“Definitive” Erythropoiesis Has Distinct Developmental Origins and Globin Expression Patterns in the Mammalian Embryo.

Abstract Abstract 2539 Poster Board II-516 A transient wave of primitive erythropoiesis begins at embryonic day 7.5 (E7.5) in the mouse as yolk sac-derived primitive erythroid progenitors (EryP-CFC) generate precursors that mature in the circulation and expand in numbers until E12.5. A second wave of erythroid progenitors (BFU-E) originates in the yolk sac beginning at E8.25 that generate definitive erythroid cells in vitro. These BFU-E colonize the newly forming liver beginning at E10.5, prior to the initial appearance there of adult-repopulating hematopoietic stem cells (HSCs) between E11.5-12.5. This wave of definitive erythroid yolk sac progenitors is proposed to be the source of new blood cells required by the growing embryo after the expansion of primitive erythroid cells has ceased and before HSC-derived hematopoiesis can fulfill the erythropoietic needs of the embryo. We utilized multispectral imaging flow cytometry both to distinguish erythroid lineages and to define specific stages of erythroid precursor maturation in the mouse embryo. Consistent with this model, we found that small numbers of definitive erythrocytes first enter the embryonic circulation beginning at E11.5. All maturational stages of erythroid precursors were observed in the E11.5 liver, consistent with these first definitive erythrocytes having rapidly completed their maturation in the liver. The expression of βH1 and εy-beta globin genes is thought to be limited to primitive erythroid cells. Surprisingly, examination of globin gene expression by in situ hybridization revealed high levels of βH1-, but not εy-globin, transcripts in the parenchyma of E11.5-12.5 livers. RT-PCR analysis of globin mRNAs confirmed the expression of βH1- and adult β1-, but not εy-globin, in E11.5 liver-derived definitive (ckit+, Ter119lo) proerythroblasts sorted by flow cytometry to remove contaminating primitive (ckit-, Ter119+) erythroid cells. A similar pattern of globin gene expression was found in individual definitive erythroid colonies derived from E9.5 yolk sac and from early fetal liver. In vitro differentiation of definitive erythroid progenitors from E9.5 yolk sac revealed a maturational “switch” from βH1- and β1-globins to predominantly β1-globin. βH1-globin transcripts were not observed in proerythroblasts from bone marrow or E16.5 liver or in erythroid colonies from later fetal liver. ChIP analysis revealed that hyperacetylated domains encompass all beta globin genes in primitive erythroid cells but only the adult β1- and β2-globin genes in E16.5 liver proerythroblasts. Consistent with their unique gene expression, E11.5 liver proerythroblasts have hyperacetylated domains encompassing the βh1-, β1- and β2-, but not εy-globin genes. We also examined human globin transgene expression in mice carrying a single copy of the human beta globin locus. Because of the overlapping presence and changing proportion of primitive and definitive erythroid cells during development, we analyzed sorted cell populations whose identities were confirmed by murine globin gene expression. We confirmed that primitive erythroid cells express higher levels of γ- than ε-globin and little β-globin. E11.5 proerythroblasts and cultured E9.5 progenitors express γ- and β-, but not ε-globin. E16.5 liver proerythroblasts express β- and low levels of γ-globin, while adult marrow proerythroblasts express only β-globin transcripts. In summary, two forms of definitive erythropoiesis emerge in the murine embryo, each with distinct globin expression patterns and chromatin modifications of the β-globin locus. While both lineages predominantly express adult globins, the first, yolk sac-derived lineage uniquely expresses low levels of the embryonic βH1-globin gene as well as the human γ-globin transgene. The second definitive erythroid lineage, found in the later fetal liver and postnatal marrow, expresses only adult murine globins as well as low levels of the human γ-globin transgene only in the fetus. Our studies reveal a surprising complexity to the ontogeny of erythropoiesis. Disclosures: No relevant conflicts of interest to declare.

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Endogenous Elevations of Short Chain Fatty Acids Up-Regulate Embryonic Globin Gene Expression during Primitive Erythropoiesis.

Blood ◽

10.1182/blood.v104.11.1210.1210 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1210-1210

Author(s):

Lauren Sterner ◽

Toru Miyazaki ◽

Larry Swift ◽

Ann Dean ◽

Jane Little

Keyword(s):

Gene Expression ◽

Fatty Acids ◽

Fetal Liver ◽

Globin Gene ◽

Short Chain Fatty Acids ◽

Yolk Sac ◽

Globin Genes ◽

Wild Type ◽

Alpha Type ◽

Globin Gene Expression

Abstract We examined the effects of short chain fatty acids (SCFAs) on globin gene expression during development. We studied globin gene expression in transgenic mice that have endogenous elevations in the SCFA propionate due to a knockout (KO) of the gene for propionyl CoA carboxylase subunit A (PCCA, Miyazaki et al. JBC, 2001 Sep 21;276(38):35995–9). Serum propionate levels measured by gas chromatography were 2.5 to 3.6 mgms/ml in 2 adult PCCA KO mice and were undetectable in 2 wild type (wt) or heterozygous control adult mice. Embryonic PCCA KO offspring had propionate levels of 2.3 and 5.0 μgms/100 mgms of fetal liver, at day 16.5 (E16.5), while wt or heterozygotes at E14.5 had levels <1 μgm/100 mgms. Analysis of expression from alpha (α), beta major (βmaj), embryonic beta-type epsilon-y (εy), embryonic beta-type beta H1 (βH1) and embryonic alpha-type zeta (ζ) globin genes plus 18S ribosomal RNA as a control was undertaken using real-time PCR with gene-specific primers and taqman probes. cDNA was reverse-transcribed from the mRNA of yolk sac (YS) and fetal liver of PCCA KO and wt progeny of more than one litter from timed pregnancies. Individual PCCA embryos at E10 (n=10), E12 (n=9), and E14 (n=7) were analyzed for globin gene expression, normalized to18S expression and were compared to age-matched wt embryos (n>=4 for each time point). As expected, embryonic alpha- and beta-type globin gene expression (ζ and βH1 plus εy) predominated in E 10 YS, and definitive globin gene expression, α and βmaj, predominated in E12 or E14 fetal liver. Expression from embryonic alpha-type globin was calculated as normalized ζ/(ζ+α) and from embryonic beta-type globins as normalized (βH1+εy)/(βH1+εy+βmaj), see table. Embryonic globin gene expression was statistically significantly increased in PCCA KO E12 YS at 1.3 fold relative to wt ζ and in PCCA KO E14 YS at 1.8 fold and 2.1 fold relative to wt ζ or βH1 and εy respectively (p<.05). No increase in embryonic globin mRNA was seen in adult PCCA KO animals. We conclude that elevations of SCFAs during normal murine development causes a persistence of both embryonic alpha-type and embryonic beta-type globin gene expression during primitive, but not definitive, erythropoiesis, suggesting that SCFAs cannot reactivate silenced murine embryonic globin genes in the absence of erythroid stress. Embryonic Globin Gene Expression in Mice with Endogenous Elevations of SCFAs % Expression PCCA KO wild type p value, t test E10 ζ Yolk Sac 53+/− 2 nd E10 βH1 & ε y Yolk Sac 99 +/− 0.3 nd E12 ζ Yolk Sac 32 +/− 3 25 +/− 1 p < .05 E12 βH1 & ε y Yolk Sac 77 +/− 6 74 +/− 3 ns E14 ζ Yolk Sac 7 +/− 1.5 4 +/− 1.4 p < .05 E14 βH1 & ε y Yolk Sac 13 +/− 6 6 +/− 0.5 p < .05 E12 ζ Fetal Liver 11 +/− 4 9 +/− 2 ns E12 βH1 & ε y Fetal Liver 13 +/− 5 13+/− 3 ns E14 ζ Fetal Liver 1 +/− 0.4 0.7 +/− 0.2 ns E14 βH1 & εy Fetal Liver 6 +/− 1.8 4 +/− 1 ns

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The Human γ-Globin Gene Promoters Exhibit Markedly Different Patterns of DNA Methylation in Fetal Liver and Adult Erythroid Cells.

Blood ◽

10.1182/blood.v104.11.501.501 ◽

2004 ◽

Vol 104 (11) ◽

pp. 501-501 ◽

Cited By ~ 4

Author(s):

Christopher H. Lowrey ◽

Christine A. Richardson ◽

Kristin Johnson

Keyword(s):

Gene Expression ◽

Gene Silencing ◽

Fetal Liver ◽

Globin Gene ◽

Methylation Status ◽

Globin Genes ◽

Erythroid Cells ◽

Promoter Regions ◽

Methylation Patterns ◽

Globin Gene Expression

Abstract Despite intense investigation, the mechanisms by which human γ- to β-globin developmental gene switching occurs have yet to be fully elucidated. Based on studies in many systems, including human clinical trials with 5-Azacytidine and deoxyazacytidine, methylation has been thought to play an important, and more significantly, reversible role in γ-globin gene silencing. One mechanism by which DNA methylation is likely to effect γ-globin gene expression is through site-specific modification of CpG residues in the promoter regions of the γ-globin genes. For example, CpG methylation status has been proposed to mediate the developmentally-specific binding of Sp1 and the stage selector protein (SSP) complex to the proximal γ-globin promoters. We began this study to determine whether there were other CpG residues in the regions of the γ-globin promoters whose methylation status correlated with γ-gene silencing and thus might also serve as “molecular switches” regulating transcription factor binding, local histone acetylation and globin gene expression. To determine the methylation patterns of the γ-globin promoters, we first purified erythroid cells from human fetal liver (FL) and adult bone marrow (BM) using anti-glycophorin magnetic beads. DNA from the purified cells was subjected to bisulfite modification. The regions of the γ-globin promoters were PCR amplified and subcloned into plasmids. Individual plasmids were then sequenced to determine the methylation status of promoter regions. PCR primers were used which allowed determination of methylation status for CpGs at positions −249, −158, −52, −49. +6, +18, and +49 of the G and Aγ globin genes. An additional CpG at +210 was detectable with the Gγ primers. So far we have analyzed Gγ promoters from three FL and three BM samples. Aγ has been analyzed from two FL and three BM samples. An average of 10 clones have been sequenced for each sample. When results for samples within each condition (i.e., Gγ in FL) were combined for analysis, we see the expected increase in methylation of CpG residues in the Gγ promoter from 38% of all sites in the FL to 73% in the adult BM. This difference increases from 30% in FL to 88% adult BM when the CpGs at −158 and +210 are excluded. Combined methylation at these sites only increases from 7 to 21% between FL and BM and thus does not correlate well with changes in gene expression. Looking at the data another way shows a shift from most of the FL clones (76%) having 0 or 1 sites methylated in the Gγ promoter to 78% of the clones having 6,7 or 8 methylated sites in adult cells. While these results fit with the paradigm that methylation is associated with gene silencing, we saw a very different picture for Aγ. Because the promoter regions have nearly identical sequences, are located very close to each other and are similarly regulated, we expected their methylation patterns to be similar. However, for Aγ 13% of promoter CpGs are methylated in the FL cells but this increases to only 22% in adult erythroid cells. Maximal Aγ promoter methylation occurs at the +6 and +18 CpGs which reach only 33 and 36% methylation in adult erythroid cells. 86% of FL Aγ clones are methylated at only 0–2 promoter CpG sites. This does not change at 83% in adult cells. These results indicate differential methylation of the two human γ-globin genes and suggests that simple promoter methylation is not the primary mechanism of γ-globin gene silencing.

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Silencing of γ-Globin Gene Expression during Adult Definitive Erythropoiesis Is Mediated by a GATA-1 Repressor Complex.

Blood ◽

10.1182/blood.v110.11.271.271 ◽

2007 ◽

Vol 110 (11) ◽

pp. 271-271

Author(s):

Kenneth R. Peterson ◽

Flavia C. Costa ◽

Susanna Harju-Baker

Keyword(s):

Gene Expression ◽

Fetal Liver ◽

Globin Gene ◽

Globin Genes ◽

Erythroid Cells ◽

Nurd Complex ◽

Repressor Complex ◽

Definitive Erythropoiesis ◽

Silencer Element ◽

Globin Gene Expression

Abstract Autonomous silencing of gene expression is one mechanism operative in the control of human β-like globin gene switching. Experiments using variously truncated Aγ-globin genes linked to LCR sequences suggested that a region of the Aγ-globin gene between -730 to -378 relative to the mRNA CAP site may function as an adult stage-specific silencer element. A marked copy of the Aγ-globin gene (Aγm-globin) was inserted between LCR 5′ HS1 and the ε-globin gene in a human β-globin locus yeast artificial chromosome (Aγm 5′ ε β-YAC). The Aγm-globin gene was autonomously silenced in Aγm 5′ ε β-YAC transgenic mice, even in the absence of an adult β-globin gene. A -730 to -378 deletion of the Aγm-globin gene was introduced into the Aγm 5′ ε β-YAC to produce a Δ1s Aγm 5′ ε β-YAC. Transgenic lines containing intact β-globin loci expressed the Δ1s Aγm-globin gene in embryonic yolk sac, fetal liver, and adult blood. To further delineate the function of the Δ1s fragment, transient transfection assays and protein-DNA interaction assays were performed. The Δ1s fragment was found to act as a repressor of a constitutively active SV40 promoter in K562 cells. DNaseI footprinting analysis and electromobility shift assays demonstrated GATA-1-binding at a site -570 bp upstream of the Aγ-globin CAP site. Recently generated β-YAC transgenic mice containing a T>G point mutation at the -570 GATA site of the normally-located Aγ-globin gene displayed a HPFH phenotype. Together, these data suggested that the -730 to -378 Aγ-globin gene region contains a silencer element at the -570 GATA site that binds a GATA-1 repressor complex during the adult stage of definitive erythropoiesis to silence expression of the Aγ-globin gene. Previous studies suggested that when GATA-1 functions as a repressor, it interacts with components of the MeCp1/NuRD complex. This complex may remodel chromatin into a repressed state, leading to silenced Aγ-globin gene expression during adult definitive erythropoiesis. The presence of components of the MeCP1/NuRD complex was assessed in uninduced (γ-globin repressor present) and induced (γ-globin repressor absent) erythroid cells (K562 and KU812) and non-erythroid cells (HFF) by Western blot analysis using an antibody to Mi2, which is a component of the NuRD complex. Mi2 protein was observed in erythroid cells when the levels of γ-globin were low (uninduced K562 or KU812 cells), whereas only a weak signal was detected when γ-globin expression was induced in these cells. The Mi2 signal in the HFF cells was even weaker. Chromatin immunoprecipitation (ChIP) using fetal liver samples from day E12 and E18 conceptuses of wild-type β-YAC transgenics showed that GATA-1, FOG-1 and Mi2 proteins co-localize to the -570 GATA site of the Aγ-globin gene in samples where γ-globin is silenced (E18 fetal liver), but not in samples where γ-globin is expressed (E12 fetal liver). Our data strongly suggest that the MeCP1/NuRD complex interacts with GATA-1 protein to form a repressor that may be involved in silencing Aγ-globin gene expression. In addition, we show that GATA-1, FOG-1 and Mi2 are recruited to the analogous -567 GATA site of Gγ-globin, in a pattern that parallels that of Aγ-globin. However the binding of these proteins to Gγ-globin is weaker than that observed for Aγ-globin. These data suggest that GATA-1-mediated repression is common to both γ-globin genes, but that other mechanisms function in the differential regulation of the two γ-globin genes.

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Endogenous Elevations of Short Chain Fatty Acids (SCFAs) Can Up-Regulate Embryonic Globin Gene Expression.

Blood ◽

10.1182/blood.v110.11.1770.1770 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1770-1770

Author(s):

Himanshu Bhatia ◽

Jennifer Hallock ◽

Lauren Sterner ◽

Toru Miyazaki ◽

Ann Dean ◽

...

Keyword(s):

Gene Expression ◽

Fetal Liver ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Short Chain Fatty Acids ◽

Yolk Sac ◽

Globin Genes ◽

Key Enzyme ◽

Globin Gene Expression

Abstract Persistence of fetal hemoglobin can ameliorate adult beta (β)-globin gene disorders. Since SCFAs can affect embryonic and fetal globin gene expression, we examined their role during development. Murine globin gene expression, β-type (embryonic βH1, and epsilon-y, εY, and adult βmajor), and alpha (α)-type (embryonic zeta, ζ, >α, adult α), were compared between wildtype (wt) and transgenic mice, in which a key enzyme for SCFA metabolism, PCCA, had been knocked out (PCCA−/−, (Miyazaki et al, 2001). E10.5 PCCA−/− yolk sac (n= 9), showed increased α, βH1 and ζ gene expression, at respectively 2-, 2.6- and 1.6-fold relative to wt (n=13, p<.05), and εY gene expression, at 1.7-fold (p=0.07). The embryonic-to-adult globin gene switch was modestly delayed in yolk sacs from E12.5 PCCA−/− (n=9) vs. wt (n=4) and E 14.5 PCCA−/− (n=6) vs. wt (n=6). % embryonic β-type globin gene expression (% βH1 and εY of total β globin) was 77±6 PCCA−/− and 74±3 wt at E12.5, p=n.s., and 42±13 PCCA−/− and 21±3 wt at E14.5, p<.05; % emvbryonic α-type expression (% ζ of total α) was 32±3 PCCA−/−, 25±1wt at E12.5, p<.05 and 7±2 PCCA−/− and 4±1 wt at E14.5, p<.05). Embryonic globin gene expression in E 12.5 and 14.5 fetal livers was not different between PCCA−/− and wt embryos. Cultures of pooled E14.5 wt fetal liver cells (FLCs, n=4 separate experiments), however, suggested that embryonic globin genes can be activated in FLCs. The percent of total β-type globin gene expression that was embryonic after culture with butyrate (1mM) was 11.6±2.6%, with propionate (2.5 mM) was 3.6±0.2%, and insulin/erythropoietin or basal media was 0.03±0.03% and 0.42±0.26% respectively (p<.05 relative to SCFAs). Dose-response with propionate (n=2 seaparate experiments) suggest inadequate endogenous propionate levels for activation in PCCA −/− fetal liver, as % embryonic β-type globin gene expression rose above basal levels only at concentrations of 1 to 5 mM (2.5 mM maximal) but not at <0.6 mM. We conclude that endogenous SCFAs, at levels achievable in vivo can activate embryonic globin gene expression during development in the murine yolk-sac. However, higher levels than achievable endogenously currently are necessary to produce this effect in murine fetal livers.

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In Vivo and In Vitro Analysis of an Aγ-Globin Gene Silencer in Human β-Yac Transgenic Mice.

Blood ◽

10.1182/blood.v104.11.1220.1220 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1220-1220

Author(s):

Susanna Harju ◽

Kenneth R. Peterson

Keyword(s):

Gene Expression ◽

Transgenic Mice ◽

Fetal Liver ◽

Globin Gene ◽

Yolk Sac ◽

Luciferase Reporter ◽

Globin Genes ◽

Mobility Shift ◽

Gene Switching ◽

Globin Gene Expression

Abstract Autonomous silencing of gene expression is one mechanism operative in the control of human β-like globin gene switching and is best exemplified by the ε-globin gene. Experiments using variously truncated Aγ-globin genes linked to LCR sequences suggested that a region of the Aγ-globin gene between −730 to −378 relative to the mRNA CAP site may function as an adult stage-specific silencer element. A 5.4 Kb marked Aγ-globin gene (Aγm) inserted between LCR 5′HS1 and the ε-globin gene in a β-YAC (Aγm 5′ ε β-YAC) was silenced in transgenic mice during adult definitive erythropoiesis, even in the absence of an adult β-globin gene. In contrast, when a marked β-globin gene (βm) was inserted in this same location in another β-YAC (βm 5′ ε β-YAC), the βm-globin gene was expressed throughout ontogeny. From these data we concluded that: 1) any gene located near the LCR will be strongly expressed throughout ontogeny, unless some gene-specific silencing mechanism exists, 2) competition between the γ- and β-globin genes for interaction with the LCR is not the exclusive mechanism controlling γ- to β-globin gene switching, and 3) that the Aγm-globin gene was autonomously silenced. A -730 to -378 deletion of the Aγm-globin gene was introduced into the Aγm 5′ ε β-YAC via homologous recombination to produce a Δ1s Aγm 5′ ε β-YAC. This YAC was microinjected and six founders were obtained. Four transgenic lines were established carrying at least one full-length β-globin locus and two were established that lacked the adult β-globin gene. All founders containing an intact β-globin gene expressed the Δ1s Aγm-globin during adult erythropoiesis (45% – 122% relative to human β-globin expression). In one line examined in detail, the Δ1s Aγm-globin gene was expressed in the embryonic yolk sac, fetal liver, and adult blood. ε-globin gene expression was not detected in the embryonic yolk sac and expression of the normally located γ-globin genes was not observed at any developmental stage. β-globin gene expression was observed in the fetal liver and adult blood, although its expression was decreased. To further delineate the function of the Δ1s fragment, transient transfection assays to test silencer function and protein-DNA interaction assays were performed. Silencer activity of the352 bp Δ1s fragment was examined using a series of pGL2 luciferase reporter plasmids that were synthesized to include the Δ1s fragment; these were electroporated into various cells. Electrophoretic mobility shift assays (EMSAs) and DNAse I footprinting were employed to begin assessment of protein binding within the Δ1s fragment. A 50 bp DNA fragment spanning −713 to −664 of the Δ1s element was used in EMSAs; DNA binding activity was observed in K562 nuclear extracts. These preliminary data suggest that the −730 to −378 γ-globin gene silencer binds a repressor protein complex.

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Human β-Globin Locus Control Region Hypersensitive Site Specificity for Globin Gene Activation during Erythropoiesis.

Blood ◽

10.1182/blood.v106.11.3630.3630 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3630-3630

Author(s):

Kenneth R. Peterson ◽

Halyna Fedosyuk ◽

Susanna Harju

Keyword(s):

Gene Expression ◽

Fetal Liver ◽

Globin Gene ◽

Gene Activation ◽

Gene Promoter ◽

Yolk Sac ◽

Site Specificity ◽

Globin Genes ◽

Definitive Erythropoiesis ◽

Globin Gene Expression

Abstract Although the human β-globin locus control region (LCR) functions as a holocomplex within an active chromatin hub, we provide evidence that within the aggregate hypersensitive site (HS) activation domain of the holocomplex, the individual HSs still mediate preferential activation of the globin genes during development. A 2.9 Kb deletion of 5′HS3 (Δ5′HS3) or a 234 bp deletion of the 5′HS3 core (Δ5′HS3c) in a 213 Kb human β-globin locus yeast artificial chromosome (β-YAC) abrogate ε-globin gene expression during primitive erythropoiesis in β-YAC transgenic mice, suggesting that 5′HS3 sequences of the LCR are involved directly in ε-globin gene activation. The reduction of ε-globin gene transcription in Δ5′HS3 or Δ5′HS3c β-YAC transgenics can be explained by two hypotheses. The first is site-specificity. The interaction between the LCR and the ε-globin gene promoter involves specific sequences of 5′HS3 and specific sequences of the ε-globin gene promoter. When 5′HS3 or its core is deleted, these interactions do not take place and ε-globin gene transcription is diminished. The second hypothesis is change in conformation of the LCR. Normally, in the embryonic stage, the LCR achieves a three-dimensional conformation that favors interaction with the first gene in the complex, the ε-globin gene. When 5′HS3 is deleted, an alternate conformation is assumed that decreases the chance that there will be an interaction between the LCR and the ε-globin gene. However, the LCR interacts with the next gene in order, the γ-globin gene. In Δ5′HS3c β-YAC mice, γ-globin gene expression is normal during primitive erythropoiesis, but is extinguished in the fetal stage of definitive erythropoiesis. These data suggest that a conformational change occurs in the Δ5′HS3c LCR during the switch from embryonic to definitive erythropoiesis, from one that supports γ-globin gene expression to one that does not. Alternately, the embryonic trans-acting environment may allow the mutant LCR to interact with and activate the γ-globin genes, but the fetal trans-acting environment may not support this interaction in the absence of the 5′HS3 core. To distinguish between these possibilities, β-YAC lines were produced in which the ε-globin gene was replaced with a second marked β-globin gene (βm), coupled to either an intact LCR, a 2.9 Kb 5′HS3 deletion or a 234 bp 5′HS3 core deletion. Δ5′HS3c Δε::βm β-YAC mice expressed βm-globin throughout development beginning at day 10 in the yolk sac. γ-globin was expressed in the embryonic yolk sac, but not in the fetal liver. Some wild-type β-globin was expressed in addition to βm-globin in adult mice. The γ-globin phenotype is consistent with published data on Δ5′HS3c β-YAC mice. Although ε-globin was not expressed in Δ5′HS3c β-YAC mice, βm-globin was expressed in Δ5′HS3c Δε::βm β-YAC embryos, demonstrating that the 5′HS3 core was necessary for ε-globin expression during embryonic erythropoiesis, but not for βm-globin expression. A similar phenotype was observed in Δ5′HS3 Δε::βm β-YAC mice, except βm-globin expression was higher in the day 10 yolk sac and γ-globin expression continued into the fetal liver stage of definitive erythropoiesis consistent with results published on Δ5′HS3 β-YAC mice. These data support a site specificity model of LCR HS-globin gene interaction.

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Methyl Binding Domain Protein 2 Mediates gamma-Globin Silencing in Adult beta-YAC Transgenic Mice.

Blood ◽

10.1182/blood.v106.11.3626.3626 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3626-3626

Author(s):

Jeremy W. Rupon ◽

ShouZhen Wang ◽

Karin Gaensler ◽

Joyce Lloyd ◽

Gordon D. Ginder

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Quantitative Pcr ◽

Globin Gene ◽

Globin Genes ◽

Beta Globin ◽

Erythroid Cells ◽

Rnase Protection ◽

Gamma Globin ◽

Globin Gene Expression

Abstract The genes of the vertebrate beta-globin locus undergo a switch in expression during development whereby embryonic/fetal genes of the cluster are sequentially silenced and the adult genes are activated during erythroid development. DNA methylation has been shown to be associated with developmentally silenced globin genes, and compounds that inhibit cytosine methylation have been shown to activate transcription from developmentally silenced globin genes in several species, including humans. Previously, we have shown that the methyl domain binding protein 2 (MBD2) is involved in maintaining embryonic rho-globin gene silencing in adult avian erythroid cells. We describe here a role for MBD2 in the DNA methylation mediated silencing and maintenance of silencing of the human fetal gamma-globin gene in a transgenic mouse model. We confirmed the previously published report by Pace et al that the gamma-globin gene is reactivated, upon treatment with the DNA methyltransferase inhibitor, 5-azacytidine, of mice containing the entire beta-globin locus as a yeast artificial chromosome (BetaYAC) transgene. In order to elucidate the mechanism through which DNA methylation represses the gamma-globin gene in adult erythroid cells, betaYAC/MBD2−/− mice were generated by breeding BetaYAC mice with MBD2−/− mice. Anemic adult betaYAC/MBD2−/− mice continue to express the gamma-globin gene at a level commensurate with animals treated with 5-azacytidine, which is10–20 fold over those treated with 1-acetyl-2-phenylhydrazine alone, as measured by both quantitative PCR and by RNase protection assays. In addition, the level of gamma-globin gene expression is consistently several fold higher in MBD2−/− compared to wild type BetaYAC mice in 14.5 and 16.5 dpc fetal liver erythroblasts. Furthermore, transcriptional activation of the gamma-globin gene in adult erythroblasts is associated with a modest decrease in DNA methylation around the gamma-globin promoters and a ~4-fold enrichment of histone H3 trimethylated at lysine 4 (TriK4), as measured by chromatin immunoprecipitation assay using quantitative PCR. Finally, treatment of MBD2 null mice with 5-azacytidine induces only a small, non-additive induction of gamma-globin expression indicating that DNA methylation acts primarily through MBD2 to maintain gamma-globin suppression in adult erythroid cells. These results suggest that MBD2 is a potential target for therapeutic induction of gamma-globin gene expression in the settings of sickle cell anemia and beta-thalassemia.

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Induction of Human Fetal Globin Gene Expression by a Novel Erythroid Factor, NF-E4

Molecular and Cellular Biology ◽

10.1128/mcb.20.20.7662-7672.2000 ◽

2000 ◽

Vol 20 (20) ◽

pp. 7662-7672 ◽

Cited By ~ 51

Author(s):

Wenlai Zhou ◽

David R. Clouston ◽

Xi Wang ◽

Loretta Cerruti ◽

John M. Cunningham ◽

...

Keyword(s):

Gene Expression ◽

Fetal Liver ◽

Globin Gene ◽

K562 Cells ◽

Erythroid Cell ◽

Globin Genes ◽

Erythroid Progenitors ◽

Dimerization Domain ◽

Preferential Expression ◽

Globin Gene Expression

ABSTRACT The stage selector protein (SSP) is a heteromeric complex involved in preferential expression of the human γ-globin genes in fetal-erythroid cells. We have previously identified the ubiquitous transcription factor CP2 as a component of this complex. Using the protein dimerization domain of CP2 in a yeast two-hybrid screen, we have cloned a novel gene, NF-E4, encoding the tissue-restricted component of the SSP. NF-E4 and CP2 coimmunoprecipitate from extract derived from a fetal-erythroid cell line, and antiserum to NF-E4 ablates binding of the SSP to the γ promoter. NF-E4 is expressed in fetal liver, cord blood, and bone marrow and in the K562 and HEL cell lines, which constitutively express the fetal globin genes. Enforced expression of NF-E4 in K562 cells and primary erythroid progenitors induces endogenous fetal globin gene expression, suggesting a possible strategy for therapeutic intervention in the hemoglobinopathies.

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Mi2β-mediated silencing of the fetal γ-globin gene in adult erythroid cells

Blood ◽

10.1182/blood-2012-11-466227 ◽

2013 ◽

Vol 121 (17) ◽

pp. 3493-3501 ◽

Cited By ~ 45

Author(s):

Maria Amaya ◽

Megha Desai ◽

Merlin Nithya Gnanapragasam ◽

Shou Zhen Wang ◽

Sheng Zu Zhu ◽

...

Keyword(s):

Gene Expression ◽

Major Part ◽

Globin Gene ◽

Globin Genes ◽

Erythroid Cells ◽

Key Points ◽

Globin Gene Expression ◽

Partial Depletion

Key Points Mi2β exerts a major part of its silencing effect on embryonic and fetal globin genes by positively regulating the BCL11A and KLF1 genes. Partial depletion of Mi2β induces increased γ-globin gene expression in primary human erythroid cells without impairing differentiation.

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5'-flanking sequences mediate butyrate stimulation of embryonic globin gene expression in adult erythroid cells.

Molecular and Cellular Biology ◽

10.1128/mcb.11.9.4690 ◽

1991 ◽

Vol 11 (9) ◽

pp. 4690-4697 ◽

Cited By ~ 74

Author(s):

J G Glauber ◽

N J Wandersee ◽

J A Little ◽

G D Ginder

Keyword(s):

Gene Expression ◽

Cell Differentiation ◽

Globin Gene ◽

Beta Globin ◽

Erythroid Cells ◽

Adult Chicken ◽

Beta Globin Gene ◽

Flanking Sequences ◽

Murine Erythroleukemia ◽

Globin Gene Expression

A stable transfection assay was used to test the mechanism by which embryonic globin gene transcription is stimulated in adult erythroid cells exposed to butyric acid and its analogs. To test the appropriate expression and inducibility of chicken globin genes in murine erythroleukemia (MEL) cells, an adult chicken beta-globin gene construct was stably transfected. The chicken beta-globin gene was found to be coregulated with the endogenous adult mouse alpha-globin gene following induction of erythroid differentiation of the transfected MEL cells by incubation with either 2% dimethyl sulfoxide (DMSO) or 1 mM sodium butyrate (NaB). In contrast, a stably transfected embryonic chicken beta-type globin gene, rho, was downregulated during DMSO-induced MEL cell differentiation. However, incubation with NaB, which induces MEL cell differentiation, or alpha-amino butyrate, which does not induce differentiation of MEL cells, resulted in markedly increased levels of transcription from the stably transfected rho gene. Analysis of histone modification showed that induction of rho gene expression was not correlated with increased bulk histone acetylation. A region of 5'-flanking sequence extending from -569 to -725 bp upstream of the rho gene cap site was found to be required for both downregulation of rho gene expression during DMSO-induced differentiation and upregulation by treatment with NaB or alpha-amino butyrate. These data are support for a novel mechanism by which butyrate compounds can alter cellular gene expression through specific DNA sequences. The results reported here are also evidence that 5'-flanking sequences are involved in the suppression of embryonic globin gene expression in terminally differentiated adult erythroid cells.

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