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

Blood ◽  
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

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

Blood ◽  
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.

“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.

Enhanced Erythroid Differentiation of Murine Embryoid Bodies That Are Exposed to Short-Chain Fatty Acids during Development.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3646-3646
Author(s):  
Jennifer L. Hallock ◽  
Lauren J. Sterner ◽  
Jane A. Little
Keyword(s):  
Gene Expression ◽  
Fatty Acids ◽  
Cell Surface ◽  
Es Cells ◽  
Globin Gene ◽  
Short Chain Fatty Acids ◽  
Embryoid Bodies ◽  
Short Chain ◽  
Globin Genes ◽  
Globin Gene Expression

Abstract Embryonic/fetal-type globin genes are known to be up-regulated in models of adult erythropoiesis following exposure to short-chain fatty acids (SCFAs, butyrate or propionate), in a subset of beta-globin gene disorder patients treated with SCFAs, and in children with metabolic disorders that result in high levels of endogenous SCFAs. Here, we investigate a role for SCFAs during development, using a murine embryonic stem (ES) cell model. Globin gene expression and cell-surface marker presentation were examined in murine embryoid bodies (EBs), which differentiate from ES cells and which recapitulate many features of early murine development. ES cells were allowed to differentiate (through the removal of both LIF and contact inhibition, without cytokines), in the absence or presence of SCFAs. Expression of alpha- and beta-type globin gene RNA and transcription factor RNA in the developing EBs was analyzed daily or every-other-day for 10–14 days by real-time PCR with gene-specific primers and probes. At day +2 and +6 /7 EBs were disaggregated, labeled, and analyzed by fluorescent-activated cell sorting (FACS) with labeled antibodies that had been raised against cell-surface markers of hematopoietic and endothelial differentiation. Globin gene expression was augmented by SCFAs in all experiments, with maximal induction, when compared with untreated cells, at approximately day + 7 of differentiation; Comparative globin gene expression, between propionate-exposed and untreated, in 3 separate EB experiments and normalized to 18S expression, was variable, but was elevated for all globin genes examined, as shown in Table I. Absolute RNA expression, normalized to 18S in a representative experiment testing butyrate or propionate as inducers, indicated that alpha, zeta, and beta H1/0 were effected most by SCFAs, (in which expression ranged between 30- and 40-fold relative to 18S) while Beta major and Epsilon-y (ranging between 1- and 7-fold relative to 18S) were effected to a much lesser extent; This trend, in which alpha, zeta, and Beta H1/0 induction by SCFAs was more dramatic than that seen for Epsilon-y and beta major induction, was noted in all experiments. In a single analysis, RNA for BMP-4 and GATA-2 was up-regulated on D + 7 by SCFAs at, respectively, 7- and 18- fold, which was not true for other hematopoietic transcription factors examined, such as LMO-2, GATA-1, EKLF-1, or AML-1, FACS analyses of untreated vs. SCFA-treated EBs in three separate experiments were analyzed with a screening gate for hematopoietic cells (CKit). These experiments showed a modest increase in the hematopoietic marker CD34 in SCFA-treated vs. un-treated cells; a doubling in CKit-positive calls that expressed late erythroid markers on Day + 6, from 5.6 +/− 2% to 12.1 +/− 6.0 % TER-119 positive (P<.05), and a 3-fold, but variable increase, from 1.4 +/− 1.5 % to 4.2 +/− 4.1 % EPO-Receptor positive (n.s.). Expression of CD 45, CD 41, PECAM, and FLK-1 was not consistently different between EBs differentiated in the absence or presence of SCFAs. These data suggest that SCFAs minimally enhance hematopoiesis, but markedly enhance erythropoiesis, during differentiation in a murine developmental model; we speculate that SCFAs and related compounds could play a similar role in vivo during development. Range of globin gene expression, SCFA- vs. un-treated EBs Globin Genes Fold-expression Alpha 5- to 22-fold Zeta 4.2- to 35-fold Beta H1/0 5- to 22.5-fold Epsilon-y 9-to 96-fold Beta Major 2.5- to 22-fold

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

Blood ◽  
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.

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

Blood ◽  
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.

O-Glcnacylation Regulates γ-Globin Transcription

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1020-1020
Author(s):  
Kenneth R Peterson ◽  
Zhen Zhang ◽  
Ee Phie Tan ◽  
Anish Potnis ◽  
Nathan Bushue ◽  
...  
Keyword(s):  
Gene Expression ◽  
Sodium Butyrate ◽  
Yeast Artificial Chromosome ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Globin Genes ◽  
Globin Gene Expression ◽  
Dynamic Cycling

Abstract Patients with sickle cell disease (SCD), caused by mutation of the adult β-globin gene, are phenotypically normal if they carry compensatory mutations that result in continued expression of the fetal γ-globin genes, a condition termed hereditary persistence of fetal hemoglobin (HPFH). Thus, a logical clinical goal for treatment of SCD is to up-regulate γ-globin synthesis using compounds that are specific for increasing fetal hemoglobin (HbF) without pleiotropic effects on cellular homeostasis. Developmental regulation of the γ-globin genes is complex and normal silencing during the adult stage of erythropoiesis likely results from a combination of the loss of transcriptional activators and the gain of transcriptional repressor complexes. One mode of γ-globin silencing occurs at the GATA binding sites located at -566 or -567 relative to the Aγ-globin or Gγ-globin CAP sites respectively, and is mediated through the DNA binding moiety of GATA-1 and its recruitment of co-repressor partners, FOG-1 and Mi-2 (NuRD complex). Modifications of repressor complexes can regulate gene transcription; one such modification is O-GlcNAcylation. The O-GlcNAc post-translational modification is the attachment of a single N-acetyl-glucosamine moiety to either a serine or threonine residue on nuclear and cytoplasmic proteins. O-GlcNAc is added to proteins by O-GlcNAc transferase (OGT) and removed by O-GlcNAcase (OGA) in response to changes in extracellular signals and nutrients. A dynamic balance in protein levels also exists between these two enzymes; an increase or decrease of one results in a like compensatory change in the other. Thus, the rate of O-GlcNAc addition and removal is a dynamic cycling event that is exquisitely controlled for a given target molecule, which may offer a point of intervention in the turning off or on of gene expression. O-GlcNAcylation is involved in the regulation of many cellular processes such as stress response, cell cycle progression, and transcription. Potentially, O-GlcNAc plays a pivotal role in regulating transcription of the human γ-globin genes. We induced human erythroleukemia cell line K562 with sodium butyrate to differentiate toward the erythroid lineage and observed the expected increase of γ-globin gene expression. A robust increase of γ-globin gene expression was measured after pharmacological inhibition of OGA using Thiamet-G (TMG). Using chromatin immunoprecipitation (ChIP), we demonstrated that OGT and OGA are recruited to the -566 region of the Aγ-globin promoter, the same region occupied by the GATA-1-FOG-1-Mi-2 (NuRD) repressor complex. However, OGT recruitment to this region was decreased when O-GlcNAc levels were artificially elevated by OGA inhibition with TMG. When γ-globin expression was not induced, Mi-2 was modified with O-GlcNAc and interacted with both OGT and OGA. After induction, O-GlcNAcylation of Mi-2 was reduced and Mi2 no longer interacted with OGT. Stable K562 cells were generated in which OGA was knocked down using shRNA. Following induction of these cells with sodium butyrate, γ-globin gene expression was higher compared to control cells. These data suggest that the dynamic cycling of O-GlcNAc on the Mi-2 (NuRD) moiety contributes towards regulation of γ-globin transcription. Concurrent ChIP experiments in human β-globin locus yeast artificial chromosome (β-YAC) transgenic mice demonstrated that GATA-1, Mi2 and OGT were recruited to the -566 Aγ-globin GATA silencer site in day E18 fetal liver when γ-globin is repressed, but not in day E12 fetal liver when γ-globin is expressed. These data demonstrate that O-GlcNAc cycling is a novel mechanism regulating γ-globin gene expression and will provide new avenues to explore in how alterations in gene regulation lead to the onset, progression, and severity of hematological disease. Disclosures: No relevant conflicts of interest to declare.

Differences in the Pattern of Histone H3 (K4) and (K27) Methylation throughout the β-Globin Gene Locus in Baboon Fetal Liver and Adult Bone Marrow Erythroblasts Pre- and Post-Decitabine Treatment.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1584-1584
Author(s):  
Janet Chin ◽  
Donald Lavelle ◽  
Kestis Vaitkus ◽  
Maria Hankewych ◽  
Joseph DeSimone
Keyword(s):  
Gene Expression ◽  
Intergenic Region ◽  
Gene Locus ◽  
Fetal Liver ◽  
Globin Gene ◽  
Histone H3 ◽  
Globin Genes ◽  
Alu Sequence ◽  
Adult Bone ◽  
Globin Gene Expression

Abstract Understanding the role of chromatin structure in specifying the pattern of β-like globin gene expression during development would be important in the design of future pharmacologic therapies to increase fetal hemoglobin in patients with sickle cell disease and β-thalassemia. The baboon is an important experimental animal model to study the regulation of globin gene expression because the structure of the β-globin gene complex and developmental pattern of globin gene expression are similar to man, and HbF levels are greatly increased in baboons treated with the DNA methyltransferase inhibitor decitabine (5-aza-2′-deoxycytidine). To investigate the relationship between chromatin structure, DNA methylation, and globin gene regulation, the distribution of acetyl histone H3 (ac-H3), acetyl histone H4 (ac-H4), histone H3 (K4) dimethyl and trimethyl, and histone H3 (K27) dimethyl throughout the β-globin gene locus was determined in purified primary erythroblasts from baboon fetal liver (FL), and adult bone marrow (BM) pre- and post-decitabine treatment. Analysis was performed by chromatin immunoprecipitation (ChIP) of formaldehyde-fixed chromatin followed by real time PCR using 18 primer sets spanning the baboon β-globin gene locus from the 5′ region of the ε-globin gene to the β-globin gene. Comparison of the pattern of ac-H3 and ac-H4 suggested the presence of three subdomains of chromatin within the β-globin locus characterized by different levels of histone acetylation that exhibited a differential response to decitabine treatment. Histone H3 (K4) dimethyl was relatively enriched in the region containing the ε- and γ-globin genes and in the γ-β intergenic region 5′ to the duplicated Alu sequence in FL. Levels associated with the ε-, γ-, and γ-globin genes in adult BM were similar and relatively unaffected by decitabine treatment. In contrast, high levels of histone H3 (K4) trimethylation and pol II distribution were associated with the promoters and transcribed regions of active genes. Differences in the levels of H3 (K4) trimethylation and pol II associated with individual genes were well correlated with differences in their relative levels of expression in FL and adult BM pre- and post-decitabine treatment. The level of histone H3 (K4) trimethyl associated with the promoter of the developmentally inactive ε-globin gene was very low and not enriched compared to inactive necdin gene or the γ-β intergenic regon in adult BM suggesting that the ε-globin gene is not maintained in a “poised” transcriptional state by the presence of the histone H3 (K4) trimethyl mark near the ε-globin promoter. The pattern of histone H3 (K27) dimethyl differed in FL and adult BM. Levels of H3 (K27) dimethyl associated with the ε- and γ-globin genes in FL were 2–4 fold less than near the duplicated Alu sequence in the γ-β intergenic region, while levels were 4–10 fold higher near the ε- and γ-globin genes and γ-β intergenic region compared to the promoter and transcribed region of the β-globin gene in adult BM. Reactivation of γ-globin expression following decitabine treatment was associated with a relative decrease in the level of H3 (K27) dimethyl near the γ-globin gene. Increased H3 (K27) methylation in regions surrounding the silenced ε- and γ-globin genes suggests that the polycomb group (PcG) protein EZH2, a histone H3 (K27) methyltransferase, may be involved in globin gene silencing.

The -566 Aγ-Globin HPFH Mutation Disrupts Temporal Repression of Fetal Hemoglobin Synthesis During Fetal Liver Definitive Erythropoiesis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2019-2019
Author(s):  
Kenneth R Peterson ◽  
Halyna Fedosyuk ◽  
Flavia C Costa
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Fetal Liver ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Wild Type ◽  
Repressor Complex ◽  
Definitive Erythropoiesis ◽  
Gata Motif ◽  
Globin Gene Expression

Abstract Abstract 2019 Poster Board I-1041 Hereditary persistence of fetal hemoglobin (HPFH) is a condition associated with continued fetal hemoglobin (HbF) production in adults, where normally only very low levels of HbF are found. Sickle cell disease (SCD) patients are phenotypically normal if they carry a compensatory HPFH mutation due to the high levels of HbF. Understanding the molecular mechanisms leading to reactivation or derepression of γ-globin gene expression will lead to the development of new or better therapies to treat SCD patients. In our long-established and highly-characterized model system, transgenic mice carrying wild-type human β-globin locus yeast artificial chromosomes (β-YACs) express predominantly γ-globin and a lesser amount of γ-globin in the primitive erythroid cells of the yolk sac, mostly β-globin and some γ-globin in the definitive erythroid cells of the fetal liver and nearly exclusively β-globin in the adult definitive red blood cells, as measured both at the transcript and protein levels. We recently identified a novel Aγ-globin gene silencer motif located at -566 relative to the mRNA CAP site in a GATA motif. Repression is mediated by binding a GATA-1-FOG-1-Mi2 protein complex. Since our initial studies of this GATA-1 repressor complex were performed using β-YAC transgenic mice in which a second copy of the Aγ-globin gene was introduced between the locus control region (LCR) and the γ-globin gene, our first goal was to test if this mutation was functional at the normally-located Aγ-globin globin gene. β-YAC transgenic mice were produced with the T>G HPFH point mutation at the -566 GATA site of this gene. These mice display a mild HPFH phenotype during adult definitive erythropoiesis; γ-globin gene expression levels were increased approximately 3% compared to wild-type β-YAC mice. Expression of γ-globin is also elevated relative to wild-type β-YAC controls during primitive erythropoiesis in the embryonic yolk sac and definitive erythropoiesis in the fetal liver. Chromatin immunoprecipitation (ChIP) experiments using day E12 to E18 post-conception fetal liver samples from wild type β-YAC transgenic mice demonstrate that GATA-1 is recruited to this GATA silencer first at day E16, followed by recruitment of FOG-1 and Mi2 at day E17. In addition, ChIP experiments performed with day E18 samples from the -566 HPFH mice demonstrate that this point mutation disrupts the recruitment of GATA-1 to this site at a developmental stage when it normally binds as a repressor in wild-type β-YAC transgenic samples. GATA-2 does not bind at the -566 GATA motif when γ-globin is actively transcribed. Thus, GATA-2/GATA-1 competition does not play a role in the function of this silencer or the mechanism of HPFH at this site. In addition, BCL11A does not appear to be a component of this GATA-1 repressor complex. Taken together our data indicate that a temporal repression mechanism is operative in the silencing of γ-globin gene expression and that the presence of the -566 Aγ-globin HPFH mutation disrupts establishment of repression, resulting in continued γ-globin gene transcription during adult definitive erythropoiesis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals KLF2 is essential for primitive erythropoiesis and regulates the human and murine embryonic β-like globin genes in vivo

Blood ◽  
2005 ◽  
Vol 106 (7) ◽  
pp. 2566-2571 ◽  
Author(s):  
Priyadarshi Basu ◽  
Pamela E. Morris ◽  
Jack L. Haar ◽  
Maqsood A. Wani ◽  
Jerry B. Lingrel ◽  
...  
Keyword(s):  
Apoptotic Cell ◽  
Globin Gene ◽  
Yolk Sac ◽  
Globin Genes ◽  
Erythroid Cells ◽  
Wild Type ◽  
Evolutionarily Conserved ◽  
Primitive Erythropoiesis ◽  
Globin Gene Expression

AbstractThe Krüppel-like factors (KLFs) are a family of C2/H2 zinc finger DNA-binding proteins that are important in controlling developmental programs. Erythroid Krüppel-like factor (EKLF or KLF1) positively regulates the β-globin gene in definitive erythroid cells. KLF2 (LKLF) is closely related to EKLF and is expressed in erythroid cells. KLF2-/- mice die between embryonic day 12.5 (E12.5) and E14.5, because of severe intraembryonic hemorrhaging. They also display growth retardation and anemia. We investigated the expression of the β-like globin genes in KLF2 knockout mice. Our results show that KLF2-/- mice have a significant reduction of murine embryonic Ey- and βh1-globin but not ζ-globin gene expression in the E10.5 yolk sac, compared with wild-type mice. The expression of the adult βmaj- and βmin-globin genes is unaffected in the fetal livers of E12.5 embryos. In mice carrying the entire human globin locus, KLF2 also regulates the expression of the human embryonic ϵ-globin gene but not the adult β-globin gene, suggesting that this developmental-stage-specific role is evolutionarily conserved. KLF2 also plays a role in the maturation and/or stability of erythroid cells in the yolk sac. KLF2-/- embryos have a significantly increased number of primitive erythroid cells undergoing apoptotic cell death. (Blood. 2005;106: 2566-2571)

scholarly journals Genome Architecture of the Human β-Globin Locus Affects Developmental Regulation of Gene Expression

2005 ◽  
Vol 25 (20) ◽  
pp. 8765-8778 ◽  
Author(s):  
Susanna Harju ◽  
Patrick A. Navas ◽  
George Stamatoyannopoulos ◽  
Kenneth R. Peterson
Keyword(s):  
Gene Expression ◽  
Gene Order ◽  
Yeast Artificial Chromosome ◽  
Fetal Liver ◽  
Globin Gene ◽  
Regulation Of Gene Expression ◽  
Globin Genes ◽  
Wild Type ◽  
Globin Mrna ◽  
Definitive Erythropoiesis

ABSTRACT To test the role of gene order in globin gene expression, mutant human β-globin locus yeast artificial chromosome constructs were used, each having one additional globin gene encoding a “marked” transcript (εm, γm, or βm) integrated at different locations within the locus. When a βm-globin gene was placed between the locus control region (LCR) and the ε-globin gene, βm-globin expression dominated primitive and definitive erythropoiesis; only βm-globin mRNA was detected during the fetal and adult definitive stages of erythropoiesis. When an Aγm-globin gene was placed at the same location, Aγm-globin was expressed during embryonic erythropoiesis and the fetal liver stage of definitive erythropoiesis but was silenced during the adult stage. The downstream wild-type γ-globin genes were not expressed. When an εm-globin gene was placed between the δ- and β-globin genes, it remained silent during embryonic erythropoiesis; only the LCR-proximal wild-type ε-globin gene was expressed. Placement of a βm-globin gene upstream of the Gγ-globin gene resulted in expression of βm-globin in embryonic cells and in a significant decrease in expression of the downstream wild-type β-globin gene. These results indicate that distance from the LCR, an inherent property of spatial gene order, is a major determinant of temporal gene expression during development.

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