Molecular analysis of the human beta-globin locus activation region.

Forrester, William C.; Novak, Ulrike; Gelinas, Richard; Groudine, Mark

doi:10.1073/pnas.86.14.5439

Cited by 205 publications

(149 citation statements)

References 29 publications

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“…1B. When stably transfected into MEL cells, the construct is able to direct DNase I HS formation (14,21,38). This plasmid contains a neomycin resistance gene (neo R ) for the selection of stable transfectants and a 1.9-kb HS3 fragment, which serves as an internal control for LCR HS formation.…”

Section: Resultsmentioning

confidence: 99%

In Vivo Formation of a Human β-Globin Locus Control Region Core Element Requires Binding Sites for Multiple Factors Including GATA-1, NF-E2, Erythroid Kruppel-like Factor, and Sp1

Goodwin

McInerney

Glander

et al. 2001

Journal of Biological Chemistry

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The active elements of the ␤-globin locus control region (LCR) are located within domains of unique chromatin structure. These nuclease hypersensitive sites (HSs) are characterized by high DNase I sensitivity, erythroid specificity, similar nucleosomal structure, and evolutionarily conserved clusters of cis-acting elements that are required for the formation and function of the core elements. To determine the requirements for HS core formation in the setting of nuclear chromatin, we constructed a series of artificial HS cores containing binding sites for GATA-1, NF-E2, and Sp1. In contrast to the results of previous in vitro experiments, we found that when constructs were stably integrated in mouse erythroleukemia cells the binding sites for NF-E2, GATA-1, or Sp1 alone or in any combination were unable to form core HS structures. We subsequently identified two new cis-acting elements from the LCR HS4 core that, when combined with the NF-E2, Sp1, and tandem inverted GATA elements, result in core structure formation. Both new cis-acting elements bind Sp1, and one binds erythroid Kruppel-like factor (EKLF). We conclude that in vivo ␤-globin LCR HS core formation is more complex than previously thought and that several factors are required for this process to occur.

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Section: Resultsmentioning

confidence: 99%

In Vivo Formation of a Human β-Globin Locus Control Region Core Element Requires Binding Sites for Multiple Factors Including GATA-1, NF-E2, Erythroid Kruppel-like Factor, and Sp1

Goodwin

McInerney

Glander

et al. 2001

Journal of Biological Chemistry

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“…24 Each construct includes an intact A g-globin gene containing either an extended (1350 bp) promoter, as a HindIII-HindIII fragment (coordinates 38085-41382, GenBank humhbb), or a truncated promoter, as a StuI-HindIII fragment (39049-41382). Each g-globin cassette was linked to a recombinant form of the b-globin LCR (mLCR), 25 the HPFH2 enhancer (ScaI-NruI fragment, 5822-6544) or both. The mLCR and HPFH2 enhancers were located 5 0 and 3 0 of the A g-globin cassette, respectively.…”

Section: Plasmid Constructsmentioning

confidence: 99%

Use of the hereditary persistence of fetal hemoglobin 2 enhancer to increase the expression of oncoretrovirus vectors for human gamma-globin

et al. 2005

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The development of oncoretrovirus vectors for human g-globin has been hampered by problems of low expression and gene silencing. In order to address these problems, we investigated an enhancer element identified from individuals with deletional hereditary persistence of fetal hemoglobin 2 (HPFH2), a genetic condition characterized by elevated levels of g-globin in adults. Plasmid transfection studies in erythroid MEL (murine erythroleukemia) cells demonstrated the HPFH2 element could function synergistically with the b-globin locus control region to enhance the expression of an A g-globin gene with a truncated À382 bp promoter. A series of oncoretrovirus vectors were subsequently generated that contain an expression cassette for A g-globin linked to various combinations of the HPFH2 enhancer, the a-globin HS40 enhancer, and several versions of the promoter from A g-globin or b-globin. Expression analysis in transduced MEL cell clones revealed very high levels of promoterautonomous silencing that was at least partially abrogated by the HPFH2 enhancer. The vector containing a combination of a À201 bp A g-globin gene promoter with the Greek HPFH À117 point mutation and both the HPFH2 and HS40 enhancers exhibited no signs of vector silencing and was expressed at 248799% per copy of mouse a-globin (62% of total a-globin). This represents a significant improvement over previously reported oncoretrovirus vectors for A g-globin, and demonstrates the capacity of the HPFH2 enhancer to abrogate sequence-autonomous silencing of the A g-globin promoter in the context of a gene transfer vector.

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“…The LCR has been shown by studies of natural deletions of ␥␦␤-thalassemia (8,11,24) and by gene transfer experiments (14,18,20,51) to be indispensable for erythroid-cell-specific and high-level transcription of cis-linked globin genes and transgenes. The mechanism by which the LCR regulates transcription of the distant embryonic ε-, fetal ␥-, and adult ␤-globin genes at the respective developmental stages is not fully understood.…”

mentioning

confidence: 99%

Transcription of the HS2 Enhancer toward a cis-Linked Gene Is Independent of the Orientation, Position, and Distance of the Enhancer Relative to the Gene

Kong

Bohl

et al. 1997

Molecular and Cellular Biology

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The locus control region (LCR) of the human ␤-globin gene domain is defined by four erythroid-cell-specific DNase I-hypersensitive (HS) sites, HS1, -2, -3, and -4, located 50 to 70 kb upstream of the ␤-globin gene in a transcriptional direction: 5Ј HS4-HS3-HS2-HS1-//-ε-G ␥-A ␥-␦-␤ 3Ј (17,20,37,50). The LCR has been shown by studies of natural deletions of ␥␦␤-thalassemia (8,11,24) and by gene transfer experiments (14,18,20,51) to be indispensable for erythroid-cell-specific and high-level transcription of cis-linked globin genes and transgenes. The mechanism by which the LCR regulates transcription of the distant embryonic ε-, fetal ␥-, and adult ␤-globin genes at the respective developmental stages is not fully understood.In previous studies examining the mechanism of LCR function, a number of laboratories have investigated the ability of the individual HS sites to activate the transcription of cislinked genes in cell lines and transgenic mice. Among the LCR HS sites, HS2 has been found to possess developmental-stageindependent enhancer function (52). It is capable of stimulating the transcription of embryonic ε-, fetal ␥-, and adult ␤-globin genes in erythroid cells at the corresponding developmental stages (9,28,34,42,45,47) and may therefore constitute a major functional component of the LCR. However, a recent targeted deletion study shows that deletion of the HS2 enhancer from the murine LCR generated only mild effects on the expression of the ␤-like globin genes (15), suggesting that HS2 is not essential for LCR function and that LCR function may be due to the contribution of the other HS sites. Surprisingly, similar targeted deletion of HS3 also did not cause significant changes in the expression of the ␤-like globin genes (22). These findings suggest that LCR function is due not to the contribution of any specific HS site but to a series of interactions among its functional components, including the enhancer elements underlying the HS sites.In the above-described targeted deletion studies, deleting the HS2 or the HS3 sequence from the endogenous murine LCR and inserting in its place an independent transcription unit-a neomycin-resistant gene driven by a strong promoter-has been found to disrupt LCR function and cause severe anemia in and the deaths of homozygous transgenic mice (15,21). In the human ␤-LCR, inserting an independent transcription unit at a location between the HS2 enhancer and the downstream globin genes also disrupted LCR function (23) and caused the distantly downstream ␤-globin gene to be transcriptionally turned off. The mechanism by which the transcription of a foreign gene within the LCR might disrupt LCR function is not clearly understood (21).In an attempt to delineate the functional mechanism of the HS2 enhancer and ultimately of the LCR, we have previously analyzed the transcriptional status of the HS2 enhancer in transfected recombinant chloramphenicol acetyltransferase (CAT) plasmids (53). This earlier study showed that the transfected HS2 enhancer is itself transcribed in eryt...

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Molecular analysis of the human beta-globin locus activation region.

Cited by 205 publications

References 29 publications

In Vivo Formation of a Human β-Globin Locus Control Region Core Element Requires Binding Sites for Multiple Factors Including GATA-1, NF-E2, Erythroid Kruppel-like Factor, and Sp1

In Vivo Formation of a Human β-Globin Locus Control Region Core Element Requires Binding Sites for Multiple Factors Including GATA-1, NF-E2, Erythroid Kruppel-like Factor, and Sp1

Use of the hereditary persistence of fetal hemoglobin 2 enhancer to increase the expression of oncoretrovirus vectors for human gamma-globin

Transcription of the HS2 Enhancer toward a cis-Linked Gene Is Independent of the Orientation, Position, and Distance of the Enhancer Relative to the Gene

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