CTCF, cohesin, and histone variants: connecting the genome.
ABSTRACT During the last decades our view of the genome organization has changed. We moved from a linear view to a looped view of the genome. It is now well established that inter- and intra-connections occur between chromosomes and play a major role in gene regulations. These interconnections are mainly orchestrated by the CTCF protein, which is also known as the "master weaver" of the genome. Recent advances in sequencing and genome-wide studies revealed that CTCF binds to DNA at thousands of sites within the human genome, providing the possibility to form thousands of genomic connection hubs. Strikingly, two histone variants, namely H2A.Z and H3.3, strongly co-localize at CTCF binding sites. In this article, we will review the recent advances in CTCF biology and discuss the role of histone variants H2A.Z and H3.3 at CTCF binding sites.
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ABSTRACT: 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.Molecular and Cellular Biology 07/1996; 16(6):2802-13. · 5.37 Impact Factor
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ABSTRACT: The promoter of the amyloid beta-protein precursor (APP) gene directs high levels of cell type-specific transcription with 94 base pairs 5' to the main transcriptional start site. An essential activator domain in this proximal APP promoter is a nuclear factor binding site designated as APBbeta. The recognition domain for the APBbeta binding factor is located between position -93 and -82 relative to the main transcriptional start site. The nuclear factor that binds to the APBbeta site was partially purified by multiple steps of ion exchange and hydroxyapatite chromatography. Based on UV cross-linking results, a protein with an apparent molecular mass of 140 kDa was selected as the putative APBbeta binding protein. After the final purification step consisting of preparative SDS-polyacrylamide gel electrophoresis, partial peptide sequences were obtained that completely matched the transcriptional factor CTCF. This protein is a known regulator of c-myc and lysozyme gene expression, and it binds to a variety of diverse DNA sequences. The binding of CTCF to the APBbeta domain was further established by competition with CTCF binding oligonucleotides in mobility shift electrophoresis. The identity was also confirmed by the observation that the APBbeta binding factor is recognized by antibodies against C- and N-terminal sequences of CTCF. In addition, oligonucleotide competition during in vitro transcription affirmed that CTCF acts as a transcriptional activator in the APP gene promoter.Journal of Biological Chemistry 01/1998; 272(52):33353-9. · 4.65 Impact Factor
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ABSTRACT: Naturally occurring deletion mutations within the human beta-globin cluster lead to specific, phenotypically discrete syndromes (i.e., delta beta-thalassemias and hereditary persistence of fetal hemoglobin, HPFH), characterized by increased production of fetal hemoglobin in adult life. We have previously characterized an enhancer element, which is juxtaposed to the fetal G gamma-gene, by means of a deletion first described in a Thai family. To obtain further insights into the mechanisms involved in this deletion, we have now characterized several of its novel features. Following amplification by the polymerase chain reaction and sequencing of the 1.5-kb bridging fragment, we have shown that the 5' breakpoint of the deletion occurs 1260 bp 3' of the fetal G gamma-globin gene, whereas the 3' breakpoint lies 521 bp upstream of the EcoRI site of the enhancer element and 2845 bp upstream of the 3' breakpoint of the Chinese (A gamma delta beta) zero-thalassemia deletion. The total length of the deletion is 101 kb, which resembles that of HPFH-1 and HPFH-2 deletions and a set of two gamma delta beta-thalassemia deletions. Our data further support the hypothesis that these sets of large deletions with almost identical lengths are generated via the loss of a complete chromatin loop. To elucidate further the mechanisms leading to the deletion, we have sequenced the novel 0.5-kb region residing immediately 3' to the breakpoint and shown that it contains putative binding sites for several transcription factors, such as HNF-1, AP-1, and TFIID. Sequence comparison of the deletion breakpoints reveals no junctional homology, indicating an end-to-end joining of blunted ends; a pair of 7-nt complementary repeats adjacent to a set of a direct CCCT repeat flanks the breakpoints. This limited homology constitutes a frequent characteristic of a non-homologous recombination mechanism. All these features of the HPFH-6 deletion suggest that this mutation has resulted from a non-homologous recombination event.Human Genetics 10/1997; 100(3-4):441-5. · 4.63 Impact Factor