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• W2125154113 abstract "To explore the response of β globin locus with established chromatin domains upon their exposure to new transcriptional environments, we transferred the chromatin-packaged β globin locus of undifferentiated human embryonic stem cells (hESCs) or hESC-derived erythroblasts into an adult transcriptional environment. Distinct globin expression patterns were observed. In hESC-derived erythroblasts where both ε and γ globin were active and marked by similar chromatin modifications, ε globin was immediately silenced upon transfer, whereas γ globin continued to be expressed for months, implying that different transcriptional environments were required for their continuing expression. Whereas β globin was silent both in hESCs and in hESC-derived erythroblasts, β globin was only activated upon transfer from hESCs, but not in the presence of dominant γ globin transferred from hESC-derived erythroblasts, confirming the competing nature of γ versus β globin expression. With time, however, silencing of γ globin occurred in the adult transcriptional environment with concurrent activation of β-globin, accompanied by a drastic change in the epigenetic landscape of γ and β globin gene regions without apparent changes in the transcriptional environment. This switching process could be manipulated by overexpression or downregulation of certain transcription factors. Our studies provide important insights into the interplay between the transcription environment and existing chromatin domains, and we offer an experimental system to study the time-dependent human globin switching. To explore the response of β globin locus with established chromatin domains upon their exposure to new transcriptional environments, we transferred the chromatin-packaged β globin locus of undifferentiated human embryonic stem cells (hESCs) or hESC-derived erythroblasts into an adult transcriptional environment. Distinct globin expression patterns were observed. In hESC-derived erythroblasts where both ε and γ globin were active and marked by similar chromatin modifications, ε globin was immediately silenced upon transfer, whereas γ globin continued to be expressed for months, implying that different transcriptional environments were required for their continuing expression. Whereas β globin was silent both in hESCs and in hESC-derived erythroblasts, β globin was only activated upon transfer from hESCs, but not in the presence of dominant γ globin transferred from hESC-derived erythroblasts, confirming the competing nature of γ versus β globin expression. With time, however, silencing of γ globin occurred in the adult transcriptional environment with concurrent activation of β-globin, accompanied by a drastic change in the epigenetic landscape of γ and β globin gene regions without apparent changes in the transcriptional environment. This switching process could be manipulated by overexpression or downregulation of certain transcription factors. Our studies provide important insights into the interplay between the transcription environment and existing chromatin domains, and we offer an experimental system to study the time-dependent human globin switching. Changes in globin gene expression during development are characteristic of all species that use hemoglobin as an oxygen carrier. In most species there is only one switch from embryonic to adult, reflecting the two distinct globin expression programs of the primitive and definitive hematopoietic lineages. There are two globin switches in humans and several primates: from embryonic to fetal and from fetal to adult. The human embryonic globin program, characterized by predominant ε gene expression, is confined in the yolk sac erythropoiesis. Fetal and low levels of embryonic globin are expressed in the erythroid cells of the fetal livers of early human fetuses, but soon thereafter production of predominantly fetal and low levels of adult globin mark the program of erythroid cells of the fetal liver or the fetal bone marrow erythropoiesis [1Stamatoyannopoulos G. Control of globin gene expression during development and erythroid differentiation.Exp Hematol. 2005; 33: 259-271Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar]. Switching from fetal to adult starts in the third trimester of gestation and finishes several weeks after birth. Corresponding to the quantitative change in developmentally specific globin protein and mRNA levels, cis-linked changes occur, including histone modifications and DNA methylation surrounding the affected globin regions [2Mahajan M.C. Karmakar S. Weissman S.M. Control of beta globin genes.J Cell Biochem. 2007; 102: 801-810Crossref PubMed Scopus (30) Google Scholar, 3Kiefer C.M. Hou C. Little J.A. Dean A. Epigenetics of beta-globin gene regulation.Mutat Res. 2008; 647: 68-76Crossref PubMed Scopus (52) Google Scholar, 4Hosey A.M. Chaturvedi C.P. Brand M. Crosstalk between histone modifications maintains the developmental pattern of gene expression on a tissue-specific locus.Epigenetics. 2010; 5: 273-281Crossref PubMed Scopus (15) Google Scholar, 5Chang K.H. Fang X. Wang H. et al.Epigenetic Modifications and Chromosome Conformations of the Beta Globin Locus throughout Development.Stem Cell Rev. 2013; 9: 397-407Crossref PubMed Scopus (1) Google Scholar]. These epigenetic changes can be attributed to the actions of epigenetic modification enzymes recruited to the affected genes. For example, the recruitment of multiple epigenetic transcriptional corepressors by TR2/TR4 to the silenced globin promoters in adult erythroid cells has been reported [6Cui S. Kolodziej K.E. Obara N. et al.Nuclear receptors TR2 and TR4 recruit multiple epigenetic transcriptional corepressors that associate specifically with the embryonic beta-type globin promoters in differentiated adult erythroid cells.Mol Cell Biol. 2011; 31: 3298-3311Crossref PubMed Scopus (91) Google Scholar]. The active role of epigenetic modifications in modulating globin expressions is further illustrated by the reactivation of human fetal or mouse embryonic globin genes upon treating adult erythroid cells with pharmacologic agents that target epigenetic modification enzymes including DNA methyltransferase or histone deacetylase or with RNAi that downregulates histone modification enzyme G9a methyltransferase [4Hosey A.M. Chaturvedi C.P. Brand M. Crosstalk between histone modifications maintains the developmental pattern of gene expression on a tissue-specific locus.Epigenetics. 2010; 5: 273-281Crossref PubMed Scopus (15) Google Scholar, 7Chaturvedi C.P. Hosey A.M. Palii C. et al.Dual role for the methyltransferase G9a in the maintenance of beta-globin gene transcription in adult erythroid cells.Proc Nat Acad Sci U S A. 2009; 106: 18303-18308Crossref PubMed Scopus (64) Google Scholar]. Transfer of chromatin-packaged chromosome into a known transcriptional environment can serve as a model system for studying the interaction between transcriptional environment and established chromatin domains. Suzuki et al. [8Suzuki N. Itou T. Hasegawa Y. Okazaki T. Ikeno M. Cell to cell transfer of the chromatin-packaged human beta-globin gene cluster.Nucleic Acids Res. 2010; 38: e33Crossref PubMed Scopus (21) Google Scholar] have built an artificial mini human chromosome containing β globin locus and found that the chromatin-packaged β globin gene cluster can be activated or repressed according to the cellular environment [8Suzuki N. Itou T. Hasegawa Y. Okazaki T. Ikeno M. Cell to cell transfer of the chromatin-packaged human beta-globin gene cluster.Nucleic Acids Res. 2010; 38: e33Crossref PubMed Scopus (21) Google Scholar]. Alternatively, cell-to-cell fusion has been used to introduce chromatin-packaged β globin locus into adult type murine erythroleukemia (MEL) cells and a complex globin expression pattern dependent on both donor cell types and time in culture has been observed [9Papayannopoulou T. Brice M. Stamatoyannopoulos G. Analysis of human hemoglobin switching in MEL x human fetal erythroid cell hybrids.Cell. 1986; 46: 469-476Abstract Full Text PDF PubMed Scopus (53) Google Scholar, 10Takegawa S. Brice M. Stamatoyannopoulos G. Papayannopoulou T. Only adult hemoglobin is produced in fetal nonerythroid x MEL cell hybrids.Blood. 1986; 68: 1384-1388PubMed Google Scholar, 11Stanworth S.J. Roberts N.A. Sharpe J.A. Sloane-Stanley J.A. Wood W.G. Established epigenetic modifications determine the expression of developmentally regulated globin genes in somatic cell hybrids.Mol Cell Biol. 1995; 15: 3969-3978PubMed Google Scholar, 12Stanworth S.J. Roberts N.A. Sharpe J.A. Wood W.G. Gene expression in somatic cell hybrids derived from embryonic mice transgenic for human globin genes.Br J Haematol. 1996; 94: 631-638Crossref PubMed Scopus (3) Google Scholar]. The transferred β globin locus retains important features including the long-range interaction between the locus control region (LCR) and active globin promoters [13Kim S. Kim Y.W. Shim S.H. Kim C.G. Kim A. Chromatin structure of the LCR in the human beta-globin locus transcribing the adult delta- and beta-globin genes.Int J Biochem Cell Biol. 2012; 44: 505-513Crossref PubMed Scopus (15) Google Scholar]. The broad utility of this system has been impeded by the continuous and rapid loss of human chromosome 11 from these hybrids. To overcome this issue, we produced human embryonic stem cells (hESCs) carrying a neo resistance gene on chromosome 11. Heterospecific hybrids generated from fusing MEL cells to these hESCs or their erythroid progenies allowed us to study the response of β globin locus of two distinct chromatin states following its transfer into the adult environment of MEL cells. We confirmed that the existing chromatin domains and transcriptional environment collectively determine the initial outcome of specific globin expression, be it an adult or fetal pattern. The cells that exhibited an initial fetal pattern underwent a temporal fetal to adult switch without significant changes in the overall transcriptional environment. We showed that this system allows molecular analyses that provide insights on the sequential epigenetic changes that occur during development. We further showed that switching in this system can be modulated by drastic changes in the transcriptional environment. This system can be used in the investigation of transcriptional or pharmacologic elements for therapeutic reactivation of fetal hemoglobin in patients with β chain hemoglobinopathies. Propagation and differentiation of hESCs into erythroid lineage [14Chang K.H. Nelson A.M. Fields P.A. et al.Diverse hematopoietic potentials of five human embryonic stem cell lines.Exp Cell Res. 2008; 314: 2930-2940Crossref PubMed Scopus (25) Google Scholar], generation and characterization of heterospecific hybrids [9Papayannopoulou T. Brice M. Stamatoyannopoulos G. Analysis of human hemoglobin switching in MEL x human fetal erythroid cell hybrids.Cell. 1986; 46: 469-476Abstract Full Text PDF PubMed Scopus (53) Google Scholar], and conduction of epigenetic studies [5Chang K.H. Fang X. Wang H. et al.Epigenetic Modifications and Chromosome Conformations of the Beta Globin Locus throughout Development.Stem Cell Rev. 2013; 9: 397-407Crossref PubMed Scopus (1) Google Scholar] have been described previously. Additional details can be found in the supplemental text (online only, available at www.exphem.org). Primers for quantitative reverse transcription polymerase chain reaction (RT-PCR) are summarized in Supplementary Table E1 (online only, available at www.exphem.org). To generate an hESC line that carries a selectable marker on chromosome 11 that is expressed at all stages of differentiation, we targeted the first coding domain of general transcription factor IIH polypeptide 1 (GTF2H1), located approximately 600 kb upstream of human β globin locus, using adeno-associated virus (AAV)–mediated homologous recombination (Fig. 1A). Six neomycin-resistant H1 colonies were obtained out of 400 × 103 cells and expanded further. Two of the six clones were correctly targeted based on PCR screening (Fig. 1B) and were designated as Neo-1 and Neo-2. To test whether the targeting procedure had altered the stem cell properties of these neo-resistant hESC lines, we first determined the expression of stem cell markers and found that both lines expressed high levels of TRA-1-60, TRA-1-81, SSEA-3, and SSEA-4 (Fig. 1C), similar to parental H1 cells and other hESC lines [14Chang K.H. Nelson A.M. Fields P.A. et al.Diverse hematopoietic potentials of five human embryonic stem cell lines.Exp Cell Res. 2008; 314: 2930-2940Crossref PubMed Scopus (25) Google Scholar]. Upon differentiation, the two neo-resistant hESC lines formed cystic embryoid bodies (data not shown) and expressed comparable levels of hematopoietic markers such as CD34, CD117, CD31, CD41, CD45, glycophorin A (GlyA), and CD71 to parental H1 cells (Fig. 1D). Erythroid differentiation was studied in Neo-2 and these neomycin-resistant erythroblasts displayed a globin phenotype of high embryonic ε and fetal γ globin with almost no adult β globin (Fig. 1E), consistent with the globin expression pattern of hESC- and induced pluripotent stem cells (iPSC)-derived erythroblasts reported previously [14Chang K.H. Nelson A.M. Fields P.A. et al.Diverse hematopoietic potentials of five human embryonic stem cell lines.Exp Cell Res. 2008; 314: 2930-2940Crossref PubMed Scopus (25) Google Scholar, 15Chang K.H. Nelson A.M. Cao H. et al.Definitive-like erythroid cells derived from human embryonic stem cells coexpress high levels of embryonic and fetal globins with little or no adult globin.Blood. 2006; 108: 1515-1523Crossref PubMed Scopus (75) Google Scholar, 16Chang K.H. Huang A. Hirata R.K. Wang P.R. Russell D.W. Papayannopoulou T. Globin phenotype of erythroid cells derived from human induced pluripotent stem cells.Blood. 2010; 115: 2553-2554Crossref PubMed Scopus (42) Google Scholar]. Therefore, AAV-mediated gene targeting of GTF2H1 in hESCs did not affect their stem cell properties or their hematopoietic differentiation characteristics. We have previously reported that, based on the distribution of AcH3 and H3K4me3, the chromatin of entire β globin locus of hESCs is closed [5Chang K.H. Fang X. Wang H. et al.Epigenetic Modifications and Chromosome Conformations of the Beta Globin Locus throughout Development.Stem Cell Rev. 2013; 9: 397-407Crossref PubMed Scopus (1) Google Scholar]. This observation was further confirmed by DNase I hypersensitivity mapping showing that only a minor peak existed at hypersensitive site 2 at the LCR (Fig. 2A, Supplementary Table E2, online only, available at www.exphem.org), which was also observed in various nonerythroid cells that we have tested (data not shown). Following transfer of the inactive and DNase I resistant β globin locus into the adult erythroid environment, in all four heterospecific-hybrid lines generated, only the β globin gene was activated, which was reflected at the protein level by globin chain-specific monoclonal antibody staining (Fig. 2B, left panels), as well as at the mRNA level by RT-PCR (Fig. 2C, upper panel). Thus, upon encountering the adult erythroid environment of the MEL cells, the β globin locus of hESCs behaved like the inactive loci of other somatic cells, such as fibroblasts or lymphoblasts [10Takegawa S. Brice M. Stamatoyannopoulos G. Papayannopoulou T. Only adult hemoglobin is produced in fetal nonerythroid x MEL cell hybrids.Blood. 1986; 68: 1384-1388PubMed Google Scholar].Figure 2Response of chromatin-packaged β globin locus upon transfer from its original transcription environment into the adult erythroid transcriptional environment of MEL. (A) DNase I mapping of hESCs and hESC-derived erythroblasts. Undifferentiated hESCs had an inactive β globin locus, whereas the erythroblasts derived from hESCs showed prominent DNase I cleavages at LCR and ε and γ globin regions. (B) Globin expression patterns of hybrids derived from hESCs or from hESC erythroblasts. Representative images of immunofluorescent staining of 4–5-week hybrids with monoclonal antibodies against ε, γ, or β globin chain followed by an fluorescein isothiocyanate–conjugated anti-mouse-IgG antibody are shown. Pictures were taken with a Leica DMLB microscope with a 40× objective lens using a Leica camera with Leica LAS software (version 2.4.1R1; Leica Microsystems, Buffalo Grove, IL, USA). (C) The β locus globin mRNA expression levels of each hybrid line were determined using RT-PCR by plotting against standard curves generated using transgenic mouse tissue with known human β locus globin expression levels. Upper panel: hybrids produced by fusing MEL cells with human ES cells; notice the exclusive β globin mRNA expression. Lower panel: hybrids produced by fusing MEL cells with hESC-derived erythroblasts; notice the predominant γ globin mRNA expression. (D) DNase I mapping of the β globin locus. Comparison of the β globin locus in the prefusion hESC-derived erythroblasts to the β globin locus of hybrids derived by fusing these erythroblasts with MEL cells. Notice the silencing of ε globin gene (open arrow) and the continued expression of γ globin genes (closed arrow) following the transfer of the β globin locus of the hESC erythroblasts into the MEL cells. (E) Summary of the changes in β locus globin expression before and after transfer of the locus from its native environment into the MEL environment.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The hESC-derived erythroblasts express an embryonic/fetal globin program [15Chang K.H. Nelson A.M. Cao H. et al.Definitive-like erythroid cells derived from human embryonic stem cells coexpress high levels of embryonic and fetal globins with little or no adult globin.Blood. 2006; 108: 1515-1523Crossref PubMed Scopus (75) Google Scholar]. DNAse I mapping experiments showed that the chromatin of 5ʹ region of the β globin locus was open: the LCR as well as promoters and genes of embryonic ε and fetal γ globin were sensitive to DNaseI cleavage (Fig. 2D, Supplementary Table E3, online only, available at www.exphem.org), but there was minimal, if any, DNase I sensitivity of the β globin gene. Several changes in the profiles of DNaseI hypersensitive sites (HSs) in the LCR were observed upon transfer of this locus into the adult erythroid environment, which included narrowing of the five HSs and the increasing of the peak height for HS3, suggesting different trans-factors binding patterns in the LCR between hESC erythroblasts and the fused hybrids. The broader HS peak widths in the LCR of hESC erythroblasts might also be related to the inter-HS transcription reported previously in these cells [5Chang K.H. Fang X. Wang H. et al.Epigenetic Modifications and Chromosome Conformations of the Beta Globin Locus throughout Development.Stem Cell Rev. 2013; 9: 397-407Crossref PubMed Scopus (1) Google Scholar]. In the globin regions, transfer of the locus resulted in essential abolishment of DNase I sensitivity of the ε globin gene and its promoter (Fig. 2D, open arrow). In contrast, DNase I sensitivity of the γ globin gene region remained as high as in the parental hESC-derived erythroblasts (Fig. 2D, closed arrow) and the DNAse I sensitivity of the β gene region remained as low as in the parental hESC-derived erythroblasts (Fig. 2D and Supplementary Table E3, online only, available at www.exphem.org). Consistent with the DNaseI profiles, γ globin mRNA was the predominant globin species found in all seven hybrid lines and consisted of 85–99% of total human β locus globin mRNAs, with the remaining being β globin mRNA (Fig. 2C, lower panel). ε Globin mRNA was all but absent. At the protein level, there was a high expression of γ globin chains with minimum β globin chain and no ε globin chain expression (Fig. 2B, right panels). Thus, the main event following the transfer of chromatin-packaged β globin locus of hESC-derived erythroblasts into an adult environment is the transition of the locus from an embryonic or fetal program to a predominantly fetal program (Fig. 2E). We continued culturing the hESC-erythroblast hybrids for up to 68 weeks. Five of seven hybrid lines displayed a progressive, time-dependent γ to β globin switch in both mRNA (Fig. 3A, Supplementary Figure E1, online only, available at www.exphem.org) and protein levels (Fig. 3B and data not shown). Time-wise, this switch closely resembled the in vivo γ to β globin switch in humans and the in vitro γ to β globin switch in hybrids produced by fusing fetal erythroblasts with MEL cells [9Papayannopoulou T. Brice M. Stamatoyannopoulos G. Analysis of human hemoglobin switching in MEL x human fetal erythroid cell hybrids.Cell. 1986; 46: 469-476Abstract Full Text PDF PubMed Scopus (53) Google Scholar]. Two other hybrid cell lines showed transient reactivation of γ globin during culture before their eventual switch to a stable β globin phenotype (Fig. 3C). To determine whether the observed time-dependent γ to β globin switch was simply due to β globin-expressing cells proliferating at a higher rate relative to γ globin-expressing cells, we assessed bromodeoxyuridine (BrdU) incorporation by these two types of cells in hybrid line C3 at week 22 of culture. Similar proportion of γ and β globin-expressing cells incorporated BrdU following a 2- or 4-hour pulse (Fig. 3D), suggesting that they proliferated at a similar rate. The chromatin configurations before and after switching were examined by digital DNaseI mapping (Fig. 4A) and chromatin immunoprecipitation (ChIP)-seq for H3K27ac, H3K4me3, and H3K4me1 (Fig. 4B). The chromatin domain of LCR remained open throughout the culture period as revealed by DNaseI studies and was associated with both histone marks H3K27ac and H3K4me1, but without histone mark H3K4me3, consistent with its role as an active enhancer [17Creyghton M.P. Cheng A.W. Welstead G.G. et al.Histone H3K27ac separates active from poised enhancers and predicts developmental state.Proc Nat Acad Sci U S A. 2010; 107: 21931-21936Crossref PubMed Scopus (2361) Google Scholar]. Striking changes in chromatin profiles took place in the γ and β globin regions during the culture period. At week 7, when these hybrids expressed a predominantly fetal globin program, high peak of DNase I sensitivity was characteristic of the Aγ/Gγ genes region. At week 53, when on the basis of globin mRNA levels these hybrids have switched to adult program, the chromatin profile also changed drastically: there were no longer DNase I hypersensitivity peaks in the γ genes while there were prominent peaks in the region of β and δ genes. Similar changes were also observed with histone mark H3K4me3 (Fig. 4B). The changes in the globin programs of the hybrids could conceivably reflect changes in transcriptional environments during culture. Murine transcriptome studies were performed on hybrids before (weeks 4–8), during (week 27), or after (week 53) switch (Fig. 5A). Only four murine genes (Styx, Tmem176b, Slc22a3, and Bnip3) were found to be expressed at significantly different levels during the switch compared with early (weeks 4–8) hybrids, and seven murine genes (Bex4, Brdt, Prkcq, F2rl2, P2rx7, Angpt1, and F2r) were found to be expressed differentially when comparing the switched hybrids (week 53) to those undergoing switch (week 27). None of these genes have previously reported roles in regulating hemoglobin expression. Cytogenetic studies showed no significant linkage between switching and the loss of any particular human chromosome (Supplementary Table E4, online only, available at www.exphem.org). Human transcriptome studies confirmed the findings of cytogenetic studies (data not shown). Western blotting, which detected both human and mouse origin of transcription factors known to be critical to γ and β globin expression did not detect consistent trends of upregulation or downregulation during culture (Fig. 5B). These results suggest that the observed γ to β globin switch in the hybrid system is not due to time-related changes in the transcriptional environment. We also tested potential involvement of transcriptional factors in the two distinctly different globin expression patterns obtained following the transfer of the β globin locus from hESCs or hESC-derived erythroblasts into MEL cells (Fig. 2). mRNA and protein levels by RT-PCR and Western blots detected no significant differences in the expression levels of several known γ globin repressors and β globin activators, including EKLF [18Bieker J.J. Putting a finger on the switch.Nat Genet. 2010; 42: 733-734Crossref PubMed Scopus (19) Google Scholar, 19Tallack M.R. Perkins A.C. Three fingers on the switch: Kruppel-like factor 1 regulation of gamma-globin to beta-globin gene switching.Curr Opin Hematol. 2013; 20: 193-200Crossref PubMed Scopus (49) Google Scholar], BCL11A [20Bauer D.E. Orkin S.H. Update on fetal hemoglobin gene regulation in hemoglobinopathies.Curr Opin Pediatr. 2011; 23: 1-8Crossref PubMed Scopus (82) Google Scholar, 21Sankaran V.G. Xu J. Ragoczy T. et al.Developmental and species-divergent globin switching are driven by BCL11A.Nature. 2009; 460: 1093-1097Crossref PubMed Scopus (298) Google Scholar], MYB [22Jiang J. Best S. Menzel S. et al.cMYB is involved in the regulation of fetal hemoglobin production in adults.Blood. 2006; 108: 1077-1083Crossref PubMed Scopus (136) Google Scholar, 23Nuinoon M. Makarasara W. Mushiroda T. et al.A genome-wide association identified the common genetic variants influence disease severity in beta(0)-thalassemia/hemoglobin E.Hum Genet. 2009; Google Scholar, 24Sankaran V.G. Menne T.F. Scepanovic D. et al.MicroRNA-15a and -16-1 act via MYB to elevate fetal hemoglobin expression in human trisomy 13.Proc Nat Acad Sci U S A. 2011; 108: 1519-1524Crossref PubMed Scopus (168) Google Scholar], and SOX6 [25Xu J. Sankaran V.G. Ni M. et al.Transcriptional silencing of {gamma}-globin by BCL11A involves long-range interactions and cooperation with SOX6.Genes Dev. 2010; 24: 783-798Crossref PubMed Scopus (74) Google Scholar, 26Yi Z. Cohen-Barak O. Hagiwara N. et al.Sox6 directly silences epsilon globin expression in definitive erythropoiesis.PLoS Genet. 2006; 2: e14Crossref PubMed Scopus (89) Google Scholar], as well as γ globin activators such as HDAC9 [27Muralidhar S.A. Ramakrishnan V. Kalra I.S. Li W. Pace B.S. Histone deacetylase 9 activates gamma-globin gene expression in primary erythroid cells.J Biol Chem. 2011; 286: 2343-2353Crossref PubMed Scopus (17) Google Scholar] and KLF11 [28Emery D.W. Gavriilidis G. Asano H. Stamatoyannopoulos G. The transcription factor KLF11 can induce gamma-globin gene expression in the setting of in vivo adult erythropoiesis.J Cell Biochem. 2007; 100: 1045-1055Crossref PubMed Scopus (8) Google Scholar] between these two types of hybrids (Figs. 5C and 5D). We then studied whether globin expression in hybrids can be influenced by transcription factors, particularly EKLF [18Bieker J.J. Putting a finger on the switch.Nat Genet. 2010; 42: 733-734Crossref PubMed Scopus (19) Google Scholar, 19Tallack M.R. Perkins A.C. Three fingers on the switch: Kruppel-like factor 1 regulation of gamma-globin to beta-globin gene switching.Curr Opin Hematol. 2013; 20: 193-200Crossref PubMed Scopus (49) Google Scholar, 29Borg J. Papadopoulos P. Georgitsi M. et al.Haploinsufficiency for the erythroid transcription factor KLF1 causes hereditary persistence of fetal hemoglobin.Nat Genet. 2010; 42: 801-805Crossref PubMed Scopus (285) Google Scholar, 30Zhou D. Liu K. Sun C.W. Pawlik K.M. Townes T.M. KLF1 regulates BCL11A expression and gamma- to beta-globin gene switching.Nat Genet. 2010; 42: 742-744Crossref PubMed Scopus (272) Google Scholar] with known functions in modulating fetal and adult globin expression. We transfected hESC-erythroblast hybrid line C3 before (at week 8) or during (at week 23) switching with either wild type (WT) EKLF or EKLF with its DNA binding domain or transactivating domain removed (ΔDB and ΔTA, respectively; Fig. 6A). To allow for the identification of transfected cells and to gauge for the level of EKLF overexpression at the single cell level, polycistronic vectors were constructed using self-cleaving 2A peptides (Fig. 6A) that linked EKLF and GFP together. We found that although overexpressing mutant EKLF (both ΔDB and ΔTA) had relatively little effect on the globin expression phenotype of the hybrids, overexpressing WT EKLF drastically decreased the proportion of γ globin expressing cells and increased the proportion of β globin expressing cells, in both week-8 and week-23 hybrids (Fig. 6B). At week 8, the frequency of the γ-positive hybrids (γ+β– and γ+β+) was approximately 90% in the control hybrids with no EKLF/GFP overexpression, approximately 70% in hybrids having low EKLF overexpression and approximately 60% in hybrids with high EKLF expression. At week 23, the frequency of γ-positive hybrids was approximately 60% in the control, and approximately 35% in hybrids with either low EKLF overexpression or high EKLF overexpression. γ-Only population (γ+β–) decreased in an EKLF dose-dependent manner that reached approximately 90% in week-8 and week-23 hybrids with high EKLF overexpression. Interestingly, with low EKLF overexpression, the γ-only population decreased by approximately 50% in week 8 hybrids (from 84.97 ± 2.89 to 40.26 ± 3.61) but decreased by approximately 67% in week 23 hybrids (from 52.30 ± 2.62 to 17.09 ± 1.40), suggesting that week-23 hybrids responded more readily than week-8 hybrids did to the inhibitory effect of EKLF. These data indicate that EKLF accelerated γ to β globin switch in these hybrids in a dose-dependent fashion, and the switching hybrids more readily submitted to its effe" @default.
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• W2125154113 title "Transcriptional environment and chromatin architecture interplay dictates globin expression patterns of heterospecific hybrids derived from undifferentiated human embryonic stem cells or from their erythroid progeny" @default.
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• W2125154113 doi "https://doi.org/10.1016/j.exphem.2013.08.005" @default.
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