Role of KLF4 and KLF12 in γ-Globin Gene Regulation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4075-4075
Author(s):  
Inderdeep S Kalra ◽  
Wei Li ◽  
Shalini Muralidhar ◽  
Betty Pace
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Conflicts Of Interest ◽  
Protein Complexes ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
K562 Cells ◽  
Mrna Levels ◽  
Gene Promoters ◽  
Globin Gene Regulation

Abstract Abstract 4075 Poster Board III-1010 Kruppel-like factors (KLFs) are a family of Cys2His2 zinc-finger DNA binding proteins that regulate gene expression through CACCC/GC/GT box binding in gene promoters. The CACCC element is critical for the developmental regulation of the human γ-globin and β-globin genes and studies are being done to ferret out various factors that bind this region and modulate gene activity. We recently identified two Kruppel-like factors, KLF4 and KLF12 whose expression levels decreased based on microarray-based gene profiling, concomitantly with decreased γ-globin expression during erythroid maturation. Decreased expression of both factors was further confirmed using quantitative PCR (qPCR) analysis. KLF4 and KLF12 mRNA levels decreased 56-fold and 16-fold respectively by day 28 compared to levels in day 7 erythroid progenitors. We next determined if KLF4 and KLF12 bind the γ-globin CACC box by electrophoretic mobility shift assay (EMSA) using nuclear proteins extracted from K562 cells and a [γ-32P] labeled γ-CACC probe located between -155 to -132 relative to the γ-globin gene cap site. Three DNA-protein complexes were observed. The specificity of these interactions was confirmed by competition reactions in which preincubation with excess unlabelled γ-CACC oligonucleotide effectively abolished the formation of all DNA/protein complexes; addition of nonspecific oligonucleotide had no effect on binding activity. Addition of polyclonal KLF4 or KLF12 antibodies to the EMSA reaction resulted in a marked decrease in intensity of all DNA-protein complexes suggesting both KLF4 and KLF12 are present. Additional studies were performed to determine the effect of the known fetal hemoglobin inducer hemin on KLF gene expression in K562 cells. Hemin stimulated γ-globin transcription while increasing KLF4 and KLF12 66-fold and 4-fold respectively (p<0.05). Hemin treatment in KU812 erythroleukemia cells which actively transcribe both γ- and β-globin, also produced a 10-fold increase (p<0.05) in KLF4; KLF12 levels were not changed. Our preliminary data suggest these KLFs might play a role in γ-globin regulation. siRNA mediated gene silencing studies are underway to determine if KLF4 and/or KLF12 play a direct role in γ-globin gene regulation. This mechanism could provide important molecular targets for fetal hemoglobin reactivation. This will be highly significant towards developing therapeutic strategies for hemoglobinopathies like sickle cell anemia and β-thalassemia. Disclosures: No relevant conflicts of interest to declare.

Transcriptional Regulation of γ-Globin Gene Expression by KLF4

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 645-645
Author(s):  
Inderdeep S Kalra ◽  
Md. M Alam ◽  
Betty S Pace
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Sodium Butyrate ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
K562 Cells ◽  
Mrna Levels ◽  
Klf4 Expression ◽  
Globin Gene Regulation ◽  
Globin Gene Expression

Abstract Abstract 645 Kruppel-like factors (KLFs) are a family of Cys2His2 zinc-finger DNA binding proteins that regulate gene expression through CACCC/GC/GT box binding in various gene promoters. The CACCC element is also critical for developmental regulation of the human γ-globin and β-globin genes; therefore studies to identify transcription factors that bind the CACCC element to alter gene expression are desirable. By microarray-based gene profiling, we identified two Kruppel-like factors, KLF4 and KLF12 whose expression levels decreased simultaneously with γ-globin silencing during in vitro erythroid maturation. Subsequent reverse transcription quantitative PCR (RT-qPCR) analysis confirmed KLF4 and KLF12 mRNA levels decreased 56-fold and 16-fold respectively in erythroid progenitors from day 7 to day 28 with over 90% γ-globin gene silencing. The effects of known fetal hemoglobin inducers hemin (50μM) and sodium butyrate (2mM) on KLF factor expression was tested in K562 cells. Hemin and sodium butyrate increased KLF4 3-fold (p<0.05) and 13-fold (p<0.01) respectively while KLF12 was only induced by butyrate. Likewise, hemin treatment of KU812 leukemia cells, which actively express γ-globin and β-globin, produced a 7-fold increase in KLF4 (p<0.05) while KLF12 levels were not changed suggesting KLF4 may be directly involved in γ-globin gene regulation. To characterize its role further siRNA-mediated loss of function studies were performed in K562 cells. A 60% knockdown of KLF4 expression produced 40% attenuation of γ-globin transcription (p<0.05). To confirm this effect, rescue experiments were performed as follows: K562 cells were treated with 100nM siKLF4 alone or in combination with the pMT3-KLF4 expression vector (10 and 20μg) for 48 hrs. The 40% knockdown of γ-globin expression produced by siKLF4 was rescued to baseline levels after enforced pMT3-KLF4 expression (p<0.05). To establish whether KLF4 directly stimulates γ-globin promoter activity, we performed co-transfection of pMT3-KLF4 and the Gγ-promoter (-1500 to +36) cloned into the pGL4.17 Luc2/neo vector; a dose-dependent increase in luciferase activity (2- to 5-fold; p<0.001) was observed. Furthermore, enforced expression of pMT3-KLF4 augmented endogenous γ-globin expression 2-fold (p<0.01). Collectively, these studies suggest that KLF4 acts as a trans-activator of γ-globin gene transcription. To address the physiological relevance of these findings, studies were extended to human primary erythroid cells grown in a two-phase liquid culture system. At day 11 when γ-globin gene expression was maximal, siKLF4 treatment produced a 60% decrease in γ/β-globin mRNA levels (p<0.001). By contrast, enforced pMT3-KLF4 expression enhanced γ/β-globin 1.5-fold at day 11 and day 28 (after γ-globin silencing); HbF levels were induced 1.5-fold (p<0.05) which was demonstrated by enzyme-linked immunosorbent assay. To gain insights into the molecular mechanism of KLF4-mediated γ-globin regulation, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP) were completed. Since CREB binding protein (CBP) is known to function as a co-activator for KLF1, 4 and 13, we also tested its role in γ-globin gene regulation. EMSA performed with K562 nuclear extract and a [γ-32P] labeled γ-CACC probe (-155 to -132 relative to the γ-globin cap site) produced three DNA-protein complexes; the addition of KLF4 or CBP antibody resulted in a marked decrease in intensity of all complexes suggesting these factors bind the γ-CACC element. ChIP assay demonstrated 10-fold and 20-fold chromatin enrichment with KLF4 and CBP antibody respectively (p<0.001) confirming in vivo binding at the γ-CACC region. Lastly, co-immunoprecipitation established protein-protein interaction between KLF4 and CBP in K562 cells. Future studies will investigate the role of CBP in KLF4-mediated γ-globin regulation which will provide molecular targets for fetal hemoglobin induction and treatment of sickle cell anemia and β-thalassemia. Disclosures: No relevant conflicts of interest to declare.

Characterization of Histone Deacetyalses Involved in γ-Globin Gene Regulation.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1869-1869
Author(s):  
Shalini A Muralidhar ◽  
Sadeieh Nimer ◽  
Betty Pace
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Promoter Activity ◽  
Globin Gene ◽  
Indirect Evidence ◽  
K562 Cells ◽  
Luciferase Reporter ◽  
Globin Promoter ◽  
Globin Gene Regulation ◽  
Globin Gene Expression

Abstract Fetal hemoglobin (HbF; α2γ2) ameliorates vaso-occlusive symptoms in individuals with sickle cell disease (SCD) because of its ability to inhibit hemoglobin S polymerization. One mechanism for γ-globin reactivation likely involves chromatin modification and the release of repressor complexes in which histone deacetylases (HDACs) may be present. The objective of this study was to identify HDACs involved in γ-globin gene regulation. Experiments were performed in K562 cells to determine the ability of NaB (2mM), TSA (0.5μm) and the non-HDAC-inhibitor γ-globin activator hemin (50μM), to alter transcription levels of the HDAC genes during concomitant HbF induction. Gene expression was measured by reverse transcription (RT) of mRNA followed by quantitative PCR (qPCR) analysis using gene-specific primers. Treatment of K562 cells with TSA and NaB reduced transcription levels of both HDAC9 and HDRP (histone deacetylase related protein, a splice variant of HDAC9) from 20–80% as did the control agent hemin (p&lt;0.05). By contrast, expression of HDAC7 and HDAC10 was enhanced in the presence of both HDAC inhibitors. The altered HDAC gene expression levels provided indirect evidence for a possible role in mechanisms of γ-globin response to drug inducers. Subsequent experiments were performed to delineate whether HDAC9 and HDRP are directly involved in γ-globin regulation. We performed siRNA knockdown of HDAC9 and HDRP in K562 cells to determine the effect on expression of endogenous γ-globin. siRNA oligonucleotides were transfected using Oligofectamine (Invitrogen) for 48 hrs and expression of targeted genes were quantified by RT-qPCR. siHDAC9 and siHDRP (Dharmacon) treatment resulted in dose-dependent γ-globin silencing and transactivation respectively at 80–320nM. Experiments were then performed with 160nM of siHDAC9 or siHDRP in the K562 cell lines which were stably transfected with a luciferase reporter (pGL4.17-Luc2-neo, Promega) under the control of Gγ-globin promoter (−1500 to +36) and the pGL4.17-Luc2-neo empty vector. We likewise observed a 30% decrease in luciferase activity with siHDAC9 and a 40% increase with siHDRP suggesting that HDAC9 and its variant are directly involved in regulation of γ-promoter activity. In summary, the effects on endogenous γ globin levels and Gγ globin promoter activity through HDAC9/HDRPspecific knockdown by siRNA experiments suggest that HDAC9 molecules play a role in regulating γ-globin gene expression. We conclude that HDAC9 and HDRP have opposite regulatory effect on γ-globin gene expression and may act by a feedback mechanism.

Regulation of Globin Gene Expression and Erythroid Differentiation by Sp/KLF Factors.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4241-4241
Author(s):  
Jie Hong ◽  
George Stamatoyannopoulos ◽  
Chao-Zhong Song
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Expression System ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Regulatory Elements ◽  
Mrna Levels ◽  
Gene Promoters ◽  
Gamma Globin ◽  
Globin Gene Expression

Abstract Sp/Krüppel-like factor (KLF) family of proteins are characterized by the presence of three highly homologous Cys2His2 type zinc-fingers near the C-terminus that bind GC/CACCC boxes, which are one of the most common regulatory elements found in promoters of many cellular and viral genes. Currently, more than 20 members have been identified in the family. This family of factors plays important roles in cell growth, differentiation, development and homeostasis by regulating the expression of their target genes. The GC and GT/CACCC boxes in the globin gene promoters and the beta globin locus control region play an important role in the tissue- and developmental stage- specific expression of globin genes. We have carried out extensive studies to identify the KLF factors that regulate gamma globin expression and erythroid differentiation. Gene expression analysis revealed that most of the Sp/KLF factors are expressed, albeit at variable levels, in human fetal liver and adult blood cells. To determine the role of the Sp/KLF factors in gamma globin expression and erythroid differentiation, functional studies using systematic RNAi to knockdown selected Sp/KLF factors were performed. We used a lentiviral mediated siRNA expression system for specific silencing selected Sp/KLF factors. Effective knockdown of Sp/KLF factors was achieved as judged by a 70–90% decrease in their mRNA levels in the cells. Analyses of globin gene expression showed that the knockdown of some Sp/KLF factors resulted in changes in globin gene expression in K562 cells. We also observed that knockdown of specific Sp/KLF factors resulted in erythroid differentiation of K562 cells. These results suggest that specific Sp/KLF factors may play a role in regulation of globin gene expression and erythroid differentiation.

The Role of HDAC10 in γ-Globin Gene Regulation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5412-5412
Author(s):  
Sadeieh Nimer ◽  
Shalini A Muralidhar ◽  
Betty Pace
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Globin Gene ◽  
K562 Cells ◽  
Fold Increase ◽  
Luciferase Reporter ◽  
Sirna Treatment ◽  
Globin Gene Regulation ◽  
Globin Gene Expression

Abstract HDACs (histone deacetylases) are enzymes that cause chromatin modifications through deacetylation of histones and the recruitment of repressor complexes to mediate gene silencing. To explore this mechanism further, experiments were performed to determine if HDACs are involved in drug-mediated γ-globin gene induction. When human K562 cells were treated with the γ-globin inducer sodium butyrate, we observed 1.8-fold increase in HDAC10 transcription compared to untreated cells. This provided indirect evidence that HDAC10 may be involved in γ-globin gene regulation. To further understand the mechanism, enforced expression experiments using 10–50 μg of the expression plasmid pCMX-HDAC10 and the pCMX empty plasmid were performed by transient transfection of K562 cells via electroporation. Total RNA was isolated and subjected to reverse transcription followed by real time quantitative PCR using gene-specific primers to measure endogenous γ-globin gene levels. Enforced expression of HDAC10 resulted in dose-dependent silencing of γ-globin gene expression. To gain further evidence for a role of HDAC10 in regulating γ-globin gene expression, we performed siRNA knockdowns using SMARTpool-siHDAC10 (Dharmacon) at four concentrations (80nM–320nM) using Oligofectamine (Invitrogen). The γ-globin gene levels were not changed significantly by the siRNA treatment. We next performed enforced expression of HDAC10 in a K562 stable line established using the pGL4.17-Luc2-neo as a base vector in which the expression of luciferase reporter was driven by the Gγ-globin promoter (−1500 to +36). Control stable lines were also established with the empty vector. Preliminary studies of HDAC10 siRNA treatment of the KGγ-CRE stable lines produced a 1.2 fold increase in γ-globin gene activity. These results suggest that HDAC10 may play a role in γ-globin gene regulation during the adult development. Understanding novel mechanisms of γ-gene regulation will expand capabilities to develop therapeutics for sickle cell patients.

Structural and Functional Characterization of Csda Protein Complexes Involved In Modulation of Fetal Globin Gene Expression

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2071-2071 ◽  
Author(s):  
Sara Gaudino ◽  
Raffaella Petruzzelli ◽  
Giovanni Amendola ◽  
Raffaele Sessa ◽  
Stella Puzone ◽  
...  
Keyword(s):  
Gene Expression ◽  
Promoter Region ◽  
Protein Complexes ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
K562 Cells ◽  
Expression Levels ◽  
Isoform B ◽  
Globin Gene Expression ◽  
Chip Analysis

Abstract Abstract 2071 Impaired switching from fetal hemoglobin (HbF) to adult globin gene expression leads to hereditary persistence of fetal hemoglobin (HPFH) in adult life. This is of prime interest because elevated HbF levels ameliorate beta-thalassemia and sickle cell anemia. Fetal hemoglobin levels are regulated by complex mechanisms involving factors linked or not to the beta-globin gene locus. Recently, we reported an inverse relationship between Ggamma-globin gene (HBG2) and Cold Shock Domain Protein A (CSDA) expression levels. Based on mRNA differential display analysis, RNA interference and over-expression studies in K562 and primary erythroid cells we postulated that CSDA could contribute to regulate HBG2 expression. The putative mechanism by which CSDA modulates HBG2 expression was investigated in K562 cells by gene reporter assays on wild-type and mutant constructs of the HBG2 promoter region suspected to bind CSDA, providing experimental evidence that CSDA acts as repressor of HBG2 expression. Furthermore, chromatin immunoprecipitation (ChIP) analysis on K562 cells showed that CSDA interacts in vivo with this promoter region. In this way we were able to demonstrate that CSDA modulates HBG2 expression at least in part at the transcriptional level (Petruzzelli R et al, Br J Haematol 2010). The CSDA gene is located at position 12p13.1 and comprises 10 exons. The C-terminus (exons 6–9) is involved in protein-protein interactions. Alternative splicing of exon 6 results in two main isoforms, namely CSDA isoform a and isoform b, which show different C-terminal domains, potentially able to take part to specific protein complexes. We found that expression levels of CSDA isoform a were reduced in HPFH patients respect to isoform b. These findings suggested that isoform a could be much more involved in repression of HBG2 expression compared to isoform b. To identify putative CSDA interactors, we over-expressed these two FLAG-tagged CSDA isoforms in K562 cells. Western-blot analysis on proteins immunoprecipitated with a FLAG antibody revealed the presence of NF-kB p50 and p65 subunits and histone deacethylase 2 (HDAC2) only in samples co-immunoprecipitated with CSDA isoform a, but not with isoform b (Fig. 1). By ChIP assays with antibodies against p65, p50 and HDAC2, we demonstrated that both the NF-kB p50-p65 heterodimer and HDAC2 interact with the –200 bp region of the HBG2 promoter containing the CSDA binding site (Fig. 2). To examine the role of NF-kB and histone deacetylases on the transcriptional repression of HBG2 expression, we treated K562 cells with the proteasome inhibitor bortezomib which blocks the nuclear traslocation and transcriptional activity of the NF-kB p65-p50 complex or with the histone deacetylase inhibitor trichostatin A (TSA). Quantitative analysis by Real Time PCR showed that HBG2 expression increased following either bortezomib or TSA treatments. Furthermore, by ChIP analysis we were able to demonstrate that knock-down of CSDA abolished these interactions. To investigate if treatment with bortezomib or TSA affects the histone acetylation levels at the -200 bp region of the HBG2 promoter, we performed ChIP assays in K562 cells using an anti-acetyl-H3 antibody. Results indicated that both these drugs induces a considerable increase in H3 acetylation levels at the -200 bp region of the HBG2 promoter (Fig. 3). Taken altogether these data indicate that NF-kB and HDAC2 interact with CSDA to form a multiprotein complex which take part to the regulation of HBG2 expression by modulating local chromatin conformation. Furthermore, our study contributes to better define the role played by CSDA in fetal globin gene expression and shed novel light on the molecular mechanisms involved in globin gene switching. Disclosures: No relevant conflicts of interest to declare.

Differences in Fetal Hemoglobin Induction in Sickle Cell Disease and beta-Thalassemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 555-555 ◽  
Author(s):  
Hassana Fathallah ◽  
Ali Taher ◽  
Ali Bazarbachi ◽  
George F. Atweh
Keyword(s):  
Gene Expression ◽  
Sickle Cell Disease ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Mrna Levels ◽  
Globin Genes ◽  
Thalassemia Intermedia ◽  
Cell Disease ◽  
Globin Mrna ◽  
Globin Gene Expression

Abstract A number of therapeutic agents including hydroxyurea, butyrate and decitabine have shown considerable promise in the treatment of sickle cell disease (SCD). However, the same agents have shown less clinical activity in β-thalassemia. As a first step towards understanding the molecular basis of the different clinical responses to these agents, we have studied the mechanisms of induction of fetal hemoglobin (HbF) by butyrate in BFU-E derived cells from 5 patients with SCD and 9 patients with β-thalassemia intermedia. Exposure to butyrate resulted in a dose-dependent augmentation of γ-globin mRNA levels in erythroid cells from patients with SCD. In contrast, induction of γ-globin expression in erythroid cells from patients with β-thalassemia intermedia was only seen at a high concentration of butyrate. The increase in γ-globin mRNA levels in patients with SCD and β-thalassemia intermedia was associated with opening of the DNA structure as manifested by decreased DNA methylation at the γ-globin promoters. Interestingly, butyrate exposure had markedly different effects on the expression of the β- and α-globin genes in the two categories of patients. Butyrate decreased the level of β-globin mRNA in 4 out of 5 patients with SCD (P = 0.04), while in β-thalassemia the levels of β-globin mRNA did not change in 7 patients and decreased in 2 patients after butyrate exposure (P = 0.12). Thus in patients with SCD, the effects of the induction of the γ-globin gene on the γ/(β+γ) mRNA ratios were further enhanced by the butyrate-mediated decreased expression of the β-globin gene. As a result, γ/(β+γ) mRNA ratios increased in all patients with SCD, with a mean increase of 31% (P = 0.002). In contrast, butyrate increased γ/(β+γ) mRNA ratios only in 4 out of 9 patients with β-thalassemia, with a more modest mean increase of 12% (P = 0.004). Interestingly, the decreased β-globin expression in patients with SCD was associated with closing of the DNA configuration as manifested by hypermethylation of DNA at the promoter of the β-globin gene while methylation of the same promoter did not change following butyrate exposure in patients with β-thalassemia intermedia. More surprisingly, the expression of the α-globin genes increased following butyrate exposure in 4 out of 9 patients with β-thalassemia, while the levels of α-globin mRNA decreased in 4 out of 5 patients with SCD. As a result, the favorable effects of the butyrate-induced increase in γ-globin gene expression on the α: non-α mRNA imbalance in patients with β-thalassemia intermedia were partly neutralized by the corresponding increase in α-globin gene expression. These differences may explain, at least in part, the more favorable effects of inducers of HbF in SCD than in β-thalassemia. Further studies are necessary to fully understand the molecular bases of the different responses to agents that induce HbF in patients with these disorders.

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

Blood ◽  
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.

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.

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

Blood ◽  
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.

Sign in / Sign up

Export Citation Format

Share Document