtl2 imprinted domain on mouse chromosome 12: implications for imprinting control from comparison with Igf2-H19. 1177 1

Genomic imprinting, the differential expression of autosomal genes based on their parent of origin, is observed in all eutherian mammals that have been examined. In most instances the genes that are imprinted in one species are imprinted in others as well, suggesting that imprinting predated eutherian radiation. For example, the RNA-coding H19 gene is repressed upon paternal inheritance in all species examined to date. Thus, it is surprising that there is remarkably little sequence conservation among the cis-acting DNA regulatory elements that are required for imprinting of H19 and the tightly linked Igf2 gene. The most conserved characteristic in the imprinting control region (ICR) is the presence of multiple binding sites for the zinc finger protein CTCF, raising the possibility that CTCF binding might be sufficient for the reciprocal imprinting of H19 and Igf2. To investigate whether a human H19 transgene, harboring seven CTCF sites, is correctly recognized and imprinted in the mouse, a 100 kb transgene containing the human H19 gene was introduced into the mouse germline. The human transgene was specifically methylated after passage through the male germline in a copy number-dependent manner, but the methylation was unstable, undergoing progressive loss during development. Consequently, the transgene was highly expressed upon both maternal and paternal inheritance. These results argue that the signals for both the acquisition and maintenance of methylation imprinting are diverging rapidly.
Hum Mol Genet 2002 Feb 15
PMID:A human H19 transgene exhibits impaired paternal-specific imprint acquisition and maintenance in mice. 1185 73

Imprinting of the linked and oppositely expressed mouse H19 and Igf2 genes requires a 2-kb differentially methylated domain (DMD) that is located 2 kb upstream of H19. This element is postulated to function as a methylation-sensitive insulator. Here we test whether an additional sequence 5' of H19 is required for H19 and Igf2 imprinting. Because repetitive elements have been suggested to be important for genomic imprinting, the requirement of a G-rich repetitive element that is located immediately 3' to the DMD was first tested in two targeted deletions: a 2.9-kb deletion (Delta D MD Delta G) that removes the DMD and G-rich repeat and a 1.3-kb deletion (Delta G) removing only the latter. There are also four 21-bp GC-rich repetitive elements within the DMD that bind the insulator-associated CTCF (CCCTC-binding factor) protein and are implicated in mediating methylation-sensitive insulator activity. As three of the four repeats of the 2-kb DMD were deleted in the initial 1.6-kb Delta DMD allele, we analyzed a 3.8-kb targeted allele (Delta 3.8kb-5'H19), which deletes the entire DMD, to test the function of the fourth repeat. Comparative analysis of the 5' deletion alleles reveals that (i) the G-rich repeat element is dispensable for imprinting, (ii) the Delta DMD and Delta DMD Delta G alleles exhibit slightly more methylation upon paternal transmission, (iii) removal of the 5' CTCF site does not further perturb H19 and Igf2 imprinting, suggesting that one CTCF-binding site is insufficient to generate insulator activity in vivo, (iv) the DMD sequence is required for full activation of H19 and Igf2, and (v) deletion of the DMD disrupts H19 and Igf2 expression in a tissue-specific manner.
Mol Cell Biol 2002 Apr
PMID:Analysis of sequence upstream of the endogenous H19 gene reveals elements both essential and dispensable for imprinting. 1190 40

The 5' region of the H19 gene harbors a methylation-sensitive chromatin insulator within an imprinting control region (ICR). Insertional mutagenesis in combination with episomal assays identified nucleosome positioning sequences (NPSs) that set the stage for the remarkably precise distribution of the four target sites for the chromatin insulator protein CTCF to nucleosome linker sequences in the H19 ICR. Changing positions of the NPSs resulted in loss of both CTCF target site occupancy and insulator function, suggesting that the NPSs optimize the fidelity of the insulator function. We propose that the NPSs ensure the fidelity of the repressed status of the maternal Igf2 allele during development by constitutively maintaining availability of the CTCF target sites.
Mol Cell Biol 2002 May
PMID:Multiple nucleosome positioning sites regulate the CTCF-mediated insulator function of the H19 imprinting control region. 1197 67

The expression of integrated oncoretrovirus vectors is subject to the inhibitory effects of surrounding chromatin. A previous report from our laboratory indicated that such position effects can be overcome by flanking a reporter vector with the cHS4 chromatin insulator. To characterize this activity more thoroughly, we switched the promoter-gene combinations in the reporter vector and analyzed expression of these vectors flanked with the cHS4 fragment in both orientations following bone marrow transduction and transplantation in mice. The results indicate that the cHS4 fragment can function in both orientations and can insulate both the virus long-terminal-repeat (LTR) promoter and an internal phosphoglycerate kinase (Pgk) promoter. However, insulation of the LTR promoter diminished when the orientation of the cHS4 fragment placed the CTCF-binding core element immediately proximal to the U3 region, suggesting a minimal distance requirement. Moreover, placement of the cHS4 fragment in the U3 region of the 3' LTR dramatically decreased the level of expression from an internal Pgk promoter, presumably by blocking interaction with the 3' LTR enhancer. Finally, sorting studies suggest that the severity of position effects or autonomous promoter silencing increases as transduced progenitors differentiate into mature progeny. These findings have direct implications for the use of chromatin insulators such as cHS4 in oncoretrovirus vectors.
Mol Ther 2002 May
PMID:Topological constraints governing the use of the chicken HS4 chromatin insulator in oncoretrovirus vectors. 1199 50


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