The LCR-like α-globin positive regulatory element functions as an enhancer in transiently transfected cells during erythroid differentiation

Pondel, M.D.; George, Michael H.; Proudfoot, Nicholas J.

doi:10.1093/nar/20.2.237

Cited by 47 publications

(35 citation statements)

References 30 publications

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“…pA and poly A, polyadenylation site; ex1 to ex3, exon 1 to exon 3. was found to confer a strong (ϳ30-fold) positive effect on expression, which shows that this rabbit ␣-globin 5Ј flanking DNA segment is capable of responding to an enhancer. This result is consistent with the results of earlier studies with human ␣-globin gene constructs (31,36,47). In the test shown in Fig.…”

Section: Resultssupporting

confidence: 93%

“…1). Furthermore, reporter gene constructs promoted by the 5Ј flanking sequences of the ␣-globin gene that do show enhanced expression in the presence of HS Ϫ40 do not contain internal ␣-globin gene sequences (31,36), consistent with a role for internal sequences in enhancer-independent expression. Several possible explanations exist for the activity of this intragenic region.…”

mentioning

confidence: 70%

“…When HS Ϫ40 is added to reporter genes promoted by the proximal 5Ј flanking sequences of the human ␣-globin gene (but not including internal sequences), it enhances expression in transiently transfected erythroid cells (31,36). We also found that HS Ϫ40 enhances transient expression up to 30-fold from the rabbit ␣-globin gene 5Ј flanking sequences extending to Ϫ224.…”

Section: Discussionmentioning

confidence: 99%

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CpG Islands from the α-Globin Gene Cluster Increase Gene Expression in an Integration-Dependent Manner

Shewchuk

Hardison

1997

Molecular and Cellular Biology

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In contrast to other globin genes, the human and rabbit ␣-globin genes are expressed in transfected erythroid and nonerythroid cells in the absence of an enhancer. This enhancer-independent expression of the ␣-globin gene requires extensive sequences not only from the 5 flanking sequence but also from the intragenic region. However, the features of these internal sequences that are responsible for their positive effect are unclear. We tested several possible determinants of this activity. One possibility is that a previously identified array of discrete binding sites for known and potential regulatory proteins within the ␣-globin gene comprise an intragenic enhancer specific for the ␣-globin promoter, but directed rearrangements of the sequences show that this is not the case. Alternatively, the promoter may extend into the gene, with the function of the discrete binding sites being dependent on maintenance of their proper positions and orientations relative to the 5 flanking sequence. However, the positive effects observed in gene fusions do not localize to a discrete region of the ␣-globin gene and the results of internal deletions and point mutations argue against a required role of the targeted discrete binding sites. A third possibility is that the CpG island, which includes both the 5 flanking and intragenic regions associated with the positive activity, may itself have a more general effect on expression in transfected cells. Indeed, we show that the size of the CpG island in constructs correlates with the level of gene expression. Furthermore, the ␣-globin promoter is more active in the context of a previously inactive CpG island than in an A؉T-rich context, showing that the CpG island provides an environment more permissive for expression. These effects are seen only after integration, suggesting a possible mechanism at the level of chromatin structure.

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Section: Resultssupporting

confidence: 93%

mentioning

confidence: 70%

Section: Discussionmentioning

confidence: 99%

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CpG Islands from the α-Globin Gene Cluster Increase Gene Expression in an Integration-Dependent Manner

Shewchuk

Hardison

1997

Molecular and Cellular Biology

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“…Further remodeling and modification of the chromatin structure must occur in erythroid cells, however, since several new HS sites appear, including those at the HS-40 element and the promoter regions of the -and ␣-globin genes (54, 59). These latter sites apparently result from erythroid lineage-specific binding of nuclear factors to the above transcriptional regulatory elements, as demonstrated by previous genomic footprint analysis of 60) and of the ␣-globin upstream promoters (47).HS-40 acts as a classical enhancer in transient transfection assays (44,46,60), and it confers appropriate developmental control of the human -globin promoter activity in transgenic mice (19,23,45,56). In vitro and in vivo binding studies have shown that the HS-40 enhanceosome consists mainly of six DNA sequence motifs that are bound with nuclear factors in an erythroid lineage-specific manner: two NF-E2/AP1 motifs (5Ј-NA and 3Ј-NA), three GATA-1 motifs (b, c, and d), and a GT motif (26,50,60) (Fig.…”

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confidence: 73%

Loading of DNA-Binding Factors to an Erythroid Enhancer

Wen

Roder

et al. 2000

Molecular and Cellular Biology

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3The HS-40 enhancer is the major cis-acting regulatory element responsible for the developmental stage-and erythroid lineage-specific expression of the human ␣-like globin genes, the embryonic and the adult ␣2/␣/1. A model has been proposed in which competitive factor binding at one of the HS-40 motifs, 3-NA, modulates the capability of HS-40 to activate the embryonic -globin promoter. Furthermore, this modulation was thought to be mediated through configurational changes of the HS-40 enhanceosome during development. In this study, we have further investigated the molecular basis of this model. The interplay among the multiple nuclear factor-DNA complexes formed at the enhancers (enhanceosomes) and their cis-linked promoters (polymerase II [pol II] preinitiation complexes) is essential for the regulation of many eukaryotic genes (reviewed in references 6 and 9). Several modes of actions have been proposed for the enhanceosome function. Some enhanceosomes, such as the locus control region (LCR) of the human ␤-globin locus (reviewed in references 16, 20, and 22) may set up and/or maintain an active chromatin state of a gene or locus domain, thus allowing the formation of active pol II preinitiation complex. On the other hand, enhanceosomes could also facilitate the assembly of active pol II preinitiation complex by direct interaction with, or recruitment of, coactivators and different basal transcription factors (reference 6 and references therein).HS-40 is an element located at 40 kb upstream of the human ␣-globin locus (Fig. 1). Genetic and molecular data have indicated that it is essential for transcriptional regulation of the human embryonic -and adult ␣-globin promoters during erythroid development (21). Most of the ␣-globin gene cluster maintains an open chromatin structure in both erythroid and nonerythroid cells, possibly due to the transcription of certain ubiquitous genes (54). Further remodeling and modification of the chromatin structure must occur in erythroid cells, however, since several new HS sites appear, including those at the HS-40 element and the promoter regions of the -and ␣-globin genes (54, 59). These latter sites apparently result from erythroid lineage-specific binding of nuclear factors to the above transcriptional regulatory elements, as demonstrated by previous genomic footprint analysis of 60) and of the ␣-globin upstream promoters (47).HS-40 acts as a classical enhancer in transient transfection assays (44,46,60), and it confers appropriate developmental control of the human -globin promoter activity in transgenic mice (19,23,45,56). In vitro and in vivo binding studies have shown that the HS-40 enhanceosome consists mainly of six DNA sequence motifs that are bound with nuclear factors in an erythroid lineage-specific manner: two NF-E2/AP1 motifs (5Ј-NA and 3Ј-NA), three GATA-1 motifs (b, c, and d), and a GT motif (26,50,60) (Fig. 1A). Of these, the NF-E2/AP1 motifs could be recognized by the erythroid-enriched factor NF-E2, the ubiquitous AP1, the homodimers of small Maf family, or...

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“…Circulating blood cells from day 10.5 (d10.5) yolk sacs were isolated in phosphate-buffered saline and stained as described. 30,31 In all experiments, multiple embryos (Ͼ 10) were analyzed at each stage for each transgenic line. Analysis of wild type and each mutant line with Fisher exact test gives values between 0.061 and 0.098.…”

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confidence: 99%

Regulatory elements of the EKLF gene that direct erythroid cell-specific expression during mammalian development

Xue

Chang

Bieker

2004

Blood

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Erythroid Krü ppel-like factor (EKLF) plays an essential role in enabling ␤-globin expression during erythroid ontogeny. It is first expressed in the extraembryonic mesoderm of the yolk sac within the morphologically unique cells that give rise to the blood islands, and then later within the hepatic primordia. The BMP4/ Smad pathway plays a critical role in the induction of EKLF, and transient transfection analyses demonstrate that sequences located within less than 1 kb of its transcription initiation site are sufficient for high-level erythroid-specific transcription. We have used transgenic analyses to verify that 950 bp located adjacent to the EKLF start site of transcription is sufficient to generate lacZ expression within the blood islands as well as the fetal liver during embryonic development. Of particular importance are 3 regions, 2 of which overlap endogenous erythroid-specific DNase hypersensitive sites, and 1 of which includes the proximal promoter region. The onset of transgene expression mimics that of endogenous EKLF as it begins by day 7.5 (d7.5) to d8.0. In addition, it exhibits a strict hematopoietic specificity, localized only to these cells and not to the adjacent vasculature at all stages examined. Finally, expression is heterocellular, implying that although these elements are sufficient for tissue-specific expression, they do not shield against the position effects of adjacent chromatin. These analyses demonstrate that a surprisingly small DNA segment contains all the information needed to target a linked gene to the hematopoietic compartment at both early and later stages of development, and may be a useful cassette for this purpose. IntroductionHemoglobin production during mammalian development is characterized by a series of switches in sites of expression of the genes coding for the ␣-and ␤-globin chains. [1][2][3] The initial wave of transient primitive red cells is produced in the blood islands of the embryonic yolk sac. This is followed by generation of morphologically distinct definitive red cells produced in the fetal liver. After birth, the final switch to the bone marrow (and spleen in the mouse) completes the process. Concomitant with these changes, the ␤-like globin gene family is reprogrammed in a specific temporal sequence. The human ␤-like gene locus consists of embryonic (⑀), fetal (␥), and adult (␤) variants, while the murine ␤-like locus contains embryonic (y, bh0, bh1) and adult (␤) types. Together with a far upstream locus control region, the complete cluster is located within an open erythroid-specific chromatin domain that accommodates both long-and short-range interactions that yield developmentally correct gene expression. 4,5 Erythroid Krüppel-like factor (EKLF/KLF1 6 ) is an erythroid cell-specific transcription factor that plays a crucial role in regulation of ␤-like globin expression during erythroid development. 7,8 This is achieved by the sequence-specific interaction of its 3 zinc fingers with the CACCC element located within the adult ␤-globin promoter, 9...

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The LCR-like α-globin positive regulatory element functions as an enhancer in transiently transfected cells during erythroid differentiation

Cited by 47 publications

References 30 publications

CpG Islands from the α-Globin Gene Cluster Increase Gene Expression in an Integration-Dependent Manner

CpG Islands from the α-Globin Gene Cluster Increase Gene Expression in an Integration-Dependent Manner

Loading of DNA-Binding Factors to an Erythroid Enhancer

Regulatory elements of the EKLF gene that direct erythroid cell-specific expression during mammalian development

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