Filippova GN, Fagerlie S, Klenova EM, Myers C, Dehner Y, Goodwin G, Neiman PE, Collins SJ, Lobanenkov VVAn exceptionally conserved transcriptional repressor, CTCF, employs different combinations of zinc fingers to bind diverged promoter sequences of avian and mammalian c-myc oncogenes. Mol Cell Biol 16: 2802-2813

Fred Hutchinson Cancer Research Center, Seattle, Washington 98104, USA.
Molecular and Cellular Biology (Impact Factor: 4.78). 07/1996; 16(6):2802-13. DOI: 10.1128/MCB.16.6.2802
Source: PubMed


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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    • "To this aim, we analyzed the occupancy of the region of interest by a couple of factors known to modulate c-MYC expression in an opposite manner. In particular, we focused on CTCF and SP1, which act respectively as a negative and a positive regulator of c-MYC expression and whose consensus sequences lie in the same region that we have observed to become poly(ADP-ribosyl)ated [25], [26]. Chromatin samples from quiescent and serum-stimulated cells treated or not with the PARP inhibitor, were analyzed by ChIP-qPCR assays for CTCF and SP1 binding. "
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    ABSTRACT: Poly(ADP-ribosyl)ation is a post-translational modification of various proteins and participates in the regulation of chromatin structure and transcription through complex mechanisms not completely understood. We have previously shown that PARP-1, the major family member of poly(ADP-ribose)polymerases, plays an important role in the cell cycle reactivation of resting cells by regulating the expression of Immediate Early Response Genes, such as c-MYC, c-FOS, JUNB and EGR-1. In the present work we have investigated the molecular mechanisms by which the enzyme induces c-MYC transcription upon serum stimulation of quiescent cells. We show that PARP-1 is constitutively associated in vivo to a c-MYC promoter region recognized as biologically relevant for the transcriptional regulation of the gene. Moreover, we report that serum stimulation causes the prompt accumulation of ADP-ribose polymers on the same region and that this modification is required for chromatin decondensation and for the exchange of negative for positive transcriptional regulators. Finally we provide evidence that the inhibition of PARP activity along with serum stimulation impairs c-MYC induction by preventing the proper accumulation of histone H3 phosphoacetylation, a specific chromatin mark for the activation of Immediate Early Response Genes. These findings not only suggest a novel strategy by which PARP-1 regulates the transcriptional activity of promoters but also provide new information about the complex regulation of c-MYC expression, a critical determinant of the transition from quiescence to proliferation.
    Full-text · Article · Jul 2014 · PLoS ONE
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    • "2.1.1. CTCF CTCF is a ubiquitously expressed zinc finger DNA-binding protein that was first described as a transcription factor based on its role in regulation of c-myc gene expression [8] [9] [10]. CTCF was later shown to block the communication between enhancers and promoters (enhancer blocking activity) and also to act as a boundary to the spread of chromatin domains (barrier function), and was therefore additionally defined as an insulator protein [11] [12] [13]. "
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    ABSTRACT: The spatial organization of the nucleus results in a compartmentalized structure that affects all aspects of nuclear function. This compartmentalization involves genome organization as well as the formation of nuclear bodies and plays a role in many functions, including gene regulation, genome stability, replication, and RNA processing. Here we review the recent findings associated with the spatial organization of the nucleus and reveal that a common theme for nuclear proteins is their ability to participate in a variety of functions and pathways. We consider this multiplicity of function in terms of Crowdsourcing, a recent phenomenon in the world of information technology, and suggest that this model provides a novel way to synthesize the many intersections between nuclear organization and function.
    Full-text · Article · Jan 2014 · Biochimica et Biophysica Acta
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    • "CCCTC-binding factor (CTCF) is a highly conserved 11-zinc finger protein that is involved in multiple regulatory functions, including transcriptional activation/repression [4,5], chromatin insulation [6,7], DNA imprinting [8,9], and X chromosome inactivation [10,11]. CTCF was first identified and characterized as a transcriptional repressor of the c-myc gene in chickens, mice, and humans [5,12,13]. "
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    ABSTRACT: Acute lymphoblastic leukemia (ALL) is the most frequently occurring malignant neoplasm in children. Despite advances in treatment and outcomes for ALL patients, the pathogenesis of the disease remains unclear. Microarray analysis of samples from 100 Chinese children with ALL revealed the up-regulation of CTCF (CCCTC binding factor). CTCF is a highly conserved 11-zinc finger protein that is involved in many human cancers; however, the biological function of CTCF in pediatric ALL is unknown. The expression patterns of CTCF were evaluated in matched newly diagnosed (ND), complete remission (CR), and relapsed (RE) bone marrow samples from 28 patients. The potential oncogenic mechanism of CTCF and related pathways in leukemogenesis were investigated in leukemia cell lines. We identified significant up-regulation of CTCF in the ND samples. Importantly, the expression of CTCF returned to normal levels after CR but rebounded in the RE samples. In the pre-B ALL cell line Nalm-6, siRNA-mediated silencing of CTCF expression promoted cell apoptosis and reduced cell proliferation; accordingly, over-expression of a cDNA encoding full-length CTCF protected cells from apoptosis and enhanced cell proliferation. Furthermore, inhibition or activation of the nuclear factor-kappa B (NF-kappaB) pathway resulted in marked variations in the levels of CTCF mRNA and protein in leukemic cells, indicating that CTCF may be involved downstream of the NF-kappaB pathway. Moreover, inhibition of the NF-kappaB pathway increased cell apoptosis, which was partially rescued by ectopic over-expression of CTCF, suggesting that CTCF may play a significant role in the anti-apoptotic pathway mediated by NF-kappaB. Our results indicate that CTCF serves as both an anti-apoptotic factor and a proliferative factor in leukemic cells. It potentially contributes to leukemogenesis through the NF-kappaB pathway in pediatric ALL patients.
    Full-text · Article · Jan 2014 · Molecular Cancer
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