Globin Genes In Erythroid Cells Research Articles

The human β-globin enhancer LCR HS2 plays a role in forming a TAD by activating chromatin structure at neighboring CTCF sites.

The human β-globin locus control region (LCR) hypersensitive site 2 (HS2) is one of enhancers for transcription of the β-like globin genes in erythroid cells. Our previous study showed that the LCR HS2 has active chromatin structure before transcriptional induction of the β-globin gene, while another enhancer LCR HS3 is activated by the induction. To compare functional difference between them, we deleted each HS (ΔHS2 and ΔHS3) from the human β-globin locus in hybrid MEL/ch11 cells. Deletion of either HS2 or HS3 dramatically diminished the β-globin transcription and disrupted locus-wide histone H3K27ac and chromatin interaction between LCR HSs and gene. Surprisingly, ΔHS2 weakened interactions between CTCF sites forming the β-globin topologically associating domain (TAD), while ΔHS3 did not. CTCF occupancy and chromatin accessibility were reduced at the CTCF sites in the ΔHS2 locus. To further characterize the HS2, we deleted the maf-recognition elements for erythroid activator NF-E2 at HS2. This deletion decreased the β-globin transcription and enhancer-promoter interaction, but did not affect interactions between CTCF sites for the TAD. In light of these results, we propose that the HS2 has a role in forming a β-globin TAD by activating neighboring CTCF sites and this role is beyond typical enhancer activity.

Compound loss of function of nuclear receptors Tr2 and Tr4 leads to induction of murine embryonic β-type globin genes

AbstractThe orphan nuclear receptors TR2 and TR4 have been shown to play key roles in repressing the embryonic and fetal globin genes in erythroid cells. However, combined germline inactivation of Tr2 and Tr4 leads to periimplantation lethal demise in inbred mice. Hence, we have previously been unable to examine the consequences of their dual loss of function in adult definitive erythroid cells. To circumvent this issue, we generated conditional null mutants in both genes and performed gene inactivation in vitro in adult bone marrow cells. Compound Tr2/Tr4 loss of function led to induced expression of the embryonic εy and βh1 globins (murine counterparts of the human ε- and γ-globin genes). Additionally, TR2/TR4 function is required for terminal erythroid cell maturation. Loss of TR2/TR4 abolished their occupancy on the εy and βh1 gene promoters, and concurrently impaired co-occupancy by interacting corepressors. These data strongly support the hypothesis that the TR2/TR4 core complex is an adult stage-specific, gene-selective repressor of the embryonic globin genes. Detailed mechanistic understanding of the roles of TR2/TR4 and their cofactors in embryonic and fetal globin gene repression may ultimately enhance the discovery of novel therapeutic agents that can effectively inhibit their transcriptional activity and be safely applied to the treatment of β-globinopathies.

Stimulated stromal cells induce γ-globin gene expression in erythroid cells via nitric oxide production

We have previously shown that nitric oxide (NO) is involved in the hydroxyurea-induced increase of gamma-globin gene expression in cultured human erythroid progenitor cells and that hydroxyurea increases NO production in endothelial cells via endothelial NO synthase (NOS). We have now expanded those studies to demonstrate that stimulation of gamma-globin gene expression is also mediated by NOS induction in stromal cells within the bone marrow microenvironment. Using NO analyzer, we measured NO production in endothelial and macrophage cell cultures. In coculture studies of erythroid and stromal cells, we measured globin gene expression during stimulation by NO inducers. Hydroxyurea (30-100 microM) induced NOS-dependent production of NO in human macrophages (up to 1.2 microM). Coculture studies of human macrophages with erythroid progenitor cells also resulted in induction of gamma-globin mRNA expression (up to threefold) in the presence of hydroxyurea. NOS-dependent stimulation of NO by lipopolysaccharide (up to 0.6 microM) has been observed in human macrophages. We found that lipopolysaccharide and interferon-gamma together increased gamma-globin gene expression (up to twofold) in human macrophage/erythroid cell cocultures. Coculture of human bone marrow endothelial cells with erythroid progenitor cells also induced gamma-globin mRNA expression (2.4-fold) in the presence of hydroxyurea (40 microM). These results demonstrate an arrangement by which NO and fetal hemoglobin inducers may stimulate globin genes in erythroid cells via the common paracrine effect of bone marrow stromal cells.

A Facilitated Tracking and Transcription Mechanism of the HS2 Enhancer in the β-Globin Locus Control Region.

The 100 kb human β-globin gene locus contains the embryonic ε-, the fetal Gγ- and Aγ-, and the adult δ- and β-globin genes arranged in the transcriptional order of 5′ ε-Gγ-Aγ-δ-β 3′. The β-globin Locus Control Region (LCR), defined by DNase I hypersensitive sites HS1, 2, 3, 4 and 5 located far upstream of the globin genes, is absolutely required for transcriptional activation of the globin genes in erythroid cells. The HS2 site, located respectively 11 and 55 kb upstream of the ε- and β-globin genes, possesses prominent enhancer activity and is able to activate transcription of the globin genes over the long distance. How the HS2 enhancer acts over the long distance is not fully understood. Three mechanisms have been proposed to explain long-range enhancer function: The looping model, the protein tracking/linking model and the facilitated tracking model. We have shown earlier that a transcription mechanism of the RNA polymerase II (pol II)-complex assembled by the HS2 enhancer mediates long-range HS2 enhancer function. In the present study, we used the chicken HS4 insulator to further study the mechanism of long-range HS2 enhancer function. We created the LCR(+I) and LCR(−I) lines in human erythroid K562 cells using Cre-loxP mediated in situ recombination and in zebrafish. The integrated LCR(+I) and LCR(−I) plasmids spanned the natural 11 kb human ε-globin gene locus either with or without the chicken HS4 insulator inserted between the LCR HS2 enhancer and the ε-globin promoter. Analysis of enhancer function in the integrated plasmids and the endogenous ε-globin gene locus provided evidence for a novel facilitated tracking and transcription mechanism of long-range enhancer function: The HS2 enhancer complex containing both the enhancer DNA and the associated pol II tracked and transcribed through the 10 kb intervening DNA to loop with and activate the ε-globin promoter. The interposed insulator interrupted this facilitated tracking and transcription mechanism of the enhancer complex through the intervening DNA, thereby blocking long-range enhancer function.

Chromatin-binding in vivo of the erythroid kruppel-like factor, EKLF, in the murine globin loci

EKLF is an erythroid-specific, zinc finger-containing transcription factor essential for the activation of the mammalian beta globin gene in erythroid cells of definitive lineage. We have prepared a polyclonal anti-mouse EKLF antibody suitable for Western blotting and immunoprecipitation (IP) qualities, and used it to define the expression patterns of the EKLF protein during mouse erythroid development. We have also used this antibody for the chromatin-immunoprecipitation (ChIP) assay. EKLF was found to bind in vivo at both the mouse beta-major-globin promoter and the HS2 site of beta-LCR in the mouse erythroleukemia cells (MEL) in a DMSO-inducible manner. The DMSO-induced bindings of EKLF as well as three other proteins, namely, RNA polymerase II, acetylated histone H3, and methylated histone H3, were not abolished but significantly lowered in CB3, a MEL-derived cell line with null-expression of p45/NF-E2, an erythroid-enriched factor needed for activation of the mammalian globin loci. Interestingly, binding of EKLF in vivo was also detected in the mouse alpha-like globin locus, at the adult alpha globin promoter and its far upstream regulatory element alpha-MRE (HS26). This study provides direct evidence for EKLF-binding in vivo at the major regulatory elements of the mouse beta-like globin gene clusters the data also have interesting implications with respect to the role of EKLF-chromatin interaction in mammalian globin gene regulation.

CP2 Binding to the Promoter Is Essential for the Enhanced Transcription of Globin Genes in Erythroid Cells

CP2 Binding to the Promoter Is Essential for the Enhanced Transcription of Globin Genes in Erythroid Cells

A Long Terminal Repeat of the Human Endogenous Retrovirus ERV-9 Is Located in the 5′ Boundary Area of the Human β-Globin Locus Control Region

Transcription of the human β-like globin genes in erythroid cells is regulated by the far-upstream locus control region (LCR). In an attempt to define the 5′ border of the LCR, we have cloned and sequenced 5 kb of new upstream DNA. We found an LTR retrotransposon belonging to the ERV-9 family of human endogenous retroviruses in the apparent 5′ boundary area of the LCR. This ERV-9 LTR contains an unusual U3 enhancer region composed of 14 tandem repeats with recurrent GATA, CACCC, and CCAAT motifs. This LTR is conserved in human and gorilla, indicating its evolutionary stability in the genomes of the higher primates. In both recombinant constructs and the endogenous human genome, the LTR enhancer and promoter activate the transcription ofcis-linked DNA preferentially in erythroid cells. Our findings suggest the possibility that this LTR retrotransposon may serve a relevant host function in regulating the transcription of the β-globin LCR.

Modulatory Subdomains of the HS2 Enhancer Differentially Regulate Enhancer Activity in Erythroid Cells at Different Developmental Stages

ABSTRACT: The HS2 enhancer in the locus control region of human β-like globin genes displays developmental-stage-independent enhancer function. The mechanism by which it regulates the transcription of the globin genes in erythroid cells throughout development is not fully understood. In this paper we dissect the HS2 enhancer into an enhancer core and five modulatory subdomains M1 to M5. The enhancer core possesses developmental-stage-independent enhancer activity. The modulatory subdomains by themselves do not possess such enhancer activity, but they apparently respond to environmental signals and modulate enhancer core activity in a developmental-stage specific manner. M1 located 5' of the core strongly stimulates core activity in K562 cells at the embryonic stage. M2 and M3 located 3' of the core strongly stimulate core activity in MEL cells at the adult stage. Moreover, M3 suppresses core activity at the embryonic stage and exhibits an adult-stage-selector activity. These findings indicate that the apparent developmental-stage-independence of the HS2 enhancer is a result of multiple interactions between the core and the modulatory subdomains located both near and far from the core.

High-Level Globin Gene Expression Mediated by a Recombinant Adeno-Associated Virus Genome That Contains the 3′ γ Globin Gene Regulatory Element and Integrates as Tandem Copies in Erythroid Cells

AbstractRecombinant adeno-associated virus (rAAV) vectors are being evaluated for gene therapy applications. Using purified rAAV containing a mutationally marked globin gene (Aγ*) and sites 2, 3, and 4 from the locus control region (rHS432Aγ*), but lacking a drug-resistance gene, we investigated the relationship between multiplicity of infection (MOI), gene expression, and unselected genome integration in erythroid cells. Most primary erythroid progenitors were transduced as reflected by Aγ* mRNA in mature colonies but only at an MOI of greater than 5 × 107. Using immortalized erythroleukemia cells as a model, we found that fewer than one half of the colonies that contained the Aγ* transcript had an integrated, intact rHS432Aγ* genome. rHS432Aγ* integrated as a single copy with expression at approximately 50% the level of an endogenous γ globin gene. A second vector, rHS32Aγ*3′RE, containing the regulatory element (RE) from 3′ to the chromosomal Aγ globin gene, integrated as an intact, tandem head to tail concatamer with a median copy number of 6 with variable expression per copy ranging from approximately onefold to threefold that of an endogenous γ globin gene. These results establish that purified rAAV can be used to achieve integration and functional expression of a globin gene in erythroid cells, but only when high MOIs are used.

Production of Genetically Stable High-Titer Retroviral Vectors That Carry a Human γ-Globin Gene Under the Control of the α-Globin Locus Control Region

The ability to generate stable high-titer vectors that give rise to high levels of expression of transduced globin genes in erythroid cells is a prerequisite for effective retroviral-mediated globin gene therapy. The human beta-globin gene with its immediate flanking sequences does not contain all the regulatory elements necessary for regulated high-level and position-independent expression in erythroid cells. The regulatory element known as the beta-globin locus control region (BetaLCR) can provide a linked Beta-globin gene with these properties. However, addition of BetaLCR sequences to a retrovirus carrying a beta-globin gene increases its genetic instability. We have developed a new generation of retroviral vectors in which a human gamma-globin gene is placed under the control of the alphaLCR, the major regulatory element of the alpha-globin gene cluster. We demonstrate that these retroviruses are genetically stable in producer cell lines and can be produced at high titers that exceed 5 x 10(6) colony-forming units (CFU)/mL. In addition, we show that the transduced gamma-globin gene can be expressed in the adult erythroid environment of mouse erythroleukemia (MEL) cells at a level comparable to that of a single endogenous Betamaj-globin gene. These retroviruses can also transduce primary murine bone marrow progenitor cells as efficiently as retroviruses that carry the neomycin resistance (neor) gene. This new generation of globin retroviral vectors may prove useful for gene therapy of human beta-globin gene disorders such as sickle cell disease and beta-thalassemia.

The minimal requirements for activity in transgenic mice of hypersensitive site 3 of the beta globin locus control region.

Proper expression of the human beta-like globin genes is completely dependent on the presence of the locus control region or LCR, a region containing four DNase hypersensitive sites (HS1-4) situated 5' to the structural genes. Linkage of the LCR to a transgene results in copy number-dependent transcription, independent of the site of integration in the host genome. We have analysed a small region of the LCR (HS3) in transgenic animals to determine the minimal interactions that are required for this property. The results show that a specific combination of a G-rich sequence flanked on each side by one binding site for the transcription factor GATA1 is essential to obtain position-independent expression of a linked beta globin gene in erythroid cells. The overall transcriptional activity of HS3 is achieved through synergy with other combinations of similar binding sites.

Regulated high level expression of a human gamma-globin gene introduced into erythroid cells by an adeno-associated virus vector.

Gene therapy of severe hemoglobinopathies will require high-level expression of a transferred globin gene in erythroid cells. Distant regulatory elements flanking the beta-globin gene cluster, the locus control region, are needed for appropriate expression. We have explored the use of a human parvovirus, the adeno-associated virus (AAV), for globin gene transfer. The human A gamma-globin gene, linked to hypersensitivity site 2 from the locus control region of the beta-globin gene cluster, was subcloned into a plasmid (psub201) containing the AAV inverted terminal repeats. This construct was cotransfected with a helper plasmid containing trans-acting AAV genes into human 293 cells that had been infected with adenovirus. The recombinant AAV vector containing hypersensitivity site 2 stably introduced on average one or two unrearranged proviral copies into human K562 erythroleukemia cells. The transferred globin gene exhibited normal regulation upon hemin induction of erythroid maturation and was expressed at a level equivalent to a native chromosomal A gamma-globin gene.

A deletion of the human beta-globin locus activation region causes a major alteration in chromatin structure and replication across the entire beta-globin locus.

Naturally occurring deletions that remove sequences located approximately 60 kb upstream of the human adult beta-globin gene result in the failure to transcriptionally activate the cis-linked globin genes in erythroid cells. In addition, transfection, transgenic, and somatic cell hybrid studies have revealed that sequences within this region are essential for the developmentally regulated high-level expression of cis-linked globin genes. This regulatory region located at the 5' end of the beta-globin locus has been termed the locus activation region (LAR). Using somatic cell hybrids, we have studied the chromatin structure and timing of DNA replication of the normal human beta-globin locus and a locus containing a de novo 25-kb deletion that removes elements of the LAR. As a result of this deletion, the entire beta-globin locus and sequences approximately 100 kb 5' and 3' of the adult beta-globin gene are DNase I-resistant and do not form characteristic distant hypersensitive sites. These sequences also replicate late in S phase in an erythroid cell background. In contrast, the sequences of the normal locus are DNase I sensitive and early replicating. These results suggest that the LAR is required for both the erythroid-specific chromatin structure and timing of DNA replication over a large physical distance.

A naturally occurring gamma globin gene mutation enhances SP1 binding activity.

Transcription of the human fetal globin genes in erythroid cells is tightly regulated during different stages of development and differentiation. Two naturally occurring mutations 202 base pairs upstream of the duplicated gamma globin genes are associated with incorrectly regulated gamma globin gene gene expression; elevated levels of fetal globin are synthesized during adult life. A C-to-G base substitution upstream of the G gamma-globin gene is highly correlated with a dramatic increase in gene expression. It increases the similarity of the region to the consensus Sp1 recognition site. We determined that the mutated DNA had a 5- to 10-fold-higher affinity for Sp1 than did normal gamma globin gene sequence. We also observed a reduction in normal factor-binding activity. A different substitution at -202, C to T, upstream of the A gamma-globin gene was associated with a more moderate increase in fetal globin expression. This mutation decreased the similarity of the sequence to an Sp1 recognition site. We determined that it did not result in enhanced Sp1 binding but did alter normal factor binding. We suggest that these changes in nuclear protein-binding properties detected in vitro are responsible for the enhanced gamma globin gene expression found in -202 G gamma beta + patients with hereditary persistence of fetal hemoglobin.

A Naturally Occurring Gamma Globin Gene Mutation Enhances SP1 Binding Activity

Transcription of the human fetal globin genes in erythroid cells is tightly regulated during different stages of development and differentiation. Two naturally occurring mutations 202 base pairs upstream of the duplicated gamma globin genes are associated with incorrectly regulated gamma globin gene gene expression; elevated levels of fetal globin are synthesized during adult life. A C-to-G base substitution upstream of the G gamma-globin gene is highly correlated with a dramatic increase in gene expression. It increases the similarity of the region to the consensus Sp1 recognition site. We determined that the mutated DNA had a 5- to 10-fold-higher affinity for Sp1 than did normal gamma globin gene sequence. We also observed a reduction in normal factor-binding activity. A different substitution at -202, C to T, upstream of the A gamma-globin gene was associated with a more moderate increase in fetal globin expression. This mutation decreased the similarity of the sequence to an Sp1 recognition site. We determined that it did not result in enhanced Sp1 binding but did alter normal factor binding. We suggest that these changes in nuclear protein-binding properties detected in vitro are responsible for the enhanced gamma globin gene expression found in -202 G gamma beta + patients with hereditary persistence of fetal hemoglobin.

An erythroid-specific, developmental-stage-independent enhancer far upstream of the human "beta-like globin" genes.

We have identified an erythroid-specific enhancer element far upstream of the human "beta-like globin" genes, at 10.2-11.0 kilobases 5' of the embryonic epsilon-globin gene, and thus at 53-54 kilobases 5' of the adult beta-globin gene. It is capable of enhancing the expression of a cis-linked test gene by up to 300-fold. This enhancer element is apparently developmental-stage-independent, as it is functional at the embryonic and the adult developmental stages in erythroid cells that are expressing the respective beta-like globin genes. The enhancer and globin promoter sequences work in synergy and are capable of conferring on a cis-linked gene the high transcriptional efficiency (enhancer function), erythroid specificity (enhancer and promoter functions), and developmental-stage specificity (promoter function) that are characteristic of the in vivo transcription of the beta-like globin genes in erythroid cells.

Promoter sequences required for function of the human gamma globin gene in erythroid cells.

The human gamma- and beta-globin genes are expressed during the fetal and adult developmental periods, respectively. Differences in the sequences of their promoters may be relevant to their developmental regulation. The gamma globin gene promoter was found to be stronger than that of the beta gene. In HeLa cells co-transfected with plasmids containing the two genes, transcripts arising from the gamma promoter accumulated to a level 3-fold higher than those initiated from the beta-promoter. We have recently shown that deletion of the most distal of the conserved elements of the promoter (CACCC) reduces function to 25% of the wild-type, whereas the removal of the proximal of the two duplicated (CCAAT) elements increases promoter function 2- to 4-fold in HeLa cells during transient gene expression. Both the wild-type promoter and the truncation and linker-scanning mutants from which one of the two duplicated "CCAAT' elements had been removed, exhibited regulated expression when stably integrated into chromosomes of mouse erythroleukemia (MEL) cells. Thus, duplication of the "CCAAT' element, the feature by which the gamma promoter differs most strikingly from the beta, is not essential for function in erythroid cells.

Structure and expression of human globin genes introduced into mouse fibroblasts.

The human delta- and beta-globin genes, contained in a recombinant bacteriophage (lambda H beta G1), were introduced into mouse fibroblasts by cotransformation with a plasmid (chi 1) containing the herpes simplex thymidine kinase gene using the calcium phosphate precipitation technique. A molar ratio of lambda H eta G1 to chi 1 DNA of 3:1 was used. Four of the eleven stable transformants obtained contained intact delta- and beta-globin genes as determined by Southern blot analysis. To assess methylation in the segment of human DNA introduced into mouse cells, digestion with Hpa II or Msp I alone or with a second restriction enzyme was performed. The sites examined near the human delta- and beta-globin genes in transformed cells were not methylated. RNA extracted from the transformed cells was analyzed by RNA-cDNA hybridization; no more than 100 copies of human beta-globin mRNA/cell were found. Although hypomethylation of sites surrounding expressed globin genes in erythroid cells has been described, this property is not sufficient to ensure a high level of expression in fibroblasts.

Activation of histone gene transcription from chromatin of G 1 hela cells by s-phase non-histone chromosomal proteins

Non-histone chromosomal proteins have been implicated as having an important function in the regulation of gene expression [l-9] . Specifically, these proteins have been shown to be involved in controlling the transcription of globin genes in erythroid cells [ 1 O-l 2] and histone genes during the cell cycle [ 13-141. In HeLa cells, synthesis of histones is restricted to the period of the cell cycle during which DNA is replicated (S-phase) [15-l 81, and translation [15-17, 19-211 as well as hybridization data [22] indicate that histone mRNAs are associated with polysomes only at this time. Using a 3H-labeled single stranded complementary DNA as a probe for histone mRNA sequences [23], we have recently demonstrated that histone genes are transcribed in vitro from chromatin isolated from S-phase cells, but that chromatin isolated from Gr phase cells does not serve as a template for the transcription of histone sequences [24]. These results suggest that histone genes are transiently expressed during the period of DNA replication and that regulation of histone gene expression occurs at least in partat the transcriptional level. We have shown through chromatin in reconstitution studies that the non-histone chromosomal protein component of the genome is responsible for the difference in the in vitro transcription of histone sequences from Gi and S-phase chromatin [ 131. The present study demonstrates that when chromatin from Gr phase HeLa cells is reconstituted in the presence of nonhistone chromosomal proteins isolated from S-phase cells, a dose dependent activation of histone gene transcription is observed.