A (GATA)7 motif located in the 5? boundary area of the human ?-globin locus control region exhibits silencer activity in erythroid cells

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...

Section: Discussionmentioning

confidence: 92%

Section: Vol 23 2003 Human ␤-Globin Hs5 Insulator 8947mentioning

confidence: 86%

Section: Vol 23 2003 Human ␤-Globin Hs5 Insulator 8947mentioning

confidence: 99%

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

Tanimoto

Sugiura

Omori

et al. 2003

“…The ERV-9 transcripts may be the originating point of the exclusively nuclear LCR transcripts that are detected at different erythroid developmental stages, and it has been suggested that these transcripts may play a role in creating and/or maintaining an open chromatin configuration (1,20,31). A silencing element consisting of seven tandem GATA repeats is present in the downstream region of human 5ЈHS5 (33). In addition, the presence of a matrix attachment region (MAR) has been reported in a 3-kb fragment encompassing 5ЈHS5 (25) and two topoisomerase II recognition sites, thought to contain this MAR activity, are present just downstream of the 5ЈHS5 core (42).…”

Section: Discussionmentioning

confidence: 99%

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

Farrell

West

Felsenfeld

2002

164

155

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.

“…Silencers bind repressor protein complexes that interfere with promoter activity, thereby down-regulating gene expression (61). Recent data demonstrate that two direct repeat (DR1) elements located in the proximal ε-globin promoter bind a novel repressor activity in definitive erythroid cells called direct repeat erythroid-definitive protein (DRED) (71).…”

mentioning

confidence: 99%

Silencing of ^Aγ-Globin Gene Expression during Adult Definitive Erythropoiesis Mediated by GATA-1-FOG-1-Mi2 Complex Binding at the −566 GATA Site

Harju-Baker¹,

Costa²,

Fedosyuk³

et al. 2008

Autonomous silencing of ␥-globin transcription is an important developmental regulatory mechanism controlling globin gene switching. An adult stage-specific silencer of the A ␥-globin gene was identified between ؊730 and ؊378 relative to the mRNA start site. A marked copy of the A ␥-globin gene inserted between locus control region 5 DNase I-hypersensitive site 1 and the -globin gene was transcriptionally silenced in adult ␤-globin locus yeast artificial chromosome (␤-YAC) transgenic mice, but deletion of the 352-bp region restored expression. This fragment reduced reporter gene expression in K562 cells, and GATA-1 was shown to bind within this sequence at the ؊566 GATA site. Further, the Mi2 protein, a component of the NuRD complex, was observed in erythroid cells with low ␥-globin levels, whereas only a weak signal was detected when ␥-globin was expressed. Chromatin immunoprecipitation of fetal liver tissue from ␤-YAC transgenic mice demonstrated that GATA-1, FOG-1, and Mi2 were recruited to the A ␥-globin ؊566 or G ␥-globin ؊567 GATA site when ␥-globin expression was low (day 18) but not when ␥-globin was expressed (day 12). These data suggest that during definitive erythropoiesis, ␥-globin gene expression is silenced, in part, by binding a protein complex containing GATA-1, FOG-1, and Mi2 at the ؊566/؊567 GATA sites of the proximal ␥-globin promoters.