Conserved CTCF Insulator Elements Flank the Mouse and Human β-Globin Loci

Abstract

A binding site for the transcription factor CTCF is responsible for enhancer-blocking activity in a variety of vertebrate insulators, including the insulators at the 5 and 3 chromatin boundaries of the chicken ␤-globin locus. To date, no functional domain boundaries have been defined at mammalian ␤-globin loci, which are embedded within arrays of functional olfactory receptor genes. In an attempt to define boundary elements that could separate these gene clusters, CTCF-binding sites were searched for at the most distal DNase I-hypersensitive sites (HSs) of the mouse and human ␤-globin loci. Conserved CTCF sites were found at 5HS5 and 3HS1 of both loci. All of these sites could bind to CTCF in vitro. The sites also functioned as insulators in enhancer-blocking assays at levels correlating with CTCF-binding affinity, although enhancer-blocking activity was weak with the mouse 5HS5 site. These results show that with respect to enhancer-blocking elements, the architecture of the mouse and human ␤-globin loci is similar to that found previously for the chicken ␤-globin locus. Unlike the chicken locus, the mouse and human ␤-globin loci do not have nearby transitions in chromatin structure but the data suggest that 3HS1 and 5HS5 may function as insulators that prevent inappropriate interactions between ␤-globin regulatory elements and those of neighboring domains or subdomains, many of which possess strong enhancers.

Chromosome Conformation Capture Carbon Copy (5C): A massively parallel solution for mapping interactions between genomic elements

“…In addition, in both K562 and GM06990 cells, the LCR also interacted with the 3Ј-HS1 element and a large domain located 50-100 kb upstream. The latter region corresponds to the region around HS-62.5/ HS-60 in the murine locus (Farrell et al 2002;Bulger et al 2003). Similar longrange interactions between HSs were observed in the mouse.…”

“…Arrows indicate HSs identified in K562 cells. Red arrows indicate HSs identified in K562 cells that have been proposed to be orthologous to HS-62.5/-62.0 in the murine locus (Farrell et al 2002;Bulger et al 2003). (C) Chromatin interaction profile of HS2/3/4 of the LCR with the 400-kb region around the LCR as determined by microarray (top) and quantitative sequencing (bottom).…”

“…The clustering of these HSs has been proposed to create a chromatin hub, or a specialized nuclear compartment dedicated to the transcription of the ␤-globin genes (Tolhuis et al 2002;de Laat and Grosveld 2003). Several of the HSs in the ␤-globin locus bind the insulator binding protein CTCF (Farrell et al 2002;Bulger et al 2003) (and for human HS5, see Supplemental Fig. 6), and this protein has been proposed to mediate their interactions and formation of the chromatin hub (Patrinos et al 2004).…”

Genome imprinting regulated by the mouse Polycomb group protein Eed

“…One SNP in the region allowed for assignment of alleles recovered, and these are indicated by the base-pair change (paternal (JF1) = G, maternal (CD1) = T). One putative CTCF binding site was identified containing 11 of 16 bases identical to conserved CTCF binding sites 29 . Two EagI sites previously examined for parent-of-origin methylation are shown 30 The conditions for second-round PCR were 3 min at 95 °C and then 36 cycles of 45 s at 95°C, 60°C and 72°C.…”

CTCF: the protein, the binding partners, the binding sites and their chromatin loops

“…subsequently demonstrated to be responsible for this insulator activity of 5 0 HS4 [5]. The human and mouse b-globin loci are also located inside large chromosomal regions of inactive chromatin and are similarly flanked by CTCF-binding sites [17,18]. These were suspected to form a barrier for incoming heterochromatin, but their deletion did not lead to closing or inactivation of the b-globin locus [12,19].…”