CTCF-dependent enhancer blockers at the upstream region of the chicken  -globin gene domain

Abstract

The eukaryotic genome is partitioned into chromatin domains containing coding and intergenic regions. Insulators have been suggested to play a role in establishing and maintaining chromatin domains. Here we describe the identification and characterization of two separable enhancer blocking elements located in the 5' flanking region of the chicken alpha-globin domain, 11-16 kb upstream of the embryonic alpha-type pi gene in a DNA fragment harboring a MAR (matrix attachment region) element and three DNase I hypersensitive sites (HSs). The most upstream enhancer blocking element co-localizes with the MAR element and an erythroid-specific HS. The second enhancer blocking element roughly co-localizes with a constitutive HS. The third erythroid-specific HS present within the DNA fragment studied harbors a silencing, but not an enhancer blocking, activity. The 11 zinc-finger CCCTC-binding factor (CTCF), which plays an essential role in enhancer blocking activity in many previously characterized vertebrate insulators, is found to bind the two alpha-globin enhancer blocking elements. Detailed analysis has demonstrated that mutation of the CTCF binding site within the most upstream enhancer blocking element abolishes the enhancer blocking activity. The results are discussed with respect to special features of the tissue-specific alpha-globin gene domain located in a permanently open chromatin area.

Poly(ADP-ribosyl)ation and Epigenetics: Is CTCF PARt of the Plot?

“…By extrapolation, de-PARlation of CTCF might trigger disintegration of these interactions thus antagonizing the repressed status of the maternally inherited Igf2 promoter. A growing body of evidence pointing out at the presence of both PARP 54,55 and CTCF [56][57][58] in the loop regions provides further credibility to this model.…”

An evolutionarily conserved three-dimensional structure in the vertebrate Irx clusters facilitates enhancer sharing and coregulation

“…S5a,b ). CTCF was detected with the chick CTCF antibody 48 , which recognizes zebrafi sh CTCF 49 . Western blots were performed as reported in ref.…”

Epigenetic regulation of the human p53 gene promoter by the CTCF transcription factor in transformed cell lines

“…The electrophoretic mobility shift assay and nuclear extract preparation were performed as previously described (Schreiber et al, 1989;Valadez-Graham et al, 2004). Competitions were carried out with 200 pmol of gel-purified unlabeled oligonucleotides.…”

“…They were next mounted with Ultracruz for fluorescence with 4,6-diamidino-2-phenylindole to visualize the nucleus (Santa Cruz Biotechnology, sc-24941). Primary antibodies used were: anti-CTCF rabbit polyclonal (Valadez-Graham et al, 2004), anti-p53 goat polyclonal, anti-Rb rabbit polyclonal (Santa Cruz Biotechnology) and the histone acH3 (Upstate), H3K9me3, H3K27me3 and H4K20me3 antibodies. Primary antibodies bound to antigens were revealed using fluorescein isothiocyanate, or Texas red secondary antibodies.…”