Erythroid-specific nuclease-hypersensitive sites flanking the human beta-globin domain

Recent evidence suggests that DNA sequences from the region lying 5' of the human epsilon-globin gene are important for erythroid-specific expression of human beta-like globin genes. This region, as well as a region 20 kilobases (kb) downstream from the beta-globin gene, contains a set of developmentally stable, DNase I-superhypersensitive sites that are thought to reflect a chromatin structure supporting active globin gene expression. We have analyzed the chromatin structure in these two regions in a wide variety of nonerythroid and erythroid cells. The study included analysis of chromatin structure changes occurring during globin gene activation in mouse erythroleukemia-human nonerythroid cell hybrids. The results identified a hypersensitive site (III) 14.8 kb upstream of the epsilon-globin gene that was strictly correlated with active globin gene transcription. Interestingly, a multipotent human embryonal carcinoma cell line exhibited a hypersensitive site (IV) 18.4 kb upstream of epsilon-globin that was absent in all other nonerythroid cells examined, suggesting that chromatin structure changes at specific hypersensitive sites during embryonic development may also be important in globin gene repression.

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The coordinate replication of the human beta-globin gene domain reflects its transcriptional activity and nuclease hypersensitivity

Molecular and Cellular Biology ◽

10.1128/mcb.8.11.4958-4965.1988 ◽

1988 ◽

Vol 8 (11) ◽

pp. 4958-4965

Author(s):

V Dhar ◽

D Mager ◽

A Iqbal ◽

C L Schildkraut

Keyword(s):

Cell Lines ◽

Dna Sequences ◽

Globin Gene ◽

K562 Cells ◽

Globin Genes ◽

Beta Globin ◽

Beta Globin Gene ◽

Hypersensitive Sites ◽

Flanking Sequences ◽

Globin Gene Domain

The temporal order of replication of DNA sequences in the chromosomal domain containing the human beta-globin gene cluster and its flanking sequences (140 kilobases) was measured and compared in two different human cell lines. In human erythroleukemia (K562) cells, in which embryonic and fetal globin genes are transcribed, all of the sequences we examined from the beta-globin domain replicated early during S phase, while in HeLa cells, in which globin genes are transcriptionally silent, these sequences replicated late during S. Potential sites of initiation of DNA replication within this domain were identified. The beta-globin gene domain was also found to differ with respect to the nuclease sensitivity of the chromatin in these two cell lines. In K562 cells, hypersensitive sites for endogenous nucleases and DNase I were present in the chromatin near the earliest-replicating segments in the beta-globin domain.

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The coordinate replication of the human beta-globin gene domain reflects its transcriptional activity and nuclease hypersensitivity.

Molecular and Cellular Biology ◽

10.1128/mcb.8.11.4958 ◽

1988 ◽

Vol 8 (11) ◽

pp. 4958-4965 ◽

Cited By ~ 40

Author(s):

V Dhar ◽

D Mager ◽

A Iqbal ◽

C L Schildkraut

Keyword(s):

Cell Lines ◽

Dna Sequences ◽

Globin Gene ◽

K562 Cells ◽

Globin Genes ◽

Beta Globin ◽

Beta Globin Gene ◽

Hypersensitive Sites ◽

Flanking Sequences ◽

Globin Gene Domain

The temporal order of replication of DNA sequences in the chromosomal domain containing the human beta-globin gene cluster and its flanking sequences (140 kilobases) was measured and compared in two different human cell lines. In human erythroleukemia (K562) cells, in which embryonic and fetal globin genes are transcribed, all of the sequences we examined from the beta-globin domain replicated early during S phase, while in HeLa cells, in which globin genes are transcriptionally silent, these sequences replicated late during S. Potential sites of initiation of DNA replication within this domain were identified. The beta-globin gene domain was also found to differ with respect to the nuclease sensitivity of the chromatin in these two cell lines. In K562 cells, hypersensitive sites for endogenous nucleases and DNase I were present in the chromatin near the earliest-replicating segments in the beta-globin domain.

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Characterization of the DNase I hypersensitive site 3' of the human beta globin gene domain

Blood ◽

10.1182/blood.v81.10.2781.bloodjournal81102781 ◽

1993 ◽

Vol 81 (10) ◽

pp. 2781-2790

Author(s):

DE Fleenor ◽

RE Kaufman

Keyword(s):

Topoisomerase Ii ◽

Globin Gene ◽

Dnase I ◽

Globin Genes ◽

Beta Globin ◽

Hypersensitive Site ◽

Mobility Shift ◽

Enhancer Activity ◽

Beta Globin Gene

The members of the human beta globin gene family are flanked by strong DNase I hypersensitive sites. The collection of sites 5' to the epsilon globin gene is able to confer high levels of expression of linked globin genes, but a function has not been assigned to the site 3' to the beta globin gene (3'HS1). Our analysis of this DNase I super hypersensitive site shows that the region is composed of multiple DNase I sites. By examination of the DNA sequence, we have determined that the region is very A/T-rich and contains topoisomerase II recognition sequences, as well as several consensus binding motifs for GATA-1 and AP-1/NF-E2. Gel mobility shift assays indicate that the region can interact in vitro with GATA-1 and AP-1/NF-E2, and functional studies show that the region serves as a scaffold attachment region in both erythroid and nonerythroid cell lines. Whereas many of the physical features of 3'HS1 are shared by 5'HS2 (a component of the 5' locus control region), transient expression studies show that 3' HS1 does not share the erythroid-specific enhancer activity exhibited by 5'HS2.

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Expression of the chicken beta-globin gene cluster in mice: correct developmental expression and distributed control.

Molecular and Cellular Biology ◽

10.1128/mcb.15.1.407 ◽

1995 ◽

Vol 15 (1) ◽

pp. 407-414 ◽

Cited By ~ 29

Author(s):

M M Mason ◽

E Lee ◽

H Westphal ◽

M Reitman

Keyword(s):

Developmental Stage ◽

Globin Gene ◽

Stage Iv ◽

Gene Clusters ◽

Developmental Expression ◽

Single Element ◽

Globin Genes ◽

Beta Globin ◽

Variable Expression ◽

Hypersensitive Sites

To investigate the regulation of gene clusters, we introduced the entire chicken beta-globin cluster into mice. This 35-kb region includes the four globin genes (rho-beta H-beta A-epsilon), the four upstream hypersensitive sites, and the intergenic beta A/epsilon enhancer. The chicken globins are not arranged in order of developmental expression, which is unlike the case for the human beta-globin cluster, in which gene order plays a role in the regulation of globin expression. Mice carrying the chicken cluster expressed the transgenes with the same developmental patterns as seen in the chicken. Therefore, stage-specific erythroid transcriptional milieus existed before the divergence of birds and mammals and have been conserved since then. Mice bearing the complete cluster except for a deletion removing the beta A/epsilon enhancer displayed markedly reduced expression of the beta H, beta A, and epsilon genes with efficient (but variable) rho expression. Mice carrying the four genes and beta A/epsilon enhancer but without the upstream hypersensitive sites showed reduced expression of rho, beta H, and beta A, with variable expression of epsilon. We conclude that (i) all of the genes (except possibly rho) are under the control of both the upstream hypersensitive sites and the enhancer, (ii) the influence of the control elements can extend beyond the nearest active gene, (iii) a single element (the enhancer) can influence more than one gene in a single developmental stage, (iv) the enhancer can work bidirectionally, and (v) neither the upstream sites (as a group) nor the enhancer showed developmental stage specificity. Thus, the regulation of this cluster is achieved by interaction of two distinct control regions with each of the globin genes.

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Deletion and replacement of the mouse adult beta-globin genes by a "plug and socket" repeated targeting strategy.

Molecular and Cellular Biology ◽

10.1128/mcb.14.10.6936 ◽

1994 ◽

Vol 14 (10) ◽

pp. 6936-6943 ◽

Cited By ~ 41

Author(s):

P J Detloff ◽

J Lewis ◽

S W John ◽

W R Shehee ◽

R Langenbach ◽

...

Keyword(s):

Dna Sequences ◽

Es Cells ◽

Globin Gene ◽

Embryonic Stem ◽

Second Step ◽

Globin Genes ◽

Beta Globin ◽

Es Cell ◽

Beta Globin Gene ◽

Targeting Strategy

We describe a two-step strategy to alter any mouse locus repeatedly and efficiently by direct positive selection. Using conventional targeting for the first step, a functional neo gene and a nonfunctional HPRT minigene (the "socket") are introduced into the genome of HPRT- embryonic stem (ES) cells close to the chosen locus, in this case the beta-globin locus. For the second step, a targeting construct (the "plug") that recombines homologously with the integrated socket and supplies the remaining portion of the HPRT minigene is used; this homologous recombination generates a functional HPRT gene and makes the ES cells hypoxanthine-aminopterin-thymidine resistant. At the same time, the plug provides DNA sequences that recombine homologously with sequences in the target locus and modifies them in the desired manner; the plug is designed so that correctly targeted cells also lose the neo gene and become G418 sensitive. We have used two different plugs to make alterations in the mouse beta-globin locus starting with the same socket-containing ES cell line. One plug deleted 20 kb of DNA containing the two adult beta-globin genes. The other replaced the same region with the human beta-globin gene containing the mutation responsible for sickle cell anemia.

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Analysis of mice containing a targeted deletion of beta-globin locus control region 5' hypersensitive site 3.

Molecular and Cellular Biology ◽

10.1128/mcb.16.6.2906 ◽

1996 ◽

Vol 16 (6) ◽

pp. 2906-2912 ◽

Cited By ~ 133

Author(s):

B A Hug ◽

R L Wesselschmidt ◽

S Fiering ◽

M A Bender ◽

E Epner ◽

...

Keyword(s):

Developmental Stages ◽

Germ Line ◽

Globin Gene ◽

Globin Genes ◽

Beta Globin ◽

Erythroid Cells ◽

Hypersensitive Site ◽

Targeted Deletion ◽

Gamma Globin ◽

Gene Switching

To examine the function of murine beta-globin locus region (LCR) 5' hypersensitive site 3 (HS3) in its native chromosomal context, we deleted this site from the mouse germ line by using homologous recombination techniques. Previous experiments with human 5' HS3 in transgenic models suggested that this site independently contains at least 50% of total LCR activity and that it interacts preferentially with the human gamma-globin genes in embryonic erythroid cells. However, in this study, we demonstrate that deletion of murine 5' HS3 reduces expression of the linked embryonic epsilon y- and beta H 1-globin genes only minimally in yolk sac-derived erythroid cells and reduces output of the linked adult beta (beta major plus beta minor) globin genes by approximately 30% in adult erythrocytes. When the selectable marker PGK-neo cassette was left within the HS3 region of the LCR, a much more severe phenotype was observed at all developmental stages, suggesting that PGK-neo interferes with LCR activity when it is retained within the LCR. Collectively, these results suggest that murine 5' HS3 is not required for globin gene switching; importantly, however, it is required for approximately 30% of the total LCR activity associated with adult beta-globin gene expression in adult erythrocytes.

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The beta globin 3' enhancer element confers regulated expression on the human gamma globin gene in the human embryonic-fetal erythroleukemia cell line K562

Blood ◽

10.1182/blood.v77.4.855.bloodjournal774855 ◽

1991 ◽

Vol 77 (4) ◽

pp. 855-860

Author(s):

M Donovan-Peluso ◽

S Acuto ◽

D O'Neill ◽

A Hom ◽

A Maggio ◽

...

Keyword(s):

Developmental Stage ◽

Globin Gene ◽

K562 Cells ◽

Specific Gene ◽

Fetal Life ◽

Globin Genes ◽

Beta Globin ◽

Specific Expression ◽

Enhancer Element ◽

Gamma Globin

We have constructed fusion genes comprised of gamma and beta globin elements and globin sequences linked to neomycin resistance (neoR) genes to define the cis acting sequences responsible for developmental stage-specific expression and induction of fetal globin genes in embryonic-fetal erythroleukemia K562 cells. The results indicate that the gamma promoter is required for proper initiation of transcription. However, the accumulation of gamma globin transcripts in response to hemin induction requires the additional presence of either gamma intervening sequence 2 or the 3′ enhancer element of the beta globin gene. Thus, the gamma promoter may provide the elements for developmental stage-specific gene expression during fetal life. By contrast, the beta 3′ enhancer is erythroid-specific but not developmental stage- or gene-specific.

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Hypersensitive site 5 of the human beta locus control region functions as a chromatin insulator

Blood ◽

10.1182/blood.v84.5.1399.1399 ◽

1994 ◽

Vol 84 (5) ◽

pp. 1399-1401 ◽

Cited By ~ 76

Author(s):

Q Li ◽

G Stamatoyannopoulos

Keyword(s):

Control Region ◽

Globin Gene ◽

Locus Control Region ◽

Beta Globin ◽

Hypersensitive Site ◽

Reporter Expression ◽

Chromatin Insulator ◽

Hypersensitive Sites ◽

Globin Gene Expression ◽

Beta Gene

Abstract The human beta locus control region (LCR) consists of five DNAse I hypersensitive sites (HS), four of which are erythroid specific and one, the further upstream located 5′HS5, is constitutive. To characterize the function of 5′HS5 we analyzed globin gene expression of various constructs containing HS3 as an enhancer, HS5, and the beta gene as a reporter. Expression was analyzed in stably transfected MEL cells. We found that the enhancing effect of hypersensitive site 3 is blocked when the HS5 is interposed between HS3 and the beta globin gene. These data suggest that the human 5′HS5 has the properties of a chromatin insulator.

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Essential role of NF-E2 in remodeling of chromatin structure and transcriptional activation of the epsilon-globin gene in vivo by 5' hypersensitive site 2 of the beta-globin locus control region.

Molecular and Cellular Biology ◽

10.1128/mcb.16.11.6055 ◽

1996 ◽

Vol 16 (11) ◽

pp. 6055-6064 ◽

Cited By ~ 75

Author(s):

Q H Gong ◽

J C McDowell ◽

A Dean

Keyword(s):

Control Region ◽

Chromatin Structure ◽

Transcriptional Activation ◽

Globin Gene ◽

Locus Control Region ◽

Dnase I ◽

Beta Globin ◽

Hypersensitive Site

Much of our understanding of the process by which enhancers activate transcription has been gained from transient-transfection studies in which the DNA is not assembled with histones and other chromatin proteins as it is in the cell nucleus. To study the activation of a mammalian gene in a natural chromatin context in vivo, we constructed a minichromosome containing the human epsilon-globin gene and portions of the beta-globin locus control region (LCR). The minichromosomes replicate and are maintained at stable copy number in human erythroid cells. Expression of the minichromosomal epsilon-globin gene requires the presence of beta-globin LCR elements in cis, as is the case for the chromosomal gene. We determined the chromatin structure of the epsilon-globin gene in both the active and inactive states. The transcriptionally inactive locus is covered by an array of positioned nucleosomes extending over 1,400 bp. In minichromosomes with a (mu)LCR or DNase I-hypersensitive site 2 (HS2) which actively transcribe the epsilon-globin gene, the nucleosome at the promoter is altered or disrupted while positioning of nucleosomes in the rest of the locus is retained. All or virtually all minichromosomes are simultaneously hypersensitive to DNase I both at the promoter and at HS2. Transcriptional activation and promoter remodeling, as well as formation of the HS2 structure itself, depended on the presence of the NF-E2 binding motif in HS2. The nucleosome at the promoter which is altered upon activation is positioned over the transcriptional elements of the epsilon-globin gene, i.e., the TATA, CCAAT, and CACCC elements, and the GATA-1 site at -165. The simple availability of erythroid transcription factors that recognize these motifs is insufficient to allow expression. As in the chromosomal globin locus, regulation also occurs at the level of chromatin structure. These observations are consistent with the idea that one role of the beta-globin LCR is to maintain promoters free of nucleosomes. The restricted structural change observed upon transcriptional activation may indicate that the LCR need only make a specific contact with the proximal gene promoter to activate transcription.

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