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.

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<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.

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.

Regulation of the Human α-Globin Genes by GKLF.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1867-1867
Author(s):  
Paolo Moi ◽  
Giuseppina Maria Marini ◽  
Loredana Porcu ◽  
Isadora Asunis ◽  
Maria Giuseppina Loi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Globin Gene ◽  
Point Mutations ◽  
K562 Cells ◽  
Luciferase Reporter ◽  
Globin Genes ◽  
Small Interfering Rnas ◽  
Globin Promoter ◽  
Globin Gene Expression

Abstract EKLF and related Krueppel-like factors (KLFs) are variably implicated in the regulation of the β- and β-like globin genes. Prompted by the observation that four KLF sites are distributed in the human α-globin promoter, we investigated if any of the β-globin cluster regulating KLFs could also act to modulate the expression of the α-globin genes. We found that, among the globin regulating KLFs (EKLF, LKLF, BKLF, GKLF, KLF6, FKLF and FKLF2), only GKLF and BKLF bound specifically to three out of four KLF sites. In K562 cells, over-expressed GKLF transactivated at high levels a α-globin-luciferase reporter and its action was impaired by point mutations of the KLF sites that disrupted GKLFDNA binding. In K562 cells stably transfected with a Tet-off regulated GKLF expression cassette, GKLF induction stimulated the expression of the endogenous α-globin genes. In a complementary assay in K562 cells, knocking down GKLF expression with small interfering RNAs caused a parallel decrease in the transcription of the α-globin genes. All experiments combined support a main regulatory role of GKLF in the control of α-globin gene expression.

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.

Role of HDAC9 in γ-Globin Gene Regulation.

Blood ◽  
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.

scholarly journals Role of ZBP-89 in human globin gene regulation and erythroid differentiation

Blood ◽  
2011 ◽  
Vol 118 (13) ◽  
pp. 3684-3693 ◽  
Author(s):  
Andrew J. Woo ◽  
Jonghwan Kim ◽  
Jian Xu ◽  
Hui Huang ◽  
Alan B. Cantor
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Globin Gene ◽  
Erythroid Differentiation ◽  
Regulatory Elements ◽  
Specific Gene ◽  
Specific Gene Expression ◽  
Erythroid Maturation ◽  
Globin Gene Regulation

Abstract The molecular mechanisms underlying erythroid-specific gene regulation remain incompletely understood. Closely spaced binding sites for GATA, NF-E2/maf, and CACCC interacting transcription factors play functionally important roles in globin and other erythroid-specific gene expression. We and others recently identified the CACCC-binding transcription factor ZBP-89 as a novel GATA-1 and NF-E2/mafK interacting partner. Here, we examined the role of ZBP-89 in human globin gene regulation and erythroid maturation using a primary CD34+ cell ex vivo differentiation system. We show that ZBP-89 protein levels rise dramatically during human erythroid differentiation and that ZBP-89 occupies key cis-regulatory elements within the globin and other erythroid gene loci. ZBP-89 binding correlates strongly with RNA Pol II occupancy, active histone marks, and high-level gene expression. ZBP-89 physically associates with the histone acetyltransferases p300 and Gcn5/Trrap, and occupies common sites with Gcn5 within the human globin loci. Lentiviral short hairpin RNAs knockdown of ZBP-89 results in reduced Gcn5 occupancy, decreased acetylated histone 3 levels, lower globin and erythroid-specific gene expression, and impaired erythroid maturation. Addition of the histone deacetylase inhibitor valproic acid partially reverses the reduced globin gene expression. These findings reveal an activating role for ZBP-89 in human globin gene regulation and erythroid differentiation.

scholarly journals NRF2 Mediates Epigenetic Changes in DNA and Chromatin Structure to Regulate γ-Globin Gene Expression in a Human βYAC Transgenic Mouse Model

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1053-1053
Author(s):  
Xingguo Zhu ◽  
Alexander H. Ward ◽  
Caixia Xi ◽  
Betty S. Pace
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Gene Regulation ◽  
Globin Gene ◽  
Histone H3 ◽  
Dimethyl Fumarate ◽  
Nrf2 Knockout ◽  
Globin Gene Regulation ◽  
Globin Gene Expression

Abstract NRF2 is the master regulator for the cellular anti-oxidative stress response and previously shown to activate γ-globin gene expression in human erythroid progenitor cells. The goal of this study was to expand on these findings by exploring the in vivo function of NRF2 using the human β-globin locus YAC transgenic (βYAC) mouse carrying the entire 248kb human β-globin locus (HBB). We observed that NRF2 activation by chronic dimethyl fumarate treatment of βYAC mice, induced human γ-globin gene expression, but had no effect on the adult β-globin gene. Subsequently in a novel βYAC/NRF2 knockout mouse model established in our laboratory, we demonstrated that NRF2 loss increased mouse erythroid CD71 levels while reducing human γ-globin gene expression during erythropoiesis in mouse embryonic E13.5 and E18.5 day fetal livers and peripheral blood. Furthermore, the ability of dimethyl fumarate to induce γ-globin gene expression was abolished after NRF2 loss. By western blot analysis of nuclear protein, we confirmed that part of the mechanism of globin gene regulation by NRF2 loss involves a decline of global histone H3 lysine 4 trimethylation levels, without changing histone acetylation. Interestingly, NRF2 loss decreased the protein levels of the DNA methylcytosine dioxygenases including TET1, TET2 and TET3. Analysis of DNA methylation/hydroxymethylation levels by DNA dot-blot assay of mouse E13.5 fetal livers isolated from βYAC/NRF2 knockout mice, showed inhibition of genome wide DNA hydroxymethylation, while DNA methylation was not affected. In addition, DNA immunoprecipitation confirmed decreased hydroxymethylation in the HBB locus control region (LCR) enhancer and γ-globin gene region. These data suggest an essential role of NRF2 in modifying chromatin structure and assembling transcription complexes to regulate γ-globin gene expression. ChIP assay to assess in vivo DNA-protein interactions showed decreased associations of histone H3 lysine 4 monomethylation and trimethylation, TATA-binding protein and RNA polymerase II to the LCR and γ-globin promoter after NRF2 loss. Final studies were conducted to evaluate long-range chromatin interactions between the LCR and individual globin genes by chromosome conformation capture assay. We observed decreased interactions between the LCR and γ-globin gene promoter region after NRF2 loss while interactions in the adult β-globin was not affected suggesting NRF2 preferentially mediates γ-globin gene regulation. In conclusion, our data suggest that NRF2 alters γ-globin expression through epigenetic DNA/histone modifications in addition to direct DNA binding. Therefore, activation of NRF2 expression using small chemical compounds is an innovative strategy to induce γ-globin gene transcription for the treatment of β-hemoglobinopathies. This work was supported by funding from the National Heart, Lung, and Blood Institute to XZ through the Hemoglobinopathy Translational Research Skills Core component of U01 grant HL117684 and R01 grant HL069234 to BSP. Disclosures No relevant conflicts of interest to declare.

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.

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