Human β-Globin Locus Control Region HS5Contains CTCF- and Developmental Stage-Dependent Enhancer-BlockingActivity in ErythroidCells

Abstract

The human ␤-globin locus contains five developmentally regulated ␤-type globin genes. All five genes depend on the locus control region (LCR), located at the 5 end of the locus, for abundant globin gene transcription. The LCR is composed of five DNase I-hypersensitive sites (HSs), at least a subset of which appear to cooperate to form a holocomplex in activating genes within the locus. We previously tested the requirement for proper LCR polarity by inverting it in human ␤-globin yeast artificial chromosome transgenic mice and observed reduced expression of all the ␤-type globin genes regardless of developmental stage. This phenotype clearly demonstrated an orientation-dependent activity of the LCR, although the mechanistic basis for the observed activity was obscure. Here, we describe genetic evidence demonstrating that human HS5 includes enhancerblocking (insulator) activity that is both CTCF and developmental stage dependent. Curiously, we also observed an attenuating activity in HS5 that was specific to the -globin gene at the primitive stage and was independent of the HS5 CTCF binding site. These observations demonstrate that the phenotype observed in the LCR-inverted locus was in part attributable to placing the HS5 insulator between the LCR HS enhancers (HS1 to HS4) and the promoter of the ␤-globin gene.During the normal process of mammalian development, proper temporal and spatial expression of genetic information must be achieved. To this end, gene expression must be tightly regulated through cis-DNA elements: promoters, enhancers, and silencers. After the human genome had been completely sequenced, it became clear that the average size of intergenic regions is roughly 100 kbp. However, substantial evidence argues that enhancers can modulate promoter activity from very long distances, exceeding 100 kbp (19,20). In such a circumstance, one can envision an activity that protects a locus from neighboring gene regulatory elements to prevent improper gene regulation. Indeed, such an activity has been described: DNA insulators were first identified in Drosophila melanogaster, and similar activities were subsequently found in mammals.Insulators can protect a locus by two distinguishable means; one is a chromatin barrier function, while a second is referred to as enhancer-blocking activity. The most extensively characterized vertebrate insulator was originally identified in the chicken ␤-globin LCR, which consists of four DNase I-hypersensitive (HS) sites; the 5Ј-most is HS4. After stable transformation of K562 cells, a 1.2-kbp DNA fragment containing HS4 was found to interfere with enhancer/promoter interactions, but only when it was placed between them (10). Further analysis revealed two separable categories of activity within this 1.2-kbp DNA fragment, one of which protects stably integrated transgenes from silencing after long-term cell culture (28). This property of an insulator is referred to as its chromatin barrier activity.Chung et al. developed an enhancer-blocking assay to refine the position of a 250...

CTCF-dependent enhancer-blocking by alternative chromatin loop formation

“…Ectopic HS5 reduced globin gene transcription at all stages of mouse development as determined by RT-qPCR (Fig. S2), substantially recapitulating earlier work using semiquantitative PCR (20). Deletion of the HS5 CTCF site restored ␥-and ␤-globin expression, although it had little effect on -globin expression.…”

“…We investigated the insulator function of HS5 in mice carrying a single copy of a complete ␤-globin locus transgene with a copy of HS5 placed between the LCR and -globin, containing either an intact (HS5) or mutated CTCF site (⌬CTCF) or no HS5 (WT) (Fig. 1A) (20). CTCF occupies endogenous HS5 and 3ЈHS1 sites flanking the mouse and human ␤-globin loci in vivo (14, 21).…”

“…Earlier studies suggested that human HS5 could function as a transcriptional enhancer-blocker when placed between the LCR and the globin genes on a transgene (20). We used this system to investigate the mechanism underlying enhancer-blocking.…”

Heterogeneous nuclear ribonucleoprotein C1/C2, MeCP1, and SWI/SNF form a chromatin remodeling complex at the β-globin locus control region

“…However, their specific binding sites on the ␤-globin locus have not been determined. The role of a boundary element in controlling the spread of the heterochromatin from an upstream end of the ␤-globin LCR is well studied (3,4,35,36), but so far the isolation of any chromatin opening activity of the LCR has proved elusive. Here, we describe the structural and functional properties of a biochemically purified ␤-globin LARC.…”

“…Of these seven HS sites, the first four, HS1, HS2, HS3, and HS4, are erythroid specific (1). HS5 is present in several hematopoietic lineages and is suggested to possess insulator activity (3)(4)(5), and HS6 and HS7 are present in a variety of cell types.…”