The Role of Crowding Forces in Juxtaposing β-Globin Gene Domain Remote Regulatory Elements in Mouse Erythroid Cells

The human G gamma-globin and beta-globin genes are expressed in erythroid cells at different stages of human development, and previous studies have shown that the two cloned genes are also expressed in a differential stage-specific manner in transgenic mice. The G gamma-globin gene is expressed only in murine embryonic erythroid cells, while the beta-globin gene is active only at the fetal and adult stages. In this study, we analyzed transgenic mice carrying a series of hybrid genes in which different upstream, intragenic, or downstream sequences were contributed by the beta-globin or G gamma-globin gene. We found that hybrid 5'G gamma/3'beta globin genes containing G gamma-globin sequences upstream from the initiation codon were expressed in embryonic erythroid cells at levels similar to those of an intact G gamma-globin transgene. In contrast, beta-globin upstream sequences were insufficient for expression of 5'beta/3'G gamma hybrid globin genes or a beta-globin-metallothionein fusion gene in adult erythroid cells. However, beta-globin downstream sequences, including 212 base pairs of exon III and 1,900 base pairs of 3'-flanking DNA, were able to activate a 5'G gamma/3'beta hybrid globin gene in fetal and adult erythroid cells. These experiments suggest that positive regulatory elements upstream from the G gamma-globin and downstream from the beta-globin gene are involved in the differential expression of the two genes during development.

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The "beta-like-globin" gene domain in human erythroid cells.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.82.19.6384 ◽

1985 ◽

Vol 82 (19) ◽

pp. 6384-6388 ◽

Cited By ~ 310

Author(s):

D. Tuan ◽

W. Solomon ◽

Q. Li ◽

I. M. London

Keyword(s):

Globin Gene ◽

Erythroid Cells ◽

Globin Gene Domain

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Mechanisms controlling activation of the α-globin gene domain in chicken erythroid cells

Biochemistry (Moscow) ◽

10.1134/s000629790705001x ◽

2007 ◽

Vol 72 (5) ◽

pp. 467-470 ◽

Cited By ~ 6

Author(s):

S. V. Razin ◽

E. S. Ioudinkova

Keyword(s):

Globin Gene ◽

Erythroid Cells ◽

Globin Gene Domain

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Erythroid Specificity and Safety of Globin-Encoding Lentiviral Vectors

Blood ◽

10.1182/blood-2020-142989 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 29-29

Author(s):

Annalisa Cabriolu ◽

Ashlesha Odak ◽

Friederike Kogel ◽

Michel Sadelain

Keyword(s):

Bone Marrow ◽

Sickle Cell ◽

Research Funding ◽

Bone Marrow Cells ◽

Globin Gene ◽

Regulatory Elements ◽

Beta Globin ◽

Erythroid Cells ◽

Hematopoietic Progenitor ◽

Enhancer Blocking

The beta-thalassemias and sickle cell disease are the most common monogenic inherited blood disorders, both arising from mutations affecting the beta-globin locus. Recent clinical trials utilizing lentiviral-mediated beta-globin gene transfer in autologous CD34+ cells have shown encouraging results in patients with non-beta-zero thalassemias and sickle cell anemia. A case report of insertional mutagenesis in a beta-thalassemia patient resulted in a prolonged clonal expansion that eventually regressed without progressing to leukemic transformation. Nonetheless, this occurrence and the need to insert multiple vector copies per cell when using globin vectors that provide insufficient globin expression to achieve curative responses from a single integrated copy per cell, raise some questions about the erythroid specificity and safety of globin vectors. We thus found it imperative to investigate globin vector expression in hematopoietic progenitors and in non-erythroid cells. To this end, we investigated what regulatory elements, including locus control region (LCR) hypersensitive sites (HS) and others, achieve the highest beta-globin expression per vector copy while at the same time minimizing non-erythroid transcriptional activity. We developed an invivo assay to track the enhancer/promoter activity of different HS elements in hematopoietic progenitor and differentiated cell subsets. We designed lentiviral vectors expressing hrGFP under the control of a short β-globin promoter (137bp) controlled by a set of LCR HS elements and/or a novel biliverdin (BLV) enhancer. Analysis of hrGFP expression in bone marrow cells derived from C57BL6 mice transplanted with transduced syngeneic bone marrow cells revealed that many of the vectors encompassing elements thought to encode erythroid-specific elements were in fact expressed in long term-HSC (LT-HSC), short term-HSC (ST-HSC), multipotent progenitors (MPP), pre-GM, Granulocyte/macrophage progenitors (GMP) and megakaryocyte progenitors (MKP). These findings confirmed that ectopically integrated erythroid regulatory elements can serve as transcriptional enhancers in non-erythroid cells. This transcriptional leakiness was confirmed in therapeutic globin vectors harboring a combination of LCR elements. To avoid these effects, we flanked the globin transcription unit with a small human enhancer-blocking element called A1, kindly provided by the late George Stamatoyannopoulos (Liu, M. et al. 2015). The insulated vector showed markedly reduced non-erythroid expression in non-erythroid cells including ST-HSC, MPP, Pre-GM and GMP. Expression in mature erythroid cells was unchanged. Our findings underscore the benefits of selecting lineage-specific regulatory elements with the least lineage promiscuity for therapeutic vectors, with the eventual addition of enhancer-blocking elements. Based on these results, we are now investigating new globin expression vectors with optimal combination of HS elements. Such novel globin vectors should minimize the risk of oncogene trans-activation in hematopoietic progenitor cells and therefore improve the safety of globin gene therapy while providing lineage-specific high-level expression. Disclosures Sadelain: Mnemo: Patents & Royalties; Fate Therapeutics: Patents & Royalties, Research Funding; Atara: Patents & Royalties, Research Funding; Takeda: Patents & Royalties, Research Funding; Minerva: Other: Biotechnologies , Patents & Royalties.

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The HMG Domain Protein SSRP1/PREIIBF Is Involved in Activation of the Human Embryonic β-Like Globin Gene

Molecular and Cellular Biology ◽

10.1128/mcb.18.5.2617 ◽

1998 ◽

Vol 18 (5) ◽

pp. 2617-2628 ◽

Cited By ~ 22

Author(s):

Michael A. Dyer ◽

Patrick J. Hayes ◽

Margaret H. Baron

Keyword(s):

Developmental Stage ◽

Regulatory Element ◽

Globin Gene ◽

Regulatory Elements ◽

Minimal Promoter ◽

Erythroid Cell ◽

Expression Cloning ◽

Erythroid Cells ◽

Specific Expression ◽

Binding Factor

ABSTRACT The human embryonic β-like globin (ɛ-globin) gene is expressed in primitive erythroid cells of the yolk sac during the first few weeks of development. We have previously shown that developmental stage-specific expression of the ɛ-globin gene is mediated by multiple positive and negative regulatory elements upstream of the start of transcription. Of particular interest is one positive regulatory element, PRE II, that works together with other elements (PRE I and PRE V) to confer developmental stage- and/or tissue-specific expression on a minimal promoter. An ∼85- to 90-kDa PRE II binding factor (PREIIBF) was identified in the nuclei of erythroid cells and shown to bind specifically to a novel 19-bp region within PRE II; binding of this protein to PRE II resulted in bending of the target DNA and was required for promoter activation. In this report, we present the cDNA expression cloning of PREIIBF. The cDNA encodes a previously identified member of the HMG domain family of DNA binding proteins termed SSRP1. By a number of biochemical and immunological criteria, recombinant SSRP1 appears to be identical to the PREII binding factor from erythroid nuclei. A hallmark of HMG domain proteins is their ability to bend their target DNAs; therefore, as we speculated previously, DNA bending by SSRP1/PREIIBF may contribute to the mechanism by which PRE II synergizes with other regulatory elements located upstream and downstream. In contrast with reports from other investigators, we demonstrate that SSRP1 binds DNA with clear sequence specificity. Moreover, we show that SSRP1/PREIIBF lacks a classical activation domain but that binding by this protein to PRE II is required for activation of a minimal promoter in stable erythroid cell lines. These studies provide the first evidence that SSRP1 plays a role in transcriptional regulation. SSRP1/PREIIBF may serve an architectural function by helping to coordinate the assembly of a multiprotein complex required for stage-specific regulation of the human ɛ-globin gene.

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Human Fetal Hemoglobin Expression Is Regulated by the Developmental Stage-Specific Repressor BCL11A

Science ◽

10.1126/science.1165409 ◽

2008 ◽

Vol 322 (5909) ◽

pp. 1839-1842 ◽

Cited By ~ 487

Author(s):

Vijay G. Sankaran ◽

Tobias F. Menne ◽

Jian Xu ◽

Thomas E. Akie ◽

Guillaume Lettre ◽

...

Keyword(s):

Association Studies ◽

Globin Gene ◽

Genetic Association Studies ◽

Fetal Hemoglobin ◽

Sequence Variants ◽

Erythroid Cells ◽

Cell Disease ◽

Direct Role ◽

Globin Gene Regulation

Differences in the amount of fetal hemoglobin (HbF) that persists into adulthood affect the severity of sickle cell disease and the β-thalassemia syndromes. Genetic association studies have identified sequence variants in the gene BCL11A that influence HbF levels. Here, we examine BCL11A as a potential regulator of HbF expression. The high-HbF BCL11A genotype is associated with reduced BCL11A expression. Moreover, abundant expression of full-length forms of BCL11A is developmentally restricted to adult erythroid cells. Down-regulation of BCL11A expression in primary adult erythroid cells leads to robust HbF expression. Consistent with a direct role of BCL11A in globin gene regulation, we find that BCL11A occupies several discrete sites in the β-globin gene cluster. BCL11A emerges as a therapeutic target for reactivation of HbF in β-hemoglobin disorders.

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The role of human globin gene promoters in the expression of hybrid genes in erythroid and non-erythroid cells

Biochemical and Biophysical Research Communications ◽

10.1016/0006-291x(87)90365-2 ◽

1987 ◽

Vol 143 (3) ◽

pp. 1099-1106 ◽

Cited By ~ 7

Author(s):

Santina Acuto ◽

Maryann Donovan-Peluso ◽

Nino Giambona ◽

Arthur Bank

Keyword(s):

Globin Gene ◽

Gene Promoters ◽

Erythroid Cells ◽

Hybrid Genes

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Function of normal and mutated gamma-globin gene promoters in electroporated K562 erythroleukemia cells

Blood ◽

10.1182/blood.v75.4.990.990 ◽

1990 ◽

Vol 75 (4) ◽

pp. 990-999 ◽

Cited By ~ 46

Author(s):

MJ Ulrich ◽

TJ Ley

Keyword(s):

Transcription Initiation ◽

Globin Gene ◽

Regulatory Elements ◽

Promoter Strength ◽

Gene Promoters ◽

Erythroid Cells ◽

Cis Acting ◽

Promoter Function ◽

Gamma Globin ◽

Globin Promoter

Abstract We examined the importance of cis-acting regulatory elements of the human gamma-globin gene promoter in a cell line (K562) where this gene normally functions. A gamma-Globin promoter fragments were fused to the neomycin phosphotransferase (neoR) gene in a plasmid-based vector and transiently transfected by electroporation into K562 cells. Correctly initiated “A gamma-neo” transcripts were detected with an S1 nuclease protection assay that was internally controlled for transfection efficiency and RNA content. We first optimized the conditions for electroporation, and then determined input DNA concentrations that permitted study of gamma-promoter function in the linear range of the assay. We discovered that a gamma-globin promoter fragment extending from -299 to +36 (with respect to the transcription initiation site) was active in this transient transfection assay, and that the expression of this promoter was increased by the SV40 enhancer. Deletion of the gamma-globin promoter to position -199 did not significantly affect gamma-globin promoter function. However, deletion to -160 reduced gamma promoter strength to 70% that of control, deletion to position -130 to 19% that of control, and deletion to position -61 to 8.7% that of control. Three gamma-globin promoters containing mutations associated with hereditary persistence of fetal hemoglobin (-202 C----G, -196 C----T, and -117 G----A) were not overexpressed in the K562 cell environment, consistent with the hypothesis that these promoters are not overexpressed in fetal erythroblasts, only adult erythroid cells. This system will allow us to further dissect the roles of regulatory globin cis-acting DNA elements in fetal erythroid cells.

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Regulated expression of human alpha- and beta-globin genes in transient heterokaryons.

Molecular and Cellular Biology ◽

10.1128/mcb.11.3.1239 ◽

1991 ◽

Vol 11 (3) ◽

pp. 1239-1247 ◽

Cited By ~ 14

Author(s):

M H Baron ◽

T Maniatis

Keyword(s):

Transcriptional Activation ◽

Globin Gene ◽

Regulatory Elements ◽

Erythroid Cell ◽

Globin Genes ◽

Beta Globin ◽

Erythroid Cells ◽

Aberrant Expression ◽

Previous Demonstration ◽

High Level

We have examined the expression of human alpha- and beta-like globin genes in transient heterokaryons formed by fusion of human nonerythroid cells with terminally differentiating mouse erythroleukemia (MEL) cells or with a MEL cell variant (GM979) in which the endogenous mouse embryonic beta-globin genes are activated. In both the parental MEL cells and the heterokaryons, the alpha-globin genes were activated at least 12 h earlier than the embryonic, fetal, and adult beta-globin genes. These results suggest that kinetic differences in the activation of alpha- and beta-like globin genes are not simply the result of different rates of accumulation of erythroid-specific regulatory factors but may reflect differences in the mechanisms governing the transcriptional activation of these genes during erythroid cell differentiation. In mouse GM979 x human nonerythroid heterokaryons, the human embryonic beta-globin gene was activated, consistent with our previous demonstration that erythroid cells contain stage-specific trans-acting regulators of globin gene expression. Moreover, a dramatic increase in the ratio of human fetal to adult beta-globin transcription was observed compared with that seen in MEL-human nonerythroid hybrids. This ratio change may reflect competition between the fetal and adult beta-globin genes for productive interactions with erythroid cell-specific regulatory elements. Finally, we demonstrate that the behavior of naturally occurring mutations that lead to aberrant hemoglobin switching in humans also leads to aberrant expression in transient heterokaryons. Therefore, erythroid cells must contain trans-acting factors that interact with mutated regulatory elements to induce high-level expression of the human fetal globin genes.

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The role of the polycomb complex in silencing α-globin gene expression in nonerythroid cells

Blood ◽

10.1182/blood-2008-06-161901 ◽

2008 ◽

Vol 112 (9) ◽

pp. 3889-3899 ◽

Cited By ~ 37

Author(s):

David Garrick ◽

Marco De Gobbi ◽

Vasiliki Samara ◽

Michelle Rugless ◽

Michelle Holland ◽

...

Keyword(s):

Histone Deacetylases ◽

Globin Gene ◽

Cpg Islands ◽

Gene Activation ◽

Globin Genes ◽

Erythroid Cells ◽

Polycomb Complex ◽

Globin Gene Expression

Although much is known about globin gene activation in erythroid cells, relatively little is known about how these genes are silenced in nonerythroid tissues. Here we show that the human α- and β-globin genes are silenced by fundamentally different mechanisms. The α-genes, which are surrounded by widely expressed genes in a gene dense region of the genome, are silenced very early in development via recruitment of the Polycomb (PcG) complex. By contrast, the β-globin genes, which lie in a relatively gene-poor chromosomal region, are not bound by this complex in nonerythroid cells. The PcG complex seems to be recruited to the α-cluster by sequences within the CpG islands associated with their promoters; the β-globin promoters do not lie within such islands. Chromatin associated with the α-globin cluster is modified by histone methylation (H3K27me3), and silencing in vivo is mediated by the localized activity of histone deacetylases (HDACs). The repressive (PcG/HDAC) machinery is removed as hematopoietic progenitors differentiate to form erythroid cells. The α- and β-globin genes thus illustrate important, contrasting mechanisms by which cell-specific hematopoietic genes (and tissue-specific genes in general) may be silenced.

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