Episomal vectors based on S/MAR and the β-globin Replicator, encoding a synthetic transcriptional activator, mediate efficient γ-globin activation in haematopoietic cells

AbstractWe report the development of episomal vectors for the specific γ-globin transcription activation in its native position by activator Zif-VP64, based on the Scaffold/Matrix Attachment Region (S/MAR) for episomal retention and the β-globin Replicator, the DNA replication-Initiation Region from the β-globin locus. Vector Zif-VP64-Ep1 containing transcription cassettes CMV- Zif-VP64 and CMV-eGFP-S/MAR transfected a)K562 cells; b)murine β-YAC bone marrow cells (BMC); c)human haematopoietic progenitor CD34+ cells, with transfection efficiencies of 46.3 ± 5.2%, 23.0 ± 2.1% and 24.2 ± 2.4% respectively. K562 transfections generated stable cell lines running for 28 weeks with and without selection, with increased levels of γ-globin mRNA by 3.3 ± 0.13, of γ-globin protein by 6.75 ± 3.25 and HbF protein by 2 ± 0.2 fold, while the vector remained episomal and non integrated. In murine β-YAC BMCs the vector mediated the activation of the silent human γ-globin gene and in CD34+ cells, increased γ-globin mRNA, albeit only transiently. A second vector Zif-VP64-Ep2, with both transcription cassettes carrying promoter SFFV instead of CMV and the addition of β-globin Replicator, transferred into CD34+ cells, produced CD34+ eGFP+ cells, that generated colonies in colony forming cell cultures. Importantly, these were 100% fluorescent, with 2.11 ± 0.13 fold increased γ-globin mRNA, compared to non-transfected cells. We consider these episomal vectors valid, safer alternatives to viral vectors.

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The Landscape of Non-Viral Gene Augmentation Strategies for Inherited Retinal Diseases

International Journal of Molecular Sciences ◽

10.3390/ijms22052318 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2318

Author(s):

Lyes Toualbi ◽

Maria Toms ◽

Mariya Moosajee

Keyword(s):

Viral Vectors ◽

Matrix Attachment Region ◽

Great Promise ◽

Viral Gene ◽

Retinal Diseases ◽

Gene Copies ◽

Augmentation Strategies ◽

Viral Gene Therapy ◽

Effective Option ◽

Attachment Region

Inherited retinal diseases (IRDs) are a heterogeneous group of disorders causing progressive loss of vision, affecting approximately one in 1000 people worldwide. Gene augmentation therapy, which typically involves using adeno-associated viral vectors for delivery of healthy gene copies to affected tissues, has shown great promise as a strategy for the treatment of IRDs. However, the use of viruses is associated with several limitations, including harmful immune responses, genome integration, and limited gene carrying capacity. Here, we review the advances in non-viral gene augmentation strategies, such as the use of plasmids with minimal bacterial backbones and scaffold/matrix attachment region (S/MAR) sequences, that have the capability to overcome these weaknesses by accommodating genes of any size and maintaining episomal transgene expression with a lower risk of eliciting an immune response. Low retinal transfection rates remain a limitation, but various strategies, including coupling the DNA with different types of chemical vehicles (nanoparticles) and the use of electrical methods such as iontophoresis and electrotransfection to aid cell entry, have shown promise in preclinical studies. Non-viral gene therapy may offer a safer and effective option for future treatment of IRDs.

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Gene Amplification and the Extrachromosomal Circular DNA

Genes ◽

10.3390/genes12101533 ◽

2021 ◽

Vol 12 (10) ◽

pp. 1533

Author(s):

Noriaki Shimizu

Keyword(s):

Gene Amplification ◽

Malignant Tumors ◽

Human Cancer ◽

Replication Initiation ◽

Matrix Attachment Region ◽

Genome Plasticity ◽

Sequence Element ◽

Circular Dna ◽

Attachment Region ◽

Human Malignant Tumors

Oncogene amplification is closely linked to the pathogenesis of a broad spectrum of human malignant tumors. The amplified genes localize either to the extrachromosomal circular DNA, which has been referred to as cytogenetically visible double minutes (DMs), or submicroscopic episome, or to the chromosomal homogeneously staining region (HSR). The extrachromosomal circle from a chromosome arm can initiate gene amplification, resulting in the formation of DMs or HSR, if it had a sequence element required for replication initiation (the replication initiation region/matrix attachment region; the IR/MAR), under a genetic background that permits gene amplification. In this article, the nature, intracellular behavior, generation, and contribution to cancer genome plasticity of such extrachromosomal circles are summarized and discussed by reviewing recent articles on these topics. Such studies are critical in the understanding and treating human cancer, and also for the production of recombinant proteins such as biopharmaceuticals by increasing the recombinant genes in the cells.

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AUF-1 and YB-1 are critical determinants of β-globin mRNA expression in erythroid cells

Blood ◽

10.1182/blood-2011-10-387316 ◽

2012 ◽

Vol 119 (4) ◽

pp. 1045-1053 ◽

Cited By ~ 9

Author(s):

Sebastiaan van Zalen ◽

Grace R. Jeschke ◽

Elizabeth O. Hexner ◽

J. Eric Russell

Keyword(s):

Posttranscriptional Regulation ◽

Rna Binding ◽

Globin Gene ◽

K562 Cells ◽

Erythroid Cells ◽

Erythroid Progenitors ◽

Globin Mrna ◽

Affinity Enrichment

Abstract The normal accumulation of β-globin protein in terminally differentiating erythroid cells is critically dependent on the high stability of its encoding mRNA. The molecular basis for this property, though, is incompletely understood. Factors that regulate β-globin mRNA within the nucleus of early erythroid progenitors are unlikely to account for the constitutively high half-life of β-globin mRNA in the cytoplasm of their anucleate erythroid progeny. We conducted in vitro protein-RNA binding analyses that identified a cytoplasm-restricted β-globin messenger ribonucleoprotein (mRNP) complex in both cultured K562 cells and erythroid-differentiated human CD34+ cells. This novel mRNP targets a specific guanine-rich pentanucleotide in a region of the β-globin 3′untranslated region that has recently been implicated as a determinant of β-globin mRNA stability. Subsequent affinity-enrichment analyses identified AUF-1 and YB-1, 2 cytoplasmic proteins with well-established roles in RNA biology, as trans-acting components of the mRNP. Factor-depletion studies conducted in vivo demonstrated the importance of the mRNP to normal steady-state levels of β-globin mRNA in erythroid precursors. These data define a previously unrecognized mechanism for the posttranscriptional regulation of β-globin mRNA during normal erythropoiesis, providing new therapeutic targets for disorders of β-globin gene expression.

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Specific globin mRNAs in human erythroleukemia (K562) cells

Blood ◽

10.1182/blood.v63.1.195.195 ◽

1984 ◽

Vol 63 (1) ◽

pp. 195-200 ◽

Cited By ~ 19

Author(s):

CW Miller ◽

K Young ◽

D Dumenil ◽

BP Alter ◽

JM Schofield ◽

...

Keyword(s):

Gene Transcription ◽

Marked Decrease ◽

Globin Gene ◽

K562 Cells ◽

Dna Probes ◽

The Other ◽

Beta Globin ◽

Globin Mrna ◽

Mrna Accumulation

Abstract Specific globin mRNA accumulation was quantitated in several lines of K562 cells in the absence and the presence of hemin. Using specific cloned DNA probes, the amounts of zeta, alpha, epsilon and gamma mRNAs were shown to be increased 2–3-fold in the presence of 20 microM hemin. No delta- or beta-globin mRNAs were detectable in any of the lines. In one line, Bos, there was a marked decrease in epsilon-globin mRNA, which increased with hemin, although still to much lower levels than in the other lines. The decreased epsilon-globin mRNA accumulation in Bos is shown to be due to decreased epsilon-globin gene transcription.

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Differing responses of globin and glycophorin gene expression to hemin in the human leukemia cell line K562

Blood ◽

10.1182/blood.v59.4.738.738 ◽

1982 ◽

Vol 59 (4) ◽

pp. 738-746 ◽

Cited By ~ 18

Author(s):

BL Tonkonow ◽

R Hoffman ◽

D Burger ◽

JT Elder ◽

EM Mazur ◽

...

Keyword(s):

Gene Expression ◽

Cell Line ◽

Globin Gene ◽

Leukemia Cell ◽

K562 Cells ◽

Leukemia Cell Line ◽

Human Leukemia ◽

Globin Mrna ◽

Human Leukemia Cell Line ◽

Globin Gene Expression

Abstract The human leukemia cell line, K562, produces embryonic and fetal hemoglobins and glycophorin A, proteins normally associated only with erythroid cells. Hemoglobin accumulation is enhanced by exposure of the cells to 0.05 mM hemin. We have examined K562 cells before and after exposure to hemin to determine whether expression of these erythroid proteins was shared by all cells or confined to specific subpopulations. Globin gene expression was examined by quantitation of globin mRNA sequences, using a 3H-globin cDNA molecular hybridization probe. Constitutive cells produced globin mRNA, the content of which was increased 3–4-fold by hemin. Cell-to-cell distribution of globin mRNA was determined by in situ hybridization of 3H-globin cDNA to constitutive and hemin-treated K562 cells. Virtually all cells in the culture exhibited grain counts above background, indicating globin gene expression by all cells, rather than a confined subpopulation. Virtually all hemin-treated cells had 3–5-fold higher grain counts, indicating uniformly increased globin gene expression. The glycophorin content of K562 cells was estimated by fluorescence-activated cell sorting (FACS) of cells labeled with fluorescein-labeled antiglycophorin antiserum. The vast majority of constitutive cells contained glycophorin, but exhibited to apparent increase in glycophorin accumulation after hemin exposure. Thus, glycophorin and globin genes exhibited differential responses to hemin. These differences could reflect normal differences in the patterns of specialized gene expression in stem cells. Alternatively, different aberrations of gene expression could be occurring in response to the determinants of the neoplastic properties of K562.

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Talen-Mediated Knock Outs Of Cis and Trans Elements Potentially Involved In Globin Gene Switching

Blood ◽

10.1182/blood.v122.21.436.436 ◽

2013 ◽

Vol 122 (21) ◽

pp. 436-436

Author(s):

Patrick A Navas ◽

Yongqi Yan ◽

Minerva E Sanchez ◽

Ericka M Johnson ◽

George Stamatoyannopoulos

Keyword(s):

Gene Expression ◽

Gene Regulation ◽

Globin Gene ◽

K562 Cells ◽

Globin Mrna ◽

Core Elements ◽

Gene Knockouts ◽

Gene Switching ◽

Cis And Trans ◽

Globin Gene Expression

Abstract Transcription activator-like effector nucleases (TALEN) are engineered proteins used for precise genome editing by generating specific DNA double strand that are repaired by homologous recombination and by non-homologous end joining. TALENs can be used to study gene regulation by deleting putative regulatory elements in the context of the native chromosome and measuring mRNA synthesis. We designed TALENs to delete individual DNAse I-hypersensitive sites (HS) of the β-globin locus control region (LCR) followed by an assessment of globin gene expression and assessment of epigenetic effects in K562 erythroleukemia cells. The β-globin LCR is composed of five HSs and functions as a powerful regulatory element responsible for appropriate levels of the five β-like globin genes during development. Introduction of plasmid DNA encoding a pair of TALENs and targeting individually the flanking region of the HS2, HS3 and HS4 core elements along with a donor 100 base single-stranded oligonucleotide resulted in the successful deletions of each of the three core elements in K562 cells. Individual K562 cells were seeded to produce clones and the mutations were screened by PCR to identify both heterozygous and homozygous clones. The TALEN-mediated 288 bp HS2 core deletion resulted 32 heterozygous (48.5%) and 6 homozygous clones (9.1%) in a total of 66 clones screened. K562 carries three copies of chromosome 11 emphasizing the robustness of TALEN technology to target each of the alleles. In the 199 bp HS3 core deletion, from 113 clones we identified 28 heterozygous (24.8%) and 3 (2.7%) homozygous clones. Lastly, the 301 bp HS4 core deletion yielded 9 homozygous (5.9%) and 12 heterozygous (7.9%) clones from 151 clones screened. Total RNA was isolated from wild-type K562 cells, and from both the heterozygous and homozygous mutant clones and subjected to RNase Protection analysis to quantitate the levels of globin mRNA. Deletion if the HS3 core in K562 cells in a ∼30% reduction in ε-globin mRNA and 2-fold reduction in γ-globin mRNA. A more dramatic effect on globin expression is observed in the HS2 core deletion, as ε- and γ-globin expression is reduced by 2- and 5-fold, respectively. These results suggest that HS2 contributes the majority of the LCR enhancer function in K562 cells. The HS4 core deletion resulted in a modest ∼20% reduction in both ε- and γ-globin expression. TALENs were designed to knockout trans-acting factors implicated to be involved in globin gene regulation and/or globin switching. TALENs bracketing the gene promoters and the first exon of 25 genes encoding either a transcription factor or histone-modifying enzyme were synthesized and post-transfection PCR screens of the transfected pool of K562 cells resulted in the successful identification of 17 gene knockouts. The 17 target genes are PRMT5, LDB1, EIF2AK3, BCL11A, HBSIL, MYB, SOX6, NFE4, NR2F2, NR2C1, NR2C2, CHTOP, NFE2, DNMT3A, RBBP4, MTA2 and MBD2. Single cell clones have been generated by limited dilution of transfected K562 pools and thus far we have identified heterozygous and homozygous clones of 8 of 17 gene knockouts, importantly all clones were identified without selection. The frequency of identifying the knockout clones, represented by the number of clones screened/ number of heterozygous clones/ number of homozygous clones, are as follows: HBS1L (63/3/0), SOX6 (68/13/2), NFE4 (56/13/7), LBD1 (300/2/0), MBD2 (301/0/1), CHTOP (288/66/6), NFE2 (712/44/5) and NR2C1 (96/40/11). The remaining nine gene knockouts and globin gene expression data will be presented at the meetings. These studies highlight a powerful TALEN-mutagenesis platform for target deletions of both cis- and trans-elements to study globin gene switching. TALENs can be synthesized in several days and the screening of the individual clones for the desired knockouts is completed within two weeks. This highly efficient mutagenesis platform will further our understanding of the molecular basis of globin switching. Disclosures: No relevant conflicts of interest to declare.

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The Effect of the Soluble Guanylyl Cyclase Stimulator Olinciguat on ƴ-Globin Gene Induction in K562 Cells

Blood ◽

10.1182/blood-2018-99-116011 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1078-1078 ◽

Cited By ~ 1

Author(s):

Joy Miyashiro ◽

Asha Pant ◽

Boris Tchernychev ◽

Todd G Milne ◽

Mark G Currie ◽

...

Keyword(s):

Cell Line ◽

Guanylyl Cyclase ◽

Mode Of Action ◽

Globin Gene ◽

Soluble Guanylyl Cyclase ◽

Fetal Hemoglobin ◽

K562 Cells ◽

Mrna Levels ◽

Globin Mrna ◽

No Signaling

Abstract Induction of fetal hemoglobin (HbF: α2ƴ2) is a recognized mode of action of hydroxyurea, the sickle cell disease (SCD) standard of care in SCD, and has been shown to prevent red blood cell (RBC) sickling. Discovery of novel HbF inducers is underway and several therapeutics with the potential to increase HbF expression are currently at different stages of preclinical and clinical development. Soluble guanylyl cyclase (sGC) is a heterodimeric heme-containing enzyme whose catalytic activity is regulated by nitric oxide (NO). Binding of NO to heme activates the catalytic domain of sGC, enabling synthesis of the second messenger cyclic guanosine monophosphate (cGMP) from guanosine triphosphate. sGC stimulators are small molecules that synergize with NO to boost signaling via the NO-sGC-cGMP pathway. This signaling pathway is involved in the regulation of many physiologic processes including inflammation, fibrosis, and blood flow. Perhaps less well-known, cGMP-mediated signaling has also been implicated in the regulation of the gene encoding the ƴ-globin subunit of fetal hemoglobin (Modulation of NO signaling by sGC stimulation, therefore, has the therapeutic potential to target the complex pathology of SCD at multiple levels. In this study, we focused on one potential mode of action of sGC stimulation-increasing HbF expression. We characterized the effects of the sGC stimulator olinciguat on ƴ-globin gene expression. Olinciguat is currently being investigated for the treatment of patients with SCD in a Phase II STRONG-SCD study (NCT03285178). The effect of olinciguat treatment on ƴ-globin mRNA levels was studied in the K562 erythroleukemic cell line. For short-term (8 hours) treatment with olinciguat, K562 cells were maintained in a serum-free media. For long-term (4 and 7 days) treatment, cell culture media contained 1% fetal bovine serum. Hydroxyurea was used as a positive control. Levels of ƴ-globin mRNA were expressed relative to mRNA levels of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase. K562 cells were treated for 8 hours with increasing concentrations of olinciguat (0.01, 0.1, 1, and 10 µM). Treatment of K562 cells with 0.1, 1, and 10 µM of olinciguat increased ƴ-globin mRNA levels by 1.43±0.08-, 1.37±0.06-, and 1.47±0.06-fold (mean±SEM), respectively. For comparison, 8 hours of treatment with hydroxyurea (800 µM) increased ƴ-globin mRNA levels by 1.25±0.03-fold. When K562 cells were cultured in the presence of olinciguat for 4 days, significant (P<0.05) induction of ƴ-globin mRNA levels was observed at 1 and 10 µM (1.13±0.03- and 1.55±0.09-fold, respectively). Induction of ƴ-globin mRNA following 4 days of incubation with hydroxyurea (800 µM) was 2.38±0.2-fold. The effects of hydroxyurea and olinciguat on ƴ-globin mRNA levels were compared following 7 days of incubation with the compounds. After 7 days of treatment of K562 cells with 0.1, 1, 3, and 10 µM of olinciguat, ƴ-globin mRNA levels were increased by 1.83±0.19-, 1.66±0.09-, 2.4±0.06-, and 2.9±0.33-fold, respectively. Treatment with 50- and 800-µM hydroxyurea increased levels of ƴ-globin mRNA by 2.33±0.15- and 3.8±0.56-fold, respectively. In conclusion, the sGC stimulator olinciguat increased the expression of mRNA for the ƴ-globin subunit of fetal hemoglobin in the erythroleukemic K562 cell line. This finding indicates that amplifying NO signaling by stimulating sGC may increase HbF expression, thereby preventing pathologic RBC sickling; this extends the potential therapeutic utility of olinciguat in SCD. Finally, the ability of olinciguat to induce HbF in SCD patients will be assessed in the ongoing Phase II STRONG-SCD study (NCT03285178). Disclosures Miyashiro: Ironwood Pharmaceuticals: Employment. Pant:Ironwood Pharmaceuticals: Employment. Tchernychev:Ironwood Pharmaceuticals: Employment, Equity Ownership. Milne:Ironwood Pharmaceutics, Inc: Employment. Currie:Ironwood Pharmaceuticals: Employment. Graul:Ironwood Pharmaceuticals, Inc: Employment. Masferrer:Ironwood Pharmaceuticals, Inc: Employment.

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Negative regulation of globin gene expression during megakaryocytic differentiation of a human erythroleukemic cell line

Molecular and Cellular Biology ◽

10.1128/mcb.11.7.3528-3536.1991 ◽

1991 ◽

Vol 11 (7) ◽

pp. 3528-3536

Author(s):

N L Lumelsky ◽

B G Forget

Keyword(s):

Globin Gene ◽

Fetal Hemoglobin ◽

K562 Cells ◽

Negative Regulation ◽

Myristate Acetate ◽

Globin Mrna ◽

Megakaryocytic Differentiation ◽

Gamma Globin ◽

Erythroleukemic Cell ◽

Erythroleukemic Cell Line

The human erythroleukemic cell line K562 was used as a model for analysis of the mechanisms responsible for alterations in gene expression during differentiation. K562 cells normally synthesize fetal hemoglobin (gamma-globin), but treatment with tumor-promoting phorbol esters (phorbol myristate acetate and tetradecanoyl phorbol acetate) results in the loss of the erythroid phenotype of the cells and causes a shift toward a megakaryocytic phenotype. This shift involves markedly decreased production of fetal hemoglobin and de novo synthesis of a number of proteins specific for megakaryocytes. The results of this work indicate that negative regulation of fetal hemoglobin during megakaryocytic differentiation of K562 cells occurs at the level of down regulation of gamma-globin mRNA accumulation. This effect consists of at least two components: reduction in the rate of transcription of the gamma-globin gene and decrease in stability of the normally very stable gamma-globin mRNA. We have developed two assay systems that permit investigation of the transcriptional and posttranscriptional effects of phorbol myristate acetate independently from each other. These assay systems make use of a heterologous reporter gene for the transcriptional analysis and a marked gamma-globin gene for the analysis of mRNA stability. The DNA sequences located in the 3' flanking region of the A gamma-globin gene were found to be responsible for the decrease in transcription rate.

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Sequences Downstream of the Erythroid Promoter Promote High-Level Expression of the Human α-Spectrin Gene by Providing Boundary Activity and Positive Regulatory Elements.

Blood ◽

10.1182/blood.v104.11.1572.1572 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1572-1572

Author(s):

Patrick G. Gallagher ◽

Douglas G. Nilson ◽

Jolinta Lin ◽

David M. Bodine

Keyword(s):

Regulatory Element ◽

Globin Gene ◽

K562 Cells ◽

Regulatory Elements ◽

Globin Mrna ◽

Intron 1 ◽

Exon 1 ◽

Globin Promoter ◽

High Level ◽

Positive Regulatory Element

Abstract Characterization of the regulatory elements that control α-spectrin (ASp) gene expression is important for understanding the pathogenesis of ASp-linked hemolytic anemia. Our previous studies demonstrated that the ASp promoter directs low levels of expression, and, addition of a downstream region of noncoding exon 1 and intron 1 containing 2 GATA-1 sites conferred a 10-fold increase in activity in transient transfection assays. Transgenic (TG) mouse lines with the ASp promoter, the Asp promoter-exon 1-intron 1, or ASp promoter-exon 1-intron 1 with mutations of both splice sites linked to the human Aγ-globin gene as reporter were created. In reticultocytes, no expression was detected in any of the 8 lines transmitting the ASp promoter-Aγ-globin transgene. TG mice with the ASp promoter-exon1-intron 1 demonstrated significant levels of Aγ-globin gene expression in reticulocytes, with levels of Aγ-globin mRNA of ~0.4% of mouse α-globin mRNA/transgene copy #. This expression was nearly position independent, as 22/24 lines expressed the transgene. Using a FACS-based assay, γ-globin protein was present in 100% of erythrocytes. Expression levels comparable to the Asp promoter-exon 1-intron 1 TG were detected in 9/9 lines with the mutated splice sites, indicating splicing did not contribute to changes in expression. DNaseI hypersensitive site (HS) mapping identified a broad, erythroid-specific HS across exon 1 and intron 1. The presence of a DNaseI HS site suggested the presence of a positive regulatory element or a chromatin modification such as a boundary element. Analysis of a positive regulatory element in vivo was sought by stably transfecting the following luciferase (luc) plasmids into K562 cells: ASp promoter, ASp promoter-exon 1-intron 1, ASp promoter-exon 1, ASp promoter-intron 1, and ASp promoter-exon 1-intron 1 with both GATA-1 sites mutated. Clones with copy # ≤5 were analyzed; ≥9 independent clones/line were analyzed. Normalized luc activity of the ASp promoter-exon 1-intron 1 was significantly higher than the ASp promoter in stably transfected cells, 86±15 v 28±3 (p<0.001). Mutation of both GATA-1 sites in the exon 1-intron 1 plasmid reduced activity to background. Normalized luc activity from the promoter-exon was 46±6; from the promoter-intron 101±31, suggesting the intron functions as a positive regulatory element. A barrier assay was performed by flanking a β-globin promoter-EGFP gene using wild type (WT) exon 1, exon 1 with the GATA site abolished, or WT intron 1, and stably transfecting the plasmids into K562 cells. The WT exon 1 and mutant exon 1 expressed GFP in 10/12 and 7/8 lines, respectively, indicating a barrier function for exon 1 independent of GATA-1 activity. Only 1/9 lines expressed EGFP when the cassette was flanked by the ASp intron and 0/8 expressed EGFP when there were no sequences flanking the β-globin promoter. TG mouse lines with the Asp promoter-exon 1 or the Asp promoter-intron 1 linked to the Aγ-globin gene were created. 1/5 TG lines with ASp promoter-exon 1 expressed at low levels and 3/7 TG lines with ASp promoter-intron 1 expressed at levels comparable to the ASp promoter-exon 1-intron 1. These results demonstrate that 2 elements downstream of the ASp promoter are required for high-level, erythroid-specific expression. Exon 1 has barrier activity and intron 1 functions as a positive regulatory element. This is an excellent candidate region for mutations associated with ASp-linked inherited hemolytic anemia.

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Role of HDAC9 in γ-Globin Gene Regulation.

Blood ◽

10.1182/blood.v114.22.4074.4074 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4074-4074

Author(s):

Shalini A Muralidhar ◽

Betty Pace

Keyword(s):

Gene Regulation ◽

Histone Deacetylase ◽

Globin Gene ◽

K562 Cells ◽

Fold Increase ◽

Mrna Levels ◽

Globin Mrna ◽

Data Support ◽

Dose Dependent

Abstract Abstract 4074 Poster Board III-1009 Strategies to induce fetal hemoglobin (HbF) synthesis for the treatment of β-hemoglobinopathies will likely involve chromatin modification in the presence of histone deacetylase (HDAC)/protein complexes to promote γ-globin gene activation. The role of various HDACs in globin transcription is not very well understood therefore, the objective of our study was to identify HDACs involved in γ-gene regulation. Screening studies were performed in K562 erythroleukemia cells to determine transcription levels for HDAC genes in the absence or presence of HbF induction. Treatment with butyrate (2mM), trichostatin A (0.5μM) and the non-HDAC inhibitor control hemin (50μM) significantly reduced mRNA levels of HDAC9 and its splice variant HDRP (histone deacetylase related protein) lending indirect evidence for their involvement in drug-mediated γ-globin regulation. Subsequent studies were performed to delineate whether HDAC9 can directly modulate γ-globin gene transcription since a role for HDAC9 in hematopoiesis has been previously demonstrated. Furthermore, consensus binding sites for GATA-1 are present in the HDAC9 gene proximal promoter. Initially, we performed siRNA knockdown using Oligofectamine (Invitrogen) in K562 cells and measured γ-globin levels by real time quantitative PCR analysis. Treatment with siHDAC9 (Dharmacon) produced dose-dependent γ-globin gene silencing over an 80-320nM range; control siRNA molecules had no effect. When HDAC9 was over-expressed in K562 cells using pTarT-HDAC9 at 10-50μg concentrations, a dose dependent 2.5-fold increase in γ-globin mRNA (p<0.05) was produced. These data support a positive regulatory role for HDAC9 in γ-gene regulation. To confirm the physiological relevance of HDAC9, similar studies were performed in human primary erythroid progenitors using a two-phase liquid culture system. The 320nM siHDAC9 concentration produced 48% and 60% decrease in γ-globin mRNA at day 11 (early progenitors) and day 28 (late progenitors) respectively. Enforced HDAC9 expression increased γ-globin by 2.5-fold (p<0.05) at both days. ELISA was performed to quantify HbF protein and cytospin preps were made to visualized hemoglobin by fluorescent staining with anti-γ-FITC antibody. HDAC9 enforced expression for 72 hrs produced a 7-fold increase in HbF and γ-FITC positive cells increased >50%. Collectively these data support a positive role for HDAC9 in γ-globin regulation. Chromatin immunoprecipitation assays will be completed to elucidate the contribution of HDAC9 in maintaining an active chromatin domain in the γ-globin promoter. We will also define interactions of GATA-1 in the HDAC9 gene to coordinate expression during erythroid maturation. Disclosures: No relevant conflicts of interest to declare.

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