Growth Factor Independence 1b (Gfi1b) Is Required for the Regulation of Fetal Globin Genes in Both Fetal and Adult Erythroid Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 350-350 ◽  
Author(s):  
Lothar Vassen ◽  
Wafaa Lemsaddek ◽  
Marie Trudel ◽  
Tarik Moroy
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Expression Array ◽  
Knock Out ◽  
Platelet Counts ◽  
Normal Platelet ◽  
Genome Wide ◽  
Globin Gene Expression

Abstract Abstract 350 Gfi1b is hematopoietic transcription factor most highly expressed in hematopoietic stem cells, megakaryocyte-erythroid precursors, megakaryocytes and throughout erythroid development. Gfi1b deficiency is lethal in mice around 13.5 dpc caused by a failure to produce functional erythrocytes, megakaryocytes and platelets, which causes severe hemorrhaging. Since this lethality has hampered further analysis of the function of Gfi1b, we used Cre-recombinase inducible conditional Gfi1b knock-out mice (Gfi1bfl/fl). The pIpC induced knock-out of Gfi1b in Gfi1bfl/flMxCre mice leads to a pronounced drop in peripheral blood platelet numbers and induces a strong extramedullary erythropoiesis in the spleen. We sorted Ter119+ bone marrow cells from wt and pIpC induced Gfi1bfl/flMxCre mice for a genome wide expression array analysis and found a significant increase in the expression levels of platelet/coagulation related genes such as PF4, vWF, F2r or Ppbp as well as of the fetal globin genes Hba-x, Hbb-ey and Hbb-ßh1, suggesting that Gfi1 regulates globin gene expression or globin switching. It remained unclear whether the disturbed erythropoiesis in Gfi1b deficient mice was caused by a bone marrow failure, or was a reaction to the anemia caused by internal bleedings as a result of low platelet counts. To clarify this and to avoid deletion of Gfi1b in megakaryocytes, we crossed Gfi1bfl/fl mice with EpoR-EGFP-Cre mice allowing a Gfi1b deletion specifically in erythroid cells at the pro-erythroblast stage. Gfi1bfl/flEpoR-EGFP-Cre embryos were paler than wt littermates, but in contrary to complete knock-outs showed no internal bleedings and had normal platelet counts. In addition, EpoR-EGFP-Cre embryos showed a mild block in terminal erythroid differentiation and a pronounced hyper-proliferation at the Ter119-,CD71+, cKit+ proerythroblast stage where Cre expression is activated. Gfi1bfl/flEpoR-Cre cells showed a strong increase of fetal Hbb-ßH1 globin gene expression and a pronounced decrease of the expression of the adult globin genes Hba, Hbb, as well as of Gata1, Foxo3a and Nfe2l2 but not Gata2. Gene expression-array analysis of fetal liver cells from wt and Gfi1bfl/flEpoR-Cre embryos from day 14.5 dpc showed that besides fetal globin genes, many genes where up-regulated that normally decrease in expression during the development between the embryonic stages 11.5 dpc to 14.5 dpc. These findings confirm that Gfi1b is required for the regulation of globin gene expression during or at the transition from embryonic/fetal to adult stages. Interestingly, Gfi1bfl/flEpoR-Cre mice were viable very likely because these animals have normal platelet counts and do not suffer from hemorrhaging like constitutive Gfi1b deficient mice. However, this also suggested that the block in erythroid development is tolerable, or that it can be overcome during maturation of the embryo. Q-PCR analysis on mRNA from sorted erythroid cells from wt and adult Gfi1bfl/flEpoR-Cre mice showed a highly increased expression of the fetal globin genes Hbb-ßh1, Hbb-ey and Hba-x but only a slight decrease of Gata1 expression, a mild increase in Nfe2l2 expression and no significant expression of Gata2 compared to age matched wild type controls. A recently published study of genome wide in vivo DNA binding of ten major hematopoietic transcription factors (Wilson et al., Cell Stem Cell, 2010) showed Gfi1b binding to hypersensitive site 2 in the globin locus control region (LCR) where also the Gfi1b interaction partner Gata1 and Nfe2 bind. From these data we conclude, that Gfi1b is required to regulate the expression of fetal globin genes during the switch from embryonic/fetal to adult stages and thereafter during adult globin expression and exerts this function by directly binding to regulatory sites in the globin locus. Since the re-expression of fetal globin genes in adult stages is a therapeutic approach for ß-thalassemia, the function of Gfi1b and its regulatory mechanisms could point to new therapeutic strategies for this disease. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽  
2013 ◽  
Vol 121 (17) ◽  
pp. 3493-3501 ◽  
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.

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

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2539-2539
Author(s):  
Kathleen E. McGrath ◽  
Jenna M Frame ◽  
George Fromm ◽  
Anne D Koniski ◽  
Paul D Kingsley ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fetal Liver ◽  
Globin Gene ◽  
Yolk Sac ◽  
Globin Genes ◽  
Beta Globin ◽  
Erythroid Cells ◽  
Erythroid Progenitors ◽  
Low Levels ◽  
Globin Gene Expression

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.

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

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 501-501 ◽  
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.

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

Blood ◽  
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.

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

Blood ◽  
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.

Analysis of DNA Methylation at the Human Alpha Globin Cluster during Hematopoiesis.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1861-1861
Author(s):  
Supachai Ekwattanakit ◽  
Helena Ayyub ◽  
Kevin Clark ◽  
Fatima Marques-Kranc ◽  
Sue Butler ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Globin Gene ◽  
Regulatory Elements ◽  
Human Cells ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Humanized Mouse ◽  
Alpha Globin ◽  
Globin Gene Expression

Abstract The analysis of globin gene regulation has elucidated many of the principles underlying mammalian gene expression. To understand how the epigenetic program that unfolds during hematopoiesis impacts on alpha (α)-globin gene expression, we analyzed DNA methylation at over 200 CpG dinucleotides in 130 kb around the human α-globin gene cluster focussing particularly on a region (12 kb) covering the α2 and α1-globin genes (HBA2 and HBA1), and the upstream regulatory sites (DNAse I hypersensitive sites (HS) -48, HS-40, HS-33, and HS-10); so-called multi-species conserved sequencesregulatory element1-4 (MCS-R1-4). Cultures of primary adult erythroid cells (at three stages: early, intermediate, and late) were analyzed together with non-erythroid human cells (neutrophils, peripheral blood mononuclear cells, and a human embryonic stem (ES) cell line). Using a recently published method involving bisulphite modification and MALDI-TOF Mass Spectrometry we quantified the level of DNA methylation across the alpha globin cluster. Most sequences outside of the CpG islands were methylated. However, the DNA methylation levels between erythroid and non-erythroid cells differed at regulatory elements. MCS-R1 contained only 1 CpG site and the average percentage of DNA methylation for erythroid and non-erythroid was 8% and 40%, respectively. While they were 8% vs. 42% (p=0.428), 11% vs. 99% (p=0.054), and 14% vs. 97% (p=0.001) in MCS-R2 to 4, respectively. To study changes in DNA methylation in a comprehensive, developmental and tissue-specific manner, we used a humanized-mouse model, in which the conserved α-globin syntenic region of mouse genomic sequence is replaced by that of human. This contains all cis-acting sequences required for fully regulated expression of the α-globin genes. Although the alpha globin cluster is expressed efficiently in this model, DNA methylation patterns differed in the 20 kb region encompassing the α-globin genes when comparing primary human cells and primary humanized cells. DNA in humanized cells were relatively hypomethylated both in erythroid and non-erythroid tissues. The methylation levels were higher in testis of the humanized mouse than in other tissues but still not as high as those in normal human non-erythroid cells. The cause of these differences is being investigated. Despite these differences, the humanized-mouse mimicked the patterns of methylation found in regulatory elements in erythroid and nonerythroid cells. This study shows that despite appropriate regulation of gene expression, epigenetic templating may differ between species (human and mouse). DNA methylation at the upstream regulatory elements might be involved in the regulation of α-globin gene expression during erythropoiesis, although it is equally possible that these changes in methylation are generated by passive demethylation secondary to transcription factor binding in erythroid cells. The mechanism(s) underlying demethylation of regulatory elements during differentiation remains to be clarified.

scholarly journals Long-Distance Control of Origin Choice and Replication Timing in the Human β-Globin Locus Are Independent of the Locus Control Region

2000 ◽  
Vol 20 (15) ◽  
pp. 5581-5591 ◽  
Author(s):  
Daniel M. Cimbora ◽  
Dirk Schübeler ◽  
Andreas Reik ◽  
Joan Hamilton ◽  
Claire Francastel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Control Region ◽  
Globin Gene ◽  
Replication Timing ◽  
S Phase ◽  
Chromatin Conformation ◽  
Erythroid Cells ◽  
Long Distance ◽  
Targeted Deletion ◽  
Globin Gene Expression

ABSTRACT DNA replication in the human β-globin locus is subject to long-distance regulation. In murine and human erythroid cells, the human locus replicates in early S phase from a bidirectional origin located near the β-globin gene. This Hispanic thalassemia deletion removes regulatory sequences located over 52 kb from the origin, resulting in replication of the locus from a different origin, a shift in replication timing to late S phase, adoption of a closed chromatin conformation, and silencing of globin gene expression in murine erythroid cells. The sequences deleted include nuclease-hypersensitive sites 2 to 5 (5′HS2-5) of the locus control region (LCR) plus an additional 27-kb upstream region. We tested a targeted deletion of 5′HS2-5 in the normal chromosomal context of the human β-globin locus to determine the role of these elements in replication origin choice and replication timing. We demonstrate that the 5′HS2-5-deleted locus initiates replication at the appropriate origin and with normal timing in murine erythroid cells, and therefore we conclude that 5′HS2-5 in the classically defined LCR do not control replication in the human β-globin locus. Recent studies also show that targeted deletion of 5′HS2-5 results in a locus that lacks globin gene expression yet retains an open chromatin conformation. Thus, the replication timing of the locus is closely correlated with nuclease sensitivity but not globin gene expression.

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

1991 ◽  
Vol 11 (9) ◽  
pp. 4690-4697 ◽  
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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