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

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

Blood ◽

10.1182/blood.v114.22.2539.2539 ◽

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.

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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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Context-Specific Roles for Erythroid Krüppel-Like Factor (EKLF) in Co-Ordinate High Level Expression of the Murine α- and β-Globin Genes.

Blood ◽

10.1182/blood.v108.11.365.365 ◽

2006 ◽

Vol 108 (11) ◽

pp. 365-365 ◽

Cited By ~ 1

Author(s):

Valerie M. Jansen ◽

Shaji Ramachandran ◽

Aurelie Desgardin ◽

Jin He ◽

Vishwas Parekh ◽

...

Keyword(s):

Gene Expression ◽

Gene Transcription ◽

Globin Gene ◽

Erythroid Cell ◽

Gene Promoters ◽

Globin Genes ◽

Context Specific ◽

High Level ◽

Kinetics Of ◽

Globin Gene Expression

Abstract Binding of EKLF to the proximal promoter CACC motif is essential for high-level tissue-specific β-globin gene expression. More recent studies have demonstrated that EKLF regulates expression of other erythroid-specific genes, suggesting a broad role for EKLF in co-ordinating gene transcription in differentiating erythroblasts. Given these observations, we hypothesized that EKLF may play a role in synchronizing α- and β-globin gene expression. Supporting this model, studies of fetal erythroblasts derived from EKLF-null embryos revealed a 3-fold reduction in murine α-globin gene expression in fetal erythroblasts when compared to wild type littermate controls. A similar reduction in primary α-globin RNA transcripts was observed in these studies. To further examine the molecular consequences of EKLF function at the α- and β-globin genes in vivo, we utilized an erythroid cell line derived from EKLF null fetal liver cells. We have demonstrated previously that introduction into these cells of the wildtype EKLF cDNA, fused in frame with a mutant estrogen response element results in tamoxifen-dependent rescue of β-globin gene expression. Consistent with our observations in primary erythroblasts, α-globin gene expression is present in the absence of functional EKLF. However, with tamoxifen induction, we observed a 3–5 fold increase in α-globin gene transcription. Interestingly, the kinetics of the changes in transcription of the α- and β-gene transcripts were similar. Enhancement in α-gene transcription was associated with EKLF binding at the α- and β-globin promoters as determined by a quantitative chromatin immunoprecipitation (ChIP) assay. Interestingly, maximal EKLF binding and α-gene transcription was observed within 2 hours of tamoxifen induction. We hypothesized that the role of EKLF may differ function at the promoters, given that a basal level of α-globin gene expression occurs in absence of EKLF binding. Supporting this hypothesis, we observed sequential recruitment of p45NF-E2, RNA polymerase II (Pol II) and the co-activator CBP to the β-promoter with tamoxifen induction. No change in GATA-1 binding was observed. In contrast, p45NF-E2 does not bind to the α-promoter and the kinetics of GATA-1 and PolII association is unchanged after tamoxifen induction. Taken together, our results demonstrate that EKLF regulates the co-ordinate high-level transcription of the α- and β-globin genes, binding in a kinetically identical manner to the gene promoters. However, the effects of EKLF on transacting factor recruitment (and chromatin modification) differ between the promoters, consistent with the idea that EKLF acts in a context-specific manner to modulate gene transcription.

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Cucurbitacin D Upregulates Fetal Hemoglobin Expression in K562 Cells and Human Erythroid Progenitors.

Blood ◽

10.1182/blood.v110.11.3833.3833 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3833-3833

Author(s):

Hongtao Xing ◽

Siwei Zhang ◽

H. Phillip Koeffler ◽

Ming Chiu Fung

Keyword(s):

Gene Expression ◽

Globin Gene ◽

Fetal Hemoglobin ◽

K562 Cells ◽

Negative Control ◽

Optimal Dosage ◽

Erythroid Progenitors ◽

Cucurbitacin D ◽

Globin Gene Expression ◽

Sodium Phenylbutyrate

Abstract The search for novel therapeutic candidates causing reactivation of fetal hemoglobin (a2g2; HbF) to reduce the imbalance of globin gene expression is important in order to develop effective approach for the clinical management of sickle cell anemia and b-thalassemia. For the first time, we have identified cucurbitacin D (CuD), a naturally occurring oxygenated tetracyclic triterpenoid, as a molecular entity inducing g-globin gene expression and HbF synthesis in K562 cells and human erythroid progenitors from either peripheral blood or bone marrow. The upregulation of HbF induced by CuD was dose- and time- dependent. CuD was compared to hydroxyurea (HU), 5-azacytidine, amifostine, recombinant human erythropoietin (rhEPO), and sodium phenylbutyrate. At their optimal dosage, CuD (12.5 ng/mL) and HU (25.0 μg/mL) induced nearly 70% K562 cells to express total hemoglobin after 6 days culture, which was higher than the induction by Amifostine (30%), 5-azacytidine (36%), rhEPO (16%), sodium phenylbutyrate (23%) at their optimal concentrations and negative control (11%). Fetal hemoglobin ELISA showed that CuD (12.5 ng/mL) and 5-azacytidine (400 ng/mL) induced higher levels of fetal hemoglobin in K562 cells (15.4 ng/μL and 29.3 ng/μL, respectively), compared to HU (10.3 ng/μL), amifostine (7.8 ng/μL), rhEPO (10.9 ng/μL), sodium phenylbutyrate (9.9 ng/μL) at their optimal concentrations and negative control (5.3 ng/μL). CuD induced a significantly higher fetal cell percentage than HU in K562 cells (65% vs 37% maximum) and primary erythroid progenitors (36% vs 21% maximum) based on the immunofluorescence imaging and flow cytometry analysis. Real-time PCR results showed that the amount of γ-globin mRNA increased from 2.5-fold in CuD-optimal-treated cells (12.5 ng/mL, 48 hours) compared with 1.5-fold in HU-optimal-treated cells (25.0 μg/mL, 48 hours). Growth inhibition assay (MTT) demonstrated that CuD at its optimal γ-globin inducing dosage (12.5 ng/mL) inhibited proliferation of K562 by less than 10% of untreated control cells; while hydroxyurea at its optimal dosage (25.0 μg/mL) inhibited 80% of cell division. The in vitro therapeutic index (calculated by dividing the dose inhibiting 50% cell growth (IC50) by dose inducing 50% maximal HbF production (ED50)) of CuD was 40-fold greater than HU. Taken together, the results suggest that CuD has the potential to be a therapeutic agent for treatment of sickle cell anemia and b-thalassemia.

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O-Glcnacylation Regulates γ-Globin Transcription

Blood ◽

10.1182/blood.v122.21.1020.1020 ◽

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.

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Regulation of globin gene expression in human K562 cells by recombinant activin A

Blood ◽

10.1182/blood.v79.3.765.bloodjournal793765 ◽

1992 ◽

Vol 79 (3) ◽

pp. 765-772 ◽

Cited By ~ 1

Author(s):

NL Jr Frigon ◽

L Shao ◽

AL Young ◽

L Maderazo ◽

J Yu

Keyword(s):

Gene Expression ◽

Transforming Growth Factor ◽

Chinese Hamster Ovary Cells ◽

Globin Gene ◽

Chinese Hamster ◽

K562 Cells ◽

Activin A ◽

Erythroid Cell ◽

Tgf Beta ◽

Globin Gene Expression

Recent studies indicate that a purified protein, activin A, belongs to the transforming growth factor beta (TGF-beta) superfamily. Similar to TGF-beta, activin A can have different biologic activities, depending on the target tissues. We used recombinant activin A to demonstrate a possible regulatory role of this protein in modulating human erythroid differentiation in the human erythroid cell line, K562. Using genomic probes containing the second exon of alpha, beta, gamma, and epsilon globins, relative abundance of various types of globin transcripts in untreated and activin-treated K562 cells was examined with S1 nuclease analysis. Despite considerable homology amongst various globin sequences, these globin probes were highly specific for their unique mRNA species in the analyses. It was shown that the abundance of specific globin probe fragments for gamma and epsilon globins (209 nucleotides) as well as alpha (180 nucleotides), which were protected from S1 digestion, increased many fold in K562 cells treated with activin A. In contrast, there were no specific transcripts of beta globin detected in either the control or activin-treated cells. The increases in the level of fetal and embryonic beta-like and alpha globin transcripts also confirmed earlier studies of Northern and slot- blot analyses using globin cDNA as probes. In addition, nuclear run-off transcription assay using isolated nuclei indicated that most of the increase in the globin transcripts after activin treatment could be attributed to the stimulation of transcription rate for globin genes. Transient transfection assays also provide evidence that activin A significantly stimulated transcriptional activity of an epsilon globin promoter in K562, but not in the nonerythroid Chinese hamster ovary cells. Therefore, it was concluded that activin A exerts its effects on globin gene expression at the level of transcription in erythroid cells.

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Correct Function of the Locus Control Region May Require Passage Through a Nonerythroid Cellular Environment

Blood ◽

10.1182/blood.v93.2.703 ◽

1999 ◽

Vol 93 (2) ◽

pp. 703-712 ◽

Cited By ~ 24

Author(s):

George Vassilopoulos ◽

Patrick A. Navas ◽

Evangelia Skarpidi ◽

Kenneth R. Peterson ◽

Chris H. Lowrey ◽

...

Keyword(s):

Gene Expression ◽

Control Region ◽

Globin Gene ◽

Locus Control Region ◽

Erythroid Cell ◽

Globin Genes ◽

Globin Mrna ◽

L Cells ◽

Correct Function ◽

Globin Gene Expression

Abstract The function of the β-globin locus control region (LCR) has been studied both in cell lines and in transgenic mice. We have previously shown that when a 248-kb β-locus YAC was first microinjected into L-cells and then transferred into MEL cells by fusion, the YAC loci of the LxMEL hybrids displayed normal expression and developmental regulation.To test whether direct transfer of a β-globin locus (β-YAC) into MEL cells could be used for studies of the function of the LCR, a 155-kb β-YAC that encompasses the entire β-globin locus was used. This YAC was retrofitted with a PGK-neo selectable marker and with two I-PpoI sites at the vector arm-cloned insert junctions, allowing detection of the intact globin loci on a single I-PpoI fragment by pulsed field gel electrophoresis (PFGE). ThePpo-155 β-YAC was used to directly lipofect MEL 585 cells. In 7 β-YAC MEL clones with at least one intact copy of the YAC, the levels of total human globin mRNA (ie, ɛ + γ + β) per copy of integrated β-YAC varied more than 97-fold between clones. These results indicated that globin gene expression was strongly influenced by the position of integration of the β-YAC into the MEL cell genome and suggested that the LCR cannot function properly when the locus is directly transferred into an erythroid cell environment as naked β-YAC DNA. To test whether passage of the β-YAC through L-cells before transfer into MEL cells was the reason for the previously observed correct developmental regulation of human globin genes in the LxMEL hybrid cells, we transfected the YAC into L-cells by lipofection. Three clones carried the intact 144-kb I-PpoI fragment and transcribed the human globin genes with a fetal-like pattern. Subsequent transfer of the YAC of these L(β-YAC) clones into MEL cells by fusion resulted in LxMEL hybrids that synthesized human globin mRNA. The variation in human β-globin mRNA (ie, ɛ + γ + β) levels between hybrids was 2.5-fold, indicating that globin gene expression was independent of position of integration of the transgene, as expected for normal LCR function. The correct function of the LCR when the YAC is first transferred into the L-cell environment raises the possibility that normal activation of the LCR requires interaction with the transcriptional environment of an uncommitted, nonerythroid cell. We propose that the activation of the LCR may represent a multistep process initiated by the binding of ubiquitous transcription factors early during the differentiation of hematopoietic stem cells and completed with the binding of erythroid type of factors in the committed erythroid progenitors.

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Cooperative Silencing of Chicken ρ- and βH- Globin Gene Expression by Chicken β-Globin Regulatory Elements: Involvement of RNAi Mediated Repression.

Blood ◽

10.1182/blood.v106.11.3627.3627 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3627-3627

Author(s):

Elliot M. Epner ◽

Jin Wang ◽

Jing Huang

Keyword(s):

Gene Expression ◽

Gene Silencing ◽

Globin Gene ◽

K562 Cells ◽

Regulatory Elements ◽

Gene Promoters ◽

Globin Genes ◽

Phenotypic Analysis ◽

Pol Ii ◽

Globin Gene Expression

Abstract The chicken β-globin locus represents a well characterized, model system where the relationship between chromatin structure, transcription and DNA replication can be studied. The locus contains several regulatory elements including an intergenic enhancer as well as upstream regulatory elements that may function either alone or in combination with the intergenic enhancer as an LCR. The availability of the recombination proficient chicken B cell line DT40 has allowed the introduction of mutations into the endogenous chicken β-globin locus and phenotypic analysis after microcell mediated chromosome transfer into human erythroleukemia (K562) cells. Using this system, we have introduced deletions in the chicken β-globin intergenic enhancer as well as 5′ HS 1,2, and 3. Expression of the embryonic ρ and fetal βH chicken globin genes were repressed by the intergenic enhancer, 5′ HS1, or 5′HS2. No ρ or βH globin gene expression was detected in K562 cells containing control chicken chromosomes, while ρ and βH mRNA were activated when the intergenic enhancer, 5′ HS1, or 5′HS2 were deleted. Chromatin immunoprecipitation (ChIP) experiments that assayed RNA polmerase II (pol II), GATA-1 and NF-E2 p45/ p18 binding at regulatory elements and gene promoters in targeted cell lines supported this hypothesis and suggested a potential role for 5′HS3 in gene activation. However, targeted deletion of 5′ HS3, unlike the other chicken β-globin regulatory elements, showed no transcriptional phenotype. Our results demonstrate the intergenic enhancer, 5′HS1, and 5′ HS2 function through a common silencing mechanism involving pol II, GATA-1, and NF-E2/P18. The recent demonstration of the involvement of Pol II in the synthesis of miRNA’s prompted us to investigate the role of miRNA’s in gene silencing in this system. A small miRNA was identified at the intergenic enhancer region. ChIP assays showed the binding of two components of the RISC (Dicer and Ago2) at the chicken globin regulatory elements. These results are consistent with the involvement of RISC and miRNA’s in gene silencing in this system.

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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 ◽

10.1182/blood.v108.11.1584.1584 ◽

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.

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Smarca5 Regulates Ctcf Recruitment to Chromatin, Including to Regulatory Loci Involved In Control of Globin Gene Expression In Erythroleukemia

Blood ◽

10.1182/blood.v116.21.5159.5159 ◽

2010 ◽

Vol 116 (21) ◽

pp. 5159-5159

Author(s):

Martina Kapalova ◽

Juraj Kokavec ◽

Nikola Curik ◽

Pavel Burda ◽

Arthur I. Skoultchi ◽

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Keyword(s):

Gene Expression ◽

Control Region ◽

Conflicts Of Interest ◽

Globin Gene ◽

K562 Cells ◽

Globin Genes ◽

Protein Levels ◽

Genetic Imprinting ◽

Regulatory Loci ◽

Globin Gene Expression

Abstract Abstract 5159 Transcription factor Ctcf (CCCTC-binding factor) represents a major regulatory component of epigenetic regulation by recognizing its unmethyled DNA binding sites, resulting in changes in expression of neighboring genes. Ctcf plays an important role in transgenerational genetic imprinting. Very little is known about its role in hematologic malignancies. Ctcf has been described to promote differentiation of human erythroleukemia K562 cells (Torano 2005). We studied Ctcf in mouse erythroleukemia (MEL) cells and found it is expressed at both the mRNA and protein levels. Using chromatin immunoprecipitation (ChIP), we found that Ctcf is recruited to the H19/Igf2 imprinting control region (ICR) and also to the promoters of the alpha globin genes (Hba-a1, Hba-a2) as well as the beta globin locus control region (LCR) in MEL cells. To determine the mechanism by which Ctcf interacts with chromatin, we tested its interaction with chromatin remodeling proteins that associate with these DNA targets, including the well known Imitation Switch (ISWI class) ATPase Smarca5 (Snf2h). Using coimmunopreciptiation and ChIP experiments we found that Smarca5 and Ctcf interact on DNA. Next, we used MEL cells expressing an inducible Smarca5 shRNA. Doxycycline induction of Smarca5 shRNA led to a 5-fold decrease in Smarca5 mRNA and protein levels within 48hrs. ChIP experiments demonstrated that depletion of Smarca5 was accompanied by loss of Ctcf from the aforementioned loci indicating Ctcf requires Smarca5 for its association with chromatin. Furthermore, this was followed by significantly decreased levels H19 RNA. Our data provide evidence that Smarca5 regulates Ctcf recruitment to chromatin, including to regulatory loci involved in controlling globin gene expression. (Grants # IGA 10310-3, MSMT 2B06077, GAUK 251070 45410, SVV-2010-254260507, NIH R01CA154239). Disclosures: No relevant conflicts of interest to declare.

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