A homeodomain protein binds to gamma-globin gene regulatory sequences.

Developmental regulation of gamma-globin gene expression probably occurs through developmental-stage-specific trans-acting factors able to promote the interaction of enhancer elements located in the far upstream locus control region with regulatory elements in the gamma gene promoters and 3' A gamma enhancer located in close proximity to the genes. We have detected a nuclear protein in K562 and baboon fetal bone marrow nuclear extracts capable of binding to A+T-rich sequences in the locus control region, gamma gene promoter, and 3' A gamma enhancer. SDS/polyacrylamide gel analysis of the purified K562 binding activity revealed a single protein of 87 kDa. A K562 cDNA clone was isolated encoding a beta-galactosidase fusion protein with a DNA binding specificity identical to that of the K562/fetal bone marrow nuclear protein. The cDNA clone encodes a homeodomain homologous to the Drosophila antennapedia protein.     

Epigenetic analysis of the human alpha- and beta-globin gene clusters

K562 erythroleukemia cells have been widely used as a model for the study of globin gene regulation. A number of agents have been shown to activate or suppress globin gene expression in these cells. However, the molecular effects of these agents on the epigenetic configuration of the alpha- and gamma-globin genes that encode HbF are not known. In this report, we investigated the relationship between globin expression and histone acetylation of the human alpha- and beta-globin clusters in the fetal erythroid environment of K562 cells. Our studies suggest that acetylation of histone H3 may be important in regulating developmental stage-specific expression of the different beta-like globin genes while acetylation of both histones H3 and H4 may be important for the regulation of tissue-specific expression of these genes. In contrast, acetylation of both histones H3 and H4 at the alpha-like globin promoters appears to be important for both developmental stage- and tissue-specific expression. Interestingly, butyrate-induced activation of alpha-globin gene expression in K562 cells is associated with significant increase in histone acetylation levels while TPA-induced inhibition is associated with decreased histone acetylation at its promoters. In contrast, changes in histone acetylation and DNA methylation do not appear to be important in the regulation of gamma-globin gene expression by the same agents. These data suggest that the butyrate-mediated induction of the fetal gamma-globin genes in K562 cells is not a direct result of its histone deacetylase inhibitor activity of butyrate on the chromatin of the gamma-globin promoters, while the induction of the alpha-globin genes could be a result of a direct effect of butyrate on chromatin at its promoters. This is another example of the important differences in the molecular mechanisms of regulation of the genes of the alpha- and beta-like globin clusters.     

Synergistic enhancement of globin gene expression by activator protein-1-like proteins.

DNA sequences corresponding to the four major DNase I hypersensitive sites upstream of the beta-globin gene cluster are essential for the achievement of high levels of globin gene expression and development regulation. In this study, we focused on one of these sites, hypersensitive site 2, which behaves as a powerful enhancer in transient expression and transgenic mouse experiments. We identified a tandem repeat of the activator protein 1 (AP-1) consensus sequence that binds AP-1-like proteins from nuclear extracts of K562 and HeLa cells. These proteins have the same binding properties as HeLa AP-1 but differ in the electrophoretic mobility and in functional assays. Transient-expression experiments in K562 of various deletion and point mutation constructs derived from hypersensitive site 2 indicate that the enhancer activity and the inducibility of a linked gamma-globin promoter are dependent upon the synergistic action of proteins bound to the tandem AP-1 repeat.     

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.     

The human beta-globin locus activation region alters the developmental fate of a human fetal globin gene in transgenic mice.

We linked a 3.3-kilobase fragment containing the entire A gamma-globin gene together with 1.3 kilobases of 5' flanking and 0.37 kilobase of 3' flanking DNA to a 2.5-kilobase fragment containing four of the developmentally stable hypersensitive sites normally located in the 5' region of the human beta-globin locus. This construct was injected into fertilized mouse eggs, and its expression was analyzed in the primitive and definitive erythroid cells, as well as the brain of 14-day embryos. All six transgenic individuals that contained intact copies of the construct expressed the transgene in an erythroid-specific fashion. Expression was observed in both primitive and definitive erythroid cells. This is in marked contrast to previous transgenic mice experiments using the same A gamma-globin gene fragment in isolation, where expression was restricted to primitive erythroid cells. Our results show that the region containing the developmentally stable globin locus hypersensitive sites changes the developmental stage specificity of a human fetal globin gene in transgenic mice. These observations imply that sequences additional to those used here are involved in the developmental control of fetal globin gene expression in vivo. The ability to express fetal globin in adult erythroid cells allows one to consider using fetal globin genes for gene therapy of sickle cell disease.     

Isolation and characterization of a K562 cell line resistant to 1-beta-D-arabinofuranosylcytosine-mediated erythroid induction

The isolation of a K562 cell line, K562(S)R, resistant to 1-beta-D-arabinofuranosylcytosine (ara-C)-mediated erythroid induction, is described. Ara-C (10-50 microM) inhibits cell growth of K562(S)R cells but is not able to activate the program of erythroid induction. This failure is associated with the lack in the increase of accumulation of epsilon-globin and gamma-globin mRNA sequences in ara-C-treated K562(S)R cells. This cell line could be of interest for studies focused on molecular mechanisms of activation of globin genes in K562 cells.     

Use of yeast artificial chromosomes (YACs) for studying control of gene expression: correct regulation of the genes of a human beta-globin locus YAC following transfer to mouse erythroleukemia cell lines.

We demonstrate that transfer of a yeast artificial chromosome (YAC) containing 230 kb of the human beta-globin locus into mouse erythroleukemia cells by fusion results in correct developmental regulation of the human beta-like globin genes. Additionally, we show that early after hybrid formation, human embryonic epsilon- and fetal gamma-globin genes are coexpressed with the adult beta gene but that after 10-20 weeks in culture, globin gene expression switches to predominantly adult. Thus, in contrast to shorter gene constructs, the globin genes of the beta-globin locus YAC are regulated like the chromosomal globin genes. These results indicate that transfer of YACs into established cell lines can be used for the analysis of the developmental control of multigenic and developmentally regulated human loci.     

Identification of novel K562 membrane proteins that adhere to bone marrow fibroblasts

Adhesion of myeloid leukemia cells to the bone marrow (BM) microenvironment is mediated in part by Beta 1 and Beta 2 integrins. Cells of the erythroleukemia line K562, derived from a patient with chronic myeloid leukemia, bind to BM fibroblasts (BMFs) but the adhesion cannot be accounted for by integrins or other known adhesion proteins including CD44 or members of the Ig or selectin families. Membrane fragments from K562 cells were radioiodinated and allowed to adhere to BMF monolayers. Adherent proteins were solubilized together with the fibroblasts, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and visualized by autoradiography. Four adherent proteins were consistently observed. Two of these, with reduced molecular weights of 52 kD and 35 to 37 kD, were prominent. Addition of soluble thrombospondin and heparin but not fibronectin inhibited binding of K562 membrane proteins to adherent BMFs and immobilized thrombospondin- and heparin-bound K562 proteins. The 52-kD protein has a multimeric structure nonreduced and has characteristics of a glycoprotein. Its adhesion to fibroblasts is divalent cation and temperature sensitive. The 35- to 37-kD protein, whose function is divalent cation but not temperature sensitive, is phosphoinositol- linked and has characteristics identical to an adherent 35- to 37-kD protein identified on murine progenitor cells. Membrane preparations from two cases of acute myeloid leukemia showed an adherent 35- to 37- kD protein and in one case an adherent 52-kD protein without other adherent bands. A K562 subclone with reduced adherence to BMFs showed reduced amounts of adherent 52-kD and 35- to 37-kD proteins. These proteins may be responsible for the adhesion of malignant and normal hematopoietic progenitor cells to the BM microenvironment.     

Development of a condensed locus control region cassette and testing in retrovirus vectors for A gamma-globin

Retrovirus vectors for A gamma-globin are being developed for the treatment of beta chain hemoglobinopathies. Toward the goal of achieving therapeutic expression levels, core elements of the beta-globin locus control region (LCR) hypersensitive sites (HS) were screened for enhancer activity in erythroid MEL and K562 cell lines using a drug-resistant colony assay. When used alone, core elements of HS1, HS3, and HS4 showed no activity and a fragment for HS2 showed only modest activity in the colony assay. However, a 1.1 kb combination of fragments for HS2, HS3, and HS4 (termed a nLCR) enhanced colony formation 17-fold in K562 cells and 94-fold in MEL cells. Addition of an HS1 fragment enhanced nLCR activity only modestly in MEL cells. When linked to a beta-globin gene, the 1.1 kb nLCR enhanced globin mRNA expression to 82% per copy of mouse alpha-globin in transfected MEL cells. Inclusion of a nLCR in retrovirus vectors containing a beta-globin promoter and various A gamma-globin gene expression cassettes resulted in extreme genetic instability and reduced titers. Specific deletions were abrogated by removing homologous sequences, but random recombinations were still observed at significant frequencies. In MEL cells containing intact provirus, A gamma-globin mRNA produced by an optimal vector containing the nLCR was only 2-fold higher (8.5% vs. 3.9% per copy of mouse alpha-globin) compared to the same vector without the nLCR. These data suggest that vector elements detract from the ability of the nLCR to enhance expression of the beta pr.A gamma cassettes.     

Accumulation of γ-globin mRNA and induction of erythroid differentiation after treatment of human leukaemic K562 cells with tallimustine

Human leukaemic K562 cells can be induced in vitro to erythroid differentiation by a variety of chemical compounds, including haemin, butyric acid, 5-azacytidine, cytosine arabinoside, mithramycin and chromomycin, cisplatin and cisplatin analogues. Differentiation of K562 cells is associated with an increase of expression of embryo-fetal globin genes, such as the zeta-, epsilon- and gamma-globin genes. The K562 cell line has been proposed as a very useful in vitro model system to determine the therapeutic potential of new differentiating compounds as well as to study the molecular mechanism(s) regulating changes in the expression of embryonic and fetal human globin genes. Inducers of erythroid differentiation stimulating gamma-globin synthesis could be considered for possible use in the therapy of haematological diseases associated with a failure in the expression of normal beta-globin genes. We have analysed the effects of tallimustine and distamycin on cell growth and differentiation of K562 cells. The results demonstrated that tallimustine is a potent inducer, while distamycin is a weak inducer, of K562 cell erythroid differentiation. Erythroid differentiation was associated with an increase of accumulation of gamma-globin mRNA and of production of both haemoglobin (Hb) Gower 1 and Hb Portland. In addition, tallimustine-mediated erythroid induction occurred in the presence of activation of the apoptotic pathway. The reasons for proposing tallimustine as an inducer of gamma-globin gene expression are strongly sustained by the finding that this compound stimulates fetal haemoglobin production in human erythroid precursor cells from normal subjects.     

In vivo protein-DNA interactions at the beta-globin gene locus.

We have investigated in vivo protein-DNA interactions in the beta-globin gene locus by dimethyl sulfate (DMS) footprinting in K562 cells, which express epsilon- and gamma-globin but not beta-globin. In the locus control region, hypersensitive site 2 (HS-2) exhibited footprints in several putative protein binding motifs. HS-3 was not footprinted. The beta promoter was also not footprinted, while extensive footprints were observed in the promoter of the active gamma-globin gene. No footprints were seen in the A gamma and beta 3' enhancers. With several motifs, additional protein interactions and alterations in binding patterns occurred with hemin induction. In HeLa cells, some footprints were observed in some of the motifs in HS-2, compatible with the finding that HS-2 has some enhancer function in HeLa cells, albeit much weaker than its activity in K562 cells. No footprint was seen in B lymphocytes. In vivo footprinting is a useful method for studying relevant protein-DNA interactions in erythroid cells.     

The DNA-binding drugs mithramycin and chromomycin are powerful inducers of erythroid differentiation of human K562 cells

The human leukaemic K562 cell line can be induced in vitro to undergo erythroid differentiation by a variety of chemical compounds, including haemin, butyric acid, 5-azacytidine and cytosine arabinoside. Differentiation of K562 cells is associated with an increased expression of embryo-fetal globin genes, such as the zeta, epsilon and gamma globin genes. Therefore the K562 cell line has been proposed as a useful in vitro model system to determine the therapeutic potential of new differentiating compounds as well as to study the molecular mechanism(s) regulating changes in the expression of embryonic and fetal human globin genes. Inducers of erythroid differentiation which stimulate gamma-globin synthesis could be considered for possible use in the experimental therapy of those haematological diseases associated with a failure in the expression of adult beta-globin genes. In this paper we demonstrated that the G + C selective DNA-binding drugs chromomycin and mithramycin were powerful inducers of erythroid differentiation of K562 cells. Erythroid differentiation was associated with an increase in the accumulation of (a) Hb Gower 1 and Hb Portland and (b) gamma-globin mRNA.     

Role of epigenetic modifications in normal globin gene regulation and butyrate-mediated induction of fetal hemoglobin

Butyrate is a prototype of histone deacetylase inhibitors that is believed to reactivate silent genes by inducing epigenetic modifications. Although butyrate was shown to induce fetal hemoglobin (HbF) production in patients with hemoglobin disorders, the mechanism of this induction has not been fully elucidated. Our studies of the epigenetic configuration of the beta-globin cluster suggest that DNA methylation and histone H3 acetylation are important for the regulation of developmental stage-specific expression of the beta-like globin genes, whereas acetylation of both histones H3 and H4 seem to be important for the regulation of tissue-specific expression. These studies suggest that DNA methylation may be important for the silencing of the beta-like globin genes in nonerythroid hematopoietic cells but may not be necessary for their silencing in nonhematopoietic cells. Furthermore, our studies demonstrate that butyrate exposure results in a true reversal of the normal developmental switch from gamma- to beta-globin expression. This is associated with increased histone acetylation and decreased DNA methylation of the gamma-globin genes, with opposite changes in the beta-globin gene. These studies provide strong support for the role of epigenetic modifications in the normal developmental and tissue-specific regulation of globin gene expression and in the butyrate-mediated pharmacologic induction of HbF production.     

The -175T----C mutation increases promoter strength in erythroid cells: correlation with evolutionary conservation of binding sites for two trans-acting factors.

A point mutation at position -175 has been detected in Agamma as well as Ggamma globin genes in individuals with hereditary persistence of fetal hemoglobin (HPFH). To prove that this single point mutation results in increased promoter strength, we transfected erythroid and nonerythroid cell lines with constructs containing normal and mutant promoters linked to the bacterial chloramphenicol acetyl transferase (CAT) gene. Differences in transfection efficiency were controlled by cotransfection of pRSVgpt. In K562 erythroleukemia cells, the -175 HPFH promoter directed three- to fourfold more CAT activity than its wild type counterpart. However, in HeLa cells the two promoters were similar in strength. The -195 to -165 region of the gamma-globin promoter contains binding sites for two proteins: a ubiquitously distributed octamer binding protein, OBP, and the erythroid-specific protein, GF-1. We find that while the GF-1 binding site is highly conserved among related primate gamma-globin genes, the octamer binding site is not. The evolutionary conservation of GF-1 as well as its erythroid-specific distribution suggest that this protein is important in gamma-globin gene expression. A role for OBP in the regulation of gamma-globin, if any, must have arisen recently in primate evolution.