A 5′ β-globin matrix-attachment region and the polyoma enhancer together confer position-independent transcription

163

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.

Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

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

Farrell

West

Felsenfeld

2002

163

155

“…The 234-bp 5ЈHS3 core mutation severely impaired or abolishes HS formation not only in the LCR but also at 5ЈHS6 (but not 5ЈHS7), approximately 28 kb 5Ј to the ε-globin gene. 5ЈHS5, which is located between 5ЈHS6 and 5ЈHS3, is a chromatin insulator (33,34,55,61,63). However, 5ЈHS5 is unable to limit the impact of the 5ЈHS3 mutations on chromatin structure to the LCR.…”

Section: Discussionmentioning

confidence: 99%

“…Classic enhancer activity of 5Ј HS site 2 (5ЈHS2) can be detected by transient transfection assays (39,59), whereas enhancer activity of 5ЈHSs 3 and 4 can only be measured by stable transfection assays or with transgenic mice (17,44,47,53), suggesting that the enhancer function of 5ЈHS3 and 4 is associated with chromatin modification. Using enhancer blocking and boundary assays, 5ЈHS5 shows chromatin insulating activity in vivo (33,34,55,61,63). However, deletion by homologous recombination of the LCR sequences including 5ЈHS5 does not have a considerable effect on globin gene expression in the mouse (3,12), suggesting that its in situ function remains unknown.…”

mentioning

confidence: 99%

Synergistic and Additive Properties of the Beta-Globin Locus Control Region (LCR) Revealed by 5′HS3 Deletion Mutations: Implication for LCR Chromatin Architecture

Fang

Sun

Xiang

et al. 2005

Deletion of the 234-bp core element of the DNase I hypersensitive site 3 (5HS3) of the locus control region (LCR) in the context of a human beta-globin locus yeast artificial chromosome (␤-YAC) results in profound effects on globin gene expression in transgenic mice. In contrast, deletion of a 2.3-kb 5HS3 region, which includes the 234-bp core sequence, has a much milder phenotype. Here we report the effects of these deletions on chromatin structure in the beta-globin locus of adult erythroblasts. The 234-bp 5HS3 deletion abolished histone acetylation throughout the ␤-globin locus; recruitment of RNA polymerase II (pol II) to the LCR and beta-globin gene promoter was reduced to a basal level; and formation of all the 5 DNase I hypersensitive sites of the LCR was disrupted. The 2.3-kb 5HS3 deletion mildly reduced the level of histone acetylation but did not change the profile across the whole locus; the 5 DNase I hypersensitive sites of the LCR were formed, but to a lesser extent; and recruitment of pol II was reduced, but only marginally. These data support the hypothesis that the LCR forms a specific chromatin structure and acts as a single entity. Based on these results we elaborate on a model of LCR chromatin architecture which accommodates the distinct phenotypes of the 5HS3 and HS3 core deletions.

“…Hypersensitivity at 5ЈHS5 is found in many nonerythroid cell lines, whereas 5ЈHS1 to 5ЈHS4 are predominantly erythroid cell specific (12,61), and 5ЈHS5 is contained on a 2.5-kb restriction fragment which was characterized as one of the potential matrix attachment regions within the ␤-globin locus (33). In addition 5ЈHS5 increases the tendency for positionindependent transcription of a linked reporter (32,68) and it has also been reported to block the activation of a promoter when placed between the promoter and an enhancer (39). It has therefore been suggested that 5ЈHS5 serves as an insulator which shields the locus from neighboring chromatin.…”

mentioning

confidence: 99%

The Locus Control Region Is Necessary for Gene Expression in the Human β-Globin Locus but Not the Maintenance of an Open Chromatin Structure in Erythroid Cells

Reik

Telling

Zitnik

et al. 1998

154

117

Studies in many systems have led to the model that the human ␤-globin locus control region (LCR) regulates the transcription, chromatin structure, and replication properties of the ␤-globin locus. However the precise mechanisms of this regulation are unknown. We have developed strategies to use homologous recombination in a tissue culture system to examine how the LCR regulates the locus in its natural chromosomal environment. Our results show that when the functional components of the LCR, as defined by transfection and transgenic studies, are deleted from the endogenous ␤-globin locus in an erythroid background, transcription of all ␤-globin genes is abolished in every cell. However, formation of the remaining hypersensitive site(s) of the LCR and the presence of a DNase I-sensitive structure of the ␤-globin locus are not affected by the deletion. In contrast, deletion of 5HS5 of the LCR, which has been suggested to serve as an insulator, has only a minor effect on ␤-globin transcription and does not influence the chromatin structure of the locus. These results show that the LCR as currently defined is not necessary to keep the locus in an "open" conformation in erythroid cells and that even in an erythroid environment an open locus is not sufficient to permit transcription of the ␤-like globin genes.The human ␤-globin locus provides a model system to study the interplay between chromatin structure and transcriptional regulation. The locus is located on chromosome 11 (11p15.5) and contains five developmentally regulated erythroid cell-specific genes arranged in the order in which they are expressed during development (5Ј-ε-G␥-A␥-␦-␤-3Ј) and an upstream regulatory region characterized by five DNase I-hypersensitive sites (HSs; see Fig. 1). By convention, these 5ЈHSs are denoted 5ЈHS1, 5ЈHS2, etc., with 5ЈHS1 being the most 3Ј and located closest to the ε-globin gene. The importance of the upstream regulatory region was established by an analysis of the effects of a naturally occurring deletion which removes 5ЈHS2 to 5ЈHS5 and 25 kb of additional sequences 5Ј of the HSs (Hispanic thalassemia). The chromosome carrying the deletion was transferred from lymphocytes of the thalassemic patient into murine erythroleukemia (MEL) cells to create the thalassemia line T-MEL. It was found that the mutation abolishes transcription of the adult human ␤-globin gene, prevents formation of 5ЈHS1 and other HSs throughout the locus, and renders the chromatin of the locus resistant to DNase I, indicative of a "closed" chromatin structure (19). In addition, the replication timing of the locus is changed from early to late in S phase (19) and a different origin of replication is used, even though the normal origin lies more than 50 kb from the site of the deletion (1, 36). The region containing the five HSs was termed the locus control region (LCR), because of its global effects on the locus. In transgenic mice, the LCR permits the expression of linked genes in all lines independent of the integration site (26), and regions with LCR...