An Exceptionally Conserved Transcriptional Repressor, CTCF, Employs Different Combinations of Zinc Fingers To Bind Diverged Promoter Sequences of Avian and Mammalian c-myc Oncogenes

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.

p300 provides a corepressor function by cooperating with YY1 and HDAC3 to repress c-Myc

“…Region V was the only other promoter region that showed an increase in the p300 and HDAC3 occupancy levels (3.3-and 9-fold, respectively). Regions IV and V contain binding sites for transcription factors MAZ (Me1a2; Izzo et al, 1999), E2F (Albert et al, 2001), CTCF (Filippova et al, 1996) that function as repressors and also TBP (TATA box; see Figure 2b). …”

CTCF terminal segments are unstructured

“…The same protein can act as a repressor, 1 activator, 2 or insulator 3 when bound to different DNA sites. The genome-wide distribution of CTCF occupancy also suggests variation in function.…”

Loss of expression of chromosome 16q genes DPEP1 and CTCF in lobular carcinoma in situ of the breast

“…The loss of CTCF protein expression has been detected in invasive ductal carcinoma [10], but expression levels in LCIS and infiltrating lobular carcinomas remain unknown. CTCF is a widely expressed transcription factor that is involved in different aspects of gene regulation including promoter activation [14] and repression [15], hormone-responsive gene silencing [16], methylation-dependent chromatin insulation and genomic imprinting [17,18]. It has also been shown that CTCF can inhibit cell growth and induce cell cycle arrest at multiple stages [19].…”