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.

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.

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.

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

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1078-1078 ◽  
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.

Exploiting the NRF2/Antioxidant Response Element Signaling Pathway to Induce γ-Globin Gene Expression and HbF Production

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2074-2074
Author(s):  
Elizabeth R. Macari ◽  
Rachel J. West ◽  
Christopher H. Lowrey
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Globin Gene ◽  
K562 Cells ◽  
Antioxidant Response ◽  
Mrna Levels ◽  
Antioxidant Response Element ◽  
Globin Mrna ◽  
Nrf2 Pathway ◽  
Globin Gene Expression

Abstract Abstract 2074 Induction of fetal hemoglobin (HbF) has proven therapeutic potential to treat sickle cell disease and β-thalassemia. However, agents known to be effective in humans, including hydroxyurea, DNMT inhibitors and butyrate derivatives are not ideal due to suppression of hematopoiesis and the possibility of long-term side effects. Two natural compounds, angelicin (Lampronti, et al, Eur J Hematol 2003) and resveratrol (Rodrigue, et al, Hematology 2001) have been found to induce γ-globin gene expression in K562 cells. These agents may be important lead compounds as they are generally non-cytotoxic and are being evaluated in ongoing human trials as cancer chemopreventative agents where these and several other agents are thought to work by activating antioxidant response pathway genes. The products of these genes are enzymes involved in antioxidant and detoxification activities and include NADPH-quinone oxireductase 1 (NQO1), glutamate-cysteine ligase (GCL) and glutathione S-transferase (GST). The activation of these genes is mediated by the transcription factor, NF-E2 related factor 2 (NRF2), which binds to a specific antioxidant response element (ARE) sequence (TGACnnnGCA) in target gene promoters. The proximal γ-globin promoter contains an ARE sequence between the two CAAT boxes, suggesting that it too may be activated by NRF2. This led us to hypothesize that drugs that activate the ARE/NRF2 pathway may provide a less toxic approach to HbF induction. To test this hypothesis, we treated K562 cells with various NRF2 pathway activators. We initially tested six compounds that are known to induce antioxidant response genes at doses that did not inhibit proliferation and found the most pronounced γ-globin induction with tert-butylhydroquinone (tBHQ) (2.8 fold). We next tested tBHQ in two different primary cell culture models: erythroid precursors isolated from normal human bone marrow and in vitro erythroid differentiation of primary human CD34+ cells. In both of these models, tBHQ treatment increased γ-globin steady state mRNA levels and induced expression of NRF2 target genes. Treatment of differentiating erythroid cells caused a dose dependent increase in γ/(γ+β) mRNA and % HbF. HPLC analysis revealed the highest non-toxic concentration of tBHQ, 5μM, produced 10% HbF while the positive control, 0.5μM of 5-Azacytidine, resulted in 12% HbF compared to the untreated control at 3%. Since similar mRNA induction was seen in K562 cells, we used these cells to characterize the mechanism of tBHQ induced γ-globin expression. First, we used siRNA to decrease NRF2 mRNA levels. This resulted in a greater than 75% knockdown of NRF2 mRNA and protein and reduced tBHQ induction of γ-globin and NQO1 gene expression by 90% and 75%, respectively, compared to samples transfected with scrambled siRNA (p < 0.01). Subsequent experiments showed that tBHQ treatment resulted in NRF2 translocation to the nucleus and binding to the NQO1 and γ-globin promoters but not at negative control sites. In addition, inducing NRF2 translocation by transiently suppressing levels of its inhibitor, KEAP1, did not result in full induction of γ-globin mRNA expression, suggesting that NRF2 translocation alone is not sufficient for γ-globin induction. However, when combined with tBHQ, suppression of KEAP1 did enhance γ-globin induction. Taken together, these results suggest that NRF2/ARE pathway activation induces γ-globin mRNA expression and HbF production in primary human erythroid cells and that this is a promising strategy for further pre-clinical development. 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.

scholarly journals Salubrinal Mediated Fetal Hemoglobin Induction through the eIF2α-ATF4 Signaling Pathway

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 32-32
Author(s):  
Nicole Hope Lopez ◽  
Biaoru Li ◽  
Xingguo Zhu ◽  
Betty S. Pace
Keyword(s):  
Flow Cytometry ◽  
Clinical Symptoms ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Fold Increase ◽  
Erythroid Progenitors ◽  
Globin Mrna ◽  
F Cells ◽  
Hbf Induction ◽  
Dose Dependent

Sickle cell disease (SCD) is a genetic disorder caused by a mutation in the adult β-globin gene, affecting ~100,000 people in the United States and millions worldwide. Clinical symptoms of SCD include anemia, pain, and progressive organ damage creating a great burden to annual healthcare costs. An effective therapeutic intervention for SCD is fetal hemoglobin (HbF) induction by pharmacologic agents to ameliorate clinical symptoms. Hydroxyurea (HU) is the only FDA-approved drug used to induce HbF in SCD, however, it is not effective in all people. Therefore, the goal of this study is to determine the ability of Salubrinal (SAL), to induce HbF. Salubrinal is a selective inhibitor of protein phosphatase 1 leading to increased levels of p-eIF2α (phosphorylated eukaryotic initiation factor 2α) and inhibition of global protein synthesis. Activating transcription factor 4 (ATF4) is a downstream target of p-eIF2α activated during oxidative stress. The main function of these signaling events is to attenuate stress to the endoplasmic reticulum. Previously we identified a Gγ-globin cAMP response element (G-CRE) that binds ATF2, a binding partner of ATF4 involved in HbF induction (Sangerman J et al. Blood 2006). Furthermore, ENCODE analysis showed ATF4 sites at -822Gγ and β-globin gene second intron. Thus, studies were performed to determine if p-eIF2α-ATF4 signaling is involved in mechanisms of HbF induction by SAL. Initial experiments involved the use of day 8 erythroid progenitors generated from human CD34+ stem cells; treatments included SAL 12, 18 and 24 µM, and 0.5% DMSO (vehicle control) for 48 h; cell viability remained &gt;90% in all drug conditions. The level of γ-globin mRNA increased 1.2-fold and 1.3-fold at SAL 18 and 24 µM respectively (p&lt;0.05). Comparable, HbF was induced by SAL 24 µM alone and combined SAL/HU treatments to 1.8-fold. To gain insights into mechanisms of HbF induction by SAL, we next quantified the level of p-eIF2α. We observed a 1.7-fold increase in p-eIF2α with SAL 12 and 24 µM and parallel increase in ATF4 (4.8-fold). Flow cytometry revealed SAL increased F-cells (HbF positive cells) from 30.9% (DMSO treated) to 90.6%. Similarly, studies were performed using sickle erythroid progenitors generated from peripheral blood mononuclear cells. On day 8, SAL (9, 18, 24 µM) dissolved in water was added for 48 h; cell viability remained &gt;90% for all drug conditions. SAL (18 μM) increased γ-globin mRNA 3.2-fold and F-cells 2.5-fold (p&lt;0.05) compared to the untreated control. We used mean fluorescence intensity (MFI) to quantify HbF protein per cells, which showed a dose-dependent increase with SAL treatment. Since sickle red blood cells are under oxidative stress, we measured the levels of reactive oxygen species (ROS) by flow cytometry. SAL 12, 18, 24 µM decreased ROS levels in a dose-dependent manner by 7.6%, 8.7% and 10% respectively. Interestingly, SAL/HU treatment decreased ROS levels by 10.2% compared to a 4.3% mediated by the nitric oxide donor HU. Western blot analysis showed a dose-dependent increase in HbF and a 3.3-fold increase in p-eIF2α (p&lt;0.05) and ATF4, without changing HbS expression. To generate data for clinical development, we utilized the Townes SCD mouse model. SCD mice (n=5 per group) were treated with SAL (3 and 5mg/kg), HU (100mg/kg; positive control) or water control (vehicle), 5 days a week for 4 weeks. Blood was drawn at week 0 (baseline), 2 and 4 at treatment completion. All data were normalized for each group and treatment response at week 2 and 4 compared to week 0 using a paired t-test and ANOVA to compare across treatment groups; statistical significance set at p&lt;0.05. All groups showed normal weight gain and no significant changes in complete blood counts, differential or reticulocyte counts. Flow cytometry of peripheral blood showed that SAL (3mg/kg) produced a 2-fold increase in F-cells by 2 and 4 weeks while SAL (5mg/kg) produced a further 3.1-fold increase in F-cells by week 4 (p&lt;0.05) without toxicity. Our initial in vitro findings, supports HbF induction by SAL involving p-eIF2α-ATF4 signaling. The interaction of ATF4 in the G-CRE and/or other predicted binding sites will be investigated. To support clinical trials, studies in the SCD preclinical model support the ability of SAL to induce HbF in vivo; additional studies are underway. Defining the mechanism of HbF induction by SAL has the potential to impact treatment for SCD. 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.

Polyomavirus enhancer contains multiple redundant sequence elements that activate both DNA replication and gene expression

1985 ◽  
Vol 5 (4) ◽  
pp. 649-658
Author(s):  
G M Veldman ◽  
S Lupton ◽  
R Kamen
Keyword(s):  
Dna Replication ◽  
Globin Gene ◽  
Mrna Levels ◽  
Beta Globin ◽  
Transcriptional Enhancer ◽  
Globin Mrna ◽  
Enhancer Region ◽  
Sequence Elements ◽  
Multiple Copies ◽  
A Site

Sequences that comprise the 244-base-pair polyomavirus enhancer region are also required in cis for viral DNA replication (Tyndall et al., Nucleic Acids Res. 9:6231-6250, 1981). We have studied the relationship between the sequences that activate replication and those that enhance transcription in two ways. One approach, recently described by de Villiers et al. (Nature [London], 312:242-246, 1984), in which the polyomavirus enhancer region was replaced with other viral or cellular transcriptional enhancers suggested that an enhancer function is required for polyomavirus DNA replication. The other approach, described in this paper, was to analyze a series of deletion mutants that functionally dissect the enhancer region and enabled us to localize four sequence elements in this region that are involved in the activation of replication. These elements, which have little sequence homology, are functionally redundant. Element A (nucleotides 5108 through 5130) was synthesized as a 26-mer with XhoI sticky ends, and one or more copies were introduced into a plasmid containing the origin of replication, but lacking the enhancer region. Whereas one copy of the 26-mer activated replication only to 2 to 5% of the wild-type level, two copies inserted in either orientation completely restored replication. We found that multiple copies of the 26-mer were also active as a transcriptional enhancer by measuring the beta-globin mRNA levels expressed from a plasmid that contained either the polyomavirus enhancer or one or more copies of the 26-mer inserted in a site 3' to the beta-globin gene. We observed a correlation between the number of inserted 26-mers and the level of beta-globin RNA expression.

Metabolism is not a Major Contributor to the Toxicity of Piperaquine, a Major Partner Antimalarial in Artemisinin-Based Combination Therapy

2021 ◽  
Vol 22 ◽  
Author(s):  
Liyuan Zhang ◽  
Zhaohua Liu ◽  
Yunrui Zhang ◽  
Yuewu Xie ◽  
Jie Xing
Keyword(s):  
Histopathological Examination ◽  
High Dose ◽  
Mrna Levels ◽  
Apoptosis Pathway ◽  
Hepatocellular Damage ◽  
Serum Biochemical ◽  
Oral Doses ◽  
Dose Dependent ◽  
Cumulative Exposures

Background: Hepatocellular damage has been reported for the antimalarial piperaquine (PQ) in the clinic after cumulative doses. Objectives: The role of metabolism in PQ toxicity was evaluated, and the mechanism mediating PQ hepatotoxicity was investigated. Method: The toxicity of PQ and its major metabolite (PQ N-oxide; M1) in mice was evaluated in terms of serum biochemical parameters. The role of metabolism in PQ toxicity was investigated in mice pretreated with an inhibitor of CYP450 (ABT) and/or FMO enzyme (MMI). The dose-dependent pharmacokinetics of PQ and M1 were studied in mice. Histopathological examination was performed to reveal the mechanism mediating PQ hepatotoxicity. Results: Serum biochemical levels (ALT and BUN) increased significantly (P < 0.05) in mice after three-day oral doses of PQ (> 200 mg/kg/day), indicating hepatotoxicity and nephrotoxicity of PQ at a high dose. Weaker toxicity was observed for M1. Pretreatment with ABT and/or MMI did not increase PQ toxicity. PQ and M1 showed linear pharmacokinetics in mice after a single oral dose, and multiple oral doses led to their cumulative exposures. Histopathological examination showed that a high dose of PQ (> 200 mg/kg/day for three days) could induce hepatocyte apoptosis. The mRNA levels of targets in NF-κB and p53 pathways could be up-regulated by 2-30-fold in mice by PQ or M1. Conclusions: PQ metabolism led to detoxification of PQ, but there was a low possibility of altered toxicity induced by metabolism inhibition. The hepatotoxicity of PQ and its N-oxidation metabolite was partly mediated by NF-κB inflammatory pathway and p53 apoptosis pathway.

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

Blood ◽  
2012 ◽  
Vol 119 (4) ◽  
pp. 1045-1053 ◽  
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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