Deciphering the Cis- and Trans-regulatory Roles of KLF6 in Primitive Hematopoiesis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4730-4730
Author(s):  
Qian Xiong ◽  
Zhaojun Zhang ◽  
Hongzhu Qu ◽  
Xiuyan Ruan ◽  
Hai Wang ◽  
...  
Keyword(s):  
Expression Pattern ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Hek293 Cells ◽  
Luciferase Reporter ◽  
Erythroid Cells ◽  
Dynamic Expression ◽  
Primitive Hematopoiesis ◽  
Cis And Trans

Abstract Abstract 4730 Krüppel-like factors (KLFs) are a conserved family of Cys2His2 zinc finger proteins which are important components of eukaryotic cellular transcriptional machinery that controls many biological processes including erythroid differentiation and development. As a transcriptional activator and a tumor suppressor, KLF6 was also involved in hematopoiesis. Klf6−/− mice is embryonic lethal by embryonic day 12.5 and associated with markedly reduced hematopoiesis as well as poorly organized yolk sac vascularization. Moreover, the expression of erythroid differentiation markers including Klf1, Gata1 and Scl are delayed and hematopoietic differentiation is impaired in klf6−/− ES cells. However, the detailed mechanism that KLF6 regulates hematopoiesis is not fully understood. To characterize the role of KLF6 in hematopoiesis, we firstly detected the dynamic expression pattern of KLF6 during erythroid differentiation by mRNA-seq in undifferentiated human embryonic stem cells (hESC), three primary erythroid cells at different developmental stages including ES-derived erythroid cells (ESER), fetal- and adult-type erythroid cells (FLER, PBER). The transcriptome analysis showed that KLF6 expressed at significantly higher level in ESER cells compared with that in other cells. Meanwhile, chromatin immunoprecipitation (ChIP) studies in human K562 cells demonstrated the enrichment of KLF6 on the promoter region of embryonic epsilon-globin gene. These results probably indicate that KLF6 play an important role in primitive hematopoiesis. To clarify whether the erythroid-specific enhancers in the genomic region of KLF6 participate in the regulation of primitive hematopoiesis, we extensively screened the erythroid-specific DNaseI hypersensitive sites (DHSs) in the KLF6 locus, from 70 kb upstream of the transcription start site to 20 kb downstream of the poly(A) site, from DNase-seq data in four erythroid cells including ESER, FLER, PBER, K562 and seven non-erythroid cells. The enhancer activity of these erythroid-specific DHSs was comprehensively characterized by dual-luciferase reporter assay in K562 cells as well as non-erythroid HeLa and HEK293 cells. Three erythroid-specific enhancers located 18–24 kb upstream of human KLF6 were finally characterized, which not only helps to understand the higher expression of KLF6 in ESER, but also hints that KLF6 could participate in primitive hematopoiesis through erythroid-specific enhancers. In conclusion, we depicted the dynamic expression pattern of KLF6 during erythroid differentiation, characterized three erythroid-specific enhancers in KLF6 gene locus, and disclosed the potential role of KLF6 in primitive hematopoiesis. Next, the overexpression and depletion of KLF6 in K562 cells will be executed to further explore whether the abnormal KLF6 will affect the expression and functions of globin genes as well as erythroid-specific transcription factors. Chromosome conformation capture (3C) analysis will be performed to evaluate the interactions between the erythroid-specific enhancers and the cis-regulatory elements of hematopoiesis related genes. Moreover, we will establish morpholino-based klf6 knockdown zebrafish model and study the target genes, interacting networks and pathways in which KLF6 involved. Collectively, these results will address the detailed cis- and trans- regulatory functions and molecular mechanism of KLF6 in regulating hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

Comprehensive Investigation of the Molecular Mechanism of Primitive Hematopoiesis Regulating by KLF3

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4729-4729
Author(s):  
Zhang Zhaojun ◽  
Xiong Qian ◽  
Wang Shaobin ◽  
Wang Hai ◽  
Zhang Qian ◽  
...  
Keyword(s):  
Target Genes ◽  
Developmental Stages ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Luciferase Reporter ◽  
P Value ◽  
Erythroid Cells ◽  
Potential Target ◽  
Primitive Hematopoiesis ◽  
Upstream Regulators

Abstract Abstract 4729 KLF3 is a member of the Krüppel-like transcription factor family. By recognizing CC/ACACCC motifs in the promoters and enhancers of its regulating genes, KLF3 plays critical roles in cell differentiation and development including B lymphocytes maturation and adipocyte differentiation. Previous studies demonstrated that KLF3-deficient mice displayed myeloproliferative disorders and abnormalities in hematopoiesis. KLF3 prefers to bind to the CACCC box in the yolk sac and fetal liver, indicating that KLF3 probably participates in the primitive hematopoiesis. However, the mechanism that KLF3 regulates primitive hematopoiesis is not fully understood. To characterize the role of KLF3 in primitive hematopoiesis, we firstly detected the expression of KLF3 during erythroid differentiation by RNA-seq in undifferentiated human embryonic stem cells (hESC) as well as three primary erythroid cells at different developmental stages including ES-derived erythroid cells (ESER), fetal- and adult-type erythroid cells (FLER, PBER). The results show that KLF3 is significantly higher expressed in ESER cells than that in other cells, which is an indicating of the role of KLF3 in primitive hematopoiesis. The expression level of KLF3 decreased at later erythroid developmental stages, which was also verified by the decreased KLF3 expression level when K562 cells induced with 50 mM of hemin for up to 72h. Secondly, to further clarify the mechanism that KLF3 regulates primitive hematopoiesis, we depleted KLF3 by shRNA interference in K562 cells, the representative of early development of erythroid cells, and performed microarray analysis to comprehensively characterize the target genes of KLF3 as well as the networks in which the target genes involved. The results indicate that down-regulated KLF3 exhibits remarkable impacts on genes expression profile in K562 cells. Total 655 (p-value<0.01, fold change>1.5) differentially expressed genes were largely disturbed and recognized as potential target genes of KLF3, in which up-regulated genes (372) were more than down-regulated genes (283). Erythroid differentiation markers including HBE, HBA1/A2, HBZ and HBD globin genes are observably up-regulated in KLF3 depleted K562 cells. These results suggest that KLF3 probably exhibits suppressive activities in primitive hematopoiesis. The IPA analysis demonstrates that the potential target genes are specifically enriched in the biofunctions of hematopoiesis and hematological system development. The IPA networks analysis demonstrates that the potential target genes are closely associated with the networks of hematological diseases and hematological system development. IPA analysis also predicted the upstream regulators to drive KLF3 in erythroid cells including GATA1 (p-value<2.83E-12) and EPO (p-value<8.51E-08) which were significantly activated. Thirdly, to clarify whether the erythroid-specific enhancers in the genomic region of KLF3 participate in the KLF3 biology of primitive hematopoiesis, we identified erythroid-specific DNaseI hypersensitive sites (DHSs) in the KLF3 locus from DNase-seq data in four erythroid cells including ESER, FLER, PBER, K562 and seven non-erythroid cells. The enhancer activity of the erythroid DHSs was comprehensively characterized by dual-luciferase reporter assays in K562 cells and non-erythroid Hela and HEK293 cells. No erythroid-specific KLF3 enhancers was finally confirmed, suggesting the regulation of primitive hematopoiesis by KLF3 could depend on the upstream regulators, downstream target genes, as well as the other cis regulatory elements (CREs), but not erythroid-specific enhancers in KLF3 locus. In conclusion, we clarified the expression pattern of KLF3 during erythroid differentiation and confirmed the important functions of KLF3 in primitive hematopoises. Moerover, we ruled out the possibility that erythroid-specific enhancers in KLF3 gene locus participate in primitive hematopoiesis. Next, ChIP and dual luciferase reporter assay will be performed to confirm the regulation of KLF3 on the target genes. The relationship between these upstream regulators and KLF3 potential target genes will be further clarified. Finally, the related observations will be verified in hematopoietic stem cells (HSCs) as well as KLF3 morpholino knockdown zebrafish to fully understand the molecular mechanism of KLF3 in regulating primitive hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

Elucidation of the Role of LMO2 (LIM-only protein 2) in Erythroid Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3443-3443
Author(s):  
AI Inoue ◽  
Tohru Fujiwara ◽  
Yoko Okitsu ◽  
Noriko Fukuhara ◽  
Yasushi Onishi ◽  
...  
Keyword(s):  
Dna Binding ◽  
Cord Blood ◽  
Western Blotting ◽  
Target Genes ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Dependent Manner ◽  
Erythroid Cells ◽  
In Erythroid Cells

Abstract Abstract 3443 Background: Developmental control mechanisms often utilize multimeric complexes containing transcription factors, coregulators, and additional non-DNA binding components. It is challenging to ascertain how such components contribute to complex function at endogenous loci. LMO2 (LIM-only protein 2) is a non-DNA binding transcriptional coregulator, and is an important regulator of hematopoietic stem cell development and erythropoiesis, as mice lacking this gene show defects in blood formation as well as fetal erythropoiesis (Warren et al. Cell. 1994). In the context of erythropoiesis, LMO2 has been demonstrated to be a part of multimetric complex, including master regulators of hematopoiesis (GATA-1 and SCL/TAL1), chromatin looping factor LDB1 and hematopoietic corepressor ETO2 (referred as GATA-SCL/TAL1 complex). As LMO2 controls hematopoiesis, its dysregulation is leukemogenic, and its influence on GATA factor function is still not evident, we investigated here the transcriptional regulatory mechanism via LMO2 in erythroid cells. Methods: For LMO2 knockdown, anti-LMO2 siRNA (Thermo Scientific Dharmacon) and pGIPZ lentiviral shRNAmir system (Open Biosystems) were used. Western blotting and Quantitative ChIP analysis were performed using antibodies for GATA-1, LMO2 (abcam), GATA-2, TAL1 and LDB1 (Santa Cruz). To obtain human primary erythroblasts, CD34-positive cells isolated from cord blood were induced in liquid suspension culture. For transcription profiling, human whole expression array was used (Agilent), and the data was analyzed with GeneSpring GX software. To induce erythroid differentiation of K562 cells, hemin was treated at a concentration of 30 uM for 24h. Results: siRNA-mediated LMO2 knockdown in hemin-treated K562 cells results in significantly decreased ratio of benzidine-staining positive cells, suggesting that LMO2 has an important role in the erythroid differentiation of K562 cells. Next, we conducted microarray analysis to characterize LMO2 target gene ensemble in K562 cells. In contrast to the predominantly repressive role of LMO2 in murine G1E-ER-GATA-1 cells (Fujiwara et al. PNAS. 2010), the analyses (n = 2) demonstrated that 177 and 78 genes were upregulated and downregulated (>1.5-fold), respectively, in the LMO2-knockdowned K562 cells. Downregulated gene ensemble contained prototypical erythroid genes such as HBB and SLC4A1 (encodes erythrocyte membrane protein band 3). To test what percentages of LMO2-regulated genes could be direct target genes of GATA-1 in K562 cells, we merged the microarray results with ChIP-seq profile (n= 5,749, Fujiwara et al. Mol Cell. 2009), and demonstrated that 26.4% and 23.1% of upregulated and downregulated genes, respectively, contained significant GATA-1 peaks in their loci. Furthermore, whereas LMO2 knockdown in K562 cells did not affect the expression of GATA-1, GATA-2 and SCL/TAL1 based on quantitative RT-PCR as well as Western blotting, the knockdown resulted in the significantly decreased chromatin occupancy of GATA-1, GATA-2, SCL/TAL1 and LDB1 at beta-globin locus control region and SLC4A1 locus. We subsequently analyzed the consequences of LMO2 knockdown in primary erythroblasts. Endogeneous LMO2 expression was upregulated along with the differentiation of cord blood cell-derived primary erythroblasts. shRNA-mediated knockdown of LMO2 in primary erythroblasts resulted in significant downregulation of HBB, HBA and SLC4A1. Conclusion: Our results suggest that LMO2 contributes to the expression of GATA-1 target genes in a context-dependent manner, through modulating the assembly of the components of GATA-SCL/TAL1 complex at endogeneous loci. Disclosures: No relevant conflicts of interest to declare.

scholarly journals MiR-27a Promotes Hemin-Induced Erythroid Differentiation of K562 Cells by Targeting CDC25B

2018 ◽  
Vol 46 (1) ◽  
pp. 365-374 ◽  
Author(s):  
Dongsheng Wang ◽  
Si Si ◽  
Qiang Wang ◽  
Guangcheng Luo ◽  
Qin Du ◽  
...  
Keyword(s):  
Cell Division ◽  
Potential Role ◽  
Globin Gene ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Luciferase Reporter ◽  
Loss Of Function ◽  
Dual Luciferase Reporter Assay ◽  
Cell Division Control

Background/Aims: MicroRNAs (miRNAs) play a crucial role in erythropoiesis. MiR-23a∼27a∼24-2 clusters have been proven to take part in erythropoiesis via some proteins. CDC25B (cell division control Cdc2 phosphostase B) is also the target of mir-27a; whether it regulates erythropoiesis and its mechanism are unknown. Methods: To evaluate the potential role of miR-27a during erythroid differentiation, we performed miR-27a gain- and loss-of-function experiments on hemin-induced K562 cells. We detected miR-27a expression after hemin stimulation at different time points. At the same time, the γ-globin gene also was measured via real-time PCR. According to the results of the chips, we screened the target protein of miR-27a through a dual-luciferase reporter assay and identified it via Western blot analyses. To evaluate the function of CDC25B, benzidine staining and flow cytometry were employed to detect the cell differentiation and cell cycle. Results: We found that miR-27a promotes hemin-induced erythroid differentiation of human K562 cells by targeting cell division cycle 25 B (CDC25B). Overexpression of miR-27a promotes the differentiation of hemin-induced K562 cells, as demonstrated by γ-globin overexpression. The inhibition of miR-27a expression suppresses erythroid differentiation, thus leading to a reduction in the γ-globin gene. CDC25B was identified as a new target of miR-27a during erythroid differentiation. Overexpression of miR-27a led to decreased CDC25B expression after hemin treatment, and CDC25B was up-regulated when miR-27a expression was inhibited. Moreover, the inhibition of CDC25B affected erythroid differentiation, as assessed by γ-globin expression. Conclusion: This study is the first report of the interaction between miR-27a and CDC25B, and it improves the understanding of miRNA functions during erythroid differentiation.

Trafficking Kinesin Binding Protein Is Essential for Human Erythropoiesis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 683-683
Author(s):  
Seung-Jae Noh ◽  
Y.Terry Lee ◽  
Colleen Byrnes ◽  
Antoinette Rabel ◽  
Jeffery L. Miller
Keyword(s):  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Annexin V ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Mitochondrial Outer Membrane ◽  
Slight Reduction ◽  
Erythroid Cells ◽  
Heme Synthesis

Abstract Abstract 683 Mitochondrial specialization in erythroblasts is important for efficient heme synthesis, with defects or reduced expression of several mitochondrial proteins causing anemia. Trafficking kinesin binding 2 (TRAK2) is known to participate in mitochondrial movement along microtubule by interacting with kinesin motor protein and making a complex with Miro that is localized on the mitochondrial outer membrane. Transcriptome data suggest that TRAK2 is highly and specifically expressed in early erythroid cells. Here the role of TRAK2 was studied among human CD34+ cells that were grown in ex vivo serum-free cultures supplemented with erythropoietin (EPO, total culture period 21 days). Quantitative PCR studies indicated that TRAK2 expression is highly regulated during erythropoiesis. Its expression pattern was nearly identical to aminolevulinate synthase 2, the erythroid specific enzyme for the committed step of the heme biosynthetic pathway, and mitoferrin 1, the erythroid specific mitochondrial iron transporter. Western analyses revealed that TRAK2 protein is detected as a doublet band with molecular weights of 130kD and 105kD. Mitochondrial co-localization of TRAK2 was verified by confocal microscopy in TRAK2-overexpressing K562 cells. To study a potential role of TRAK2 in erythropoiesis, TRAK2 expression was reduced in cultured human erythroid cells using lentiviral shRNA transduction. TRAK2 knockdown (TRAK2-KD) was confirmed by Western analysis in K562 cells. In primary erythroblasts, TRAK2-KD caused slight reduction of CD36+ immature erythroblasts at culture day 7 prior to the addition of EPO (CD36+ population 58% in control vs 40% in TRAK2-KD). After the addition of erythropoietin to the culture medium, TRAK2-KD severely restricted erythroblast proliferation (5.0 million cells/ml in control vs 0.25 million cells/ml in TRAK2-KD on culture day 18). Flow cytometric analyses showed that <1% of the CD36+ progenitors cells differentiated into glycophorin A erythroblasts compared with >90% in control cultures. Annexin-V staining indicated that more than 90% of cells had undergone apoptosis by day 14. These data suggest that TRAK2 expression is required for erythroid differentiation. As such, defects in TRAK2 expression should be considered in cases of unexplained anemia. The data also support the notion that mitochondrial location or mobility within erythroblasts may be important for iron trafficking or heme synthesis. Disclosures: No relevant conflicts of interest to declare.

Regulation of hemoglobin synthesis and proliferation of differentiating erythroid cells by heme-regulated eIF-2α kinase

Blood ◽  
2000 ◽  
Vol 96 (9) ◽  
pp. 3241-3248 ◽  
Author(s):  
John S. Crosby ◽  
Peter J. Chefalo ◽  
Irene Yeh ◽  
Shong Ying ◽  
Irving M. London ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Erythroid Differentiation ◽  
Dominant Negative ◽  
Erythroid Cells ◽  
3T3 Cells ◽  
Binding Domains ◽  
Inhibition Of Protein Synthesis ◽  
Nih 3T3 ◽  
Hemoglobin Production

Abstract Protein synthesis in reticulocytes depends on the availability of heme. In heme deficiency, inhibition of protein synthesis correlates with the activation of heme-regulated eIF-2α kinase (HRI), which blocks the initiation of protein synthesis by phosphorylating eIF-2α. HRI is a hemoprotein with 2 distinct heme-binding domains. Heme negatively regulates HRI activity by binding directly to HRI. To further study the physiological function of HRI, the wild-type (Wt) HRI and dominant-negative inactive mutants of HRI were expressed by retrovirus-mediated transfer in both non-erythroid NIH 3T3 and mouse erythroleukemic (MEL) cells. Expression of Wt HRI in 3T3 cells resulted in the inhibition of protein synthesis, a loss of proliferation, and eventually cell death. Expression of the inactive HRI mutants had no apparent effect on the growth characteristics or morphology of NIH 3T3 cells. In contrast, expression of 3 dominant-negative inactive mutants of HRI in MEL cells resulted in increased hemoglobin production and increased proliferative capacity of these cells upon dimethyl-sulfoxide induction of erythroid differentiation. These results directly demonstrate the importance of HRI in the regulation of protein synthesis in immature erythroid cells and suggest a role of HRI in the regulation of the numbers of matured erythroid cells.

The role of catechol-O-methyltransferase in catechol-enhanced erythroid differentiation of K562 cells

2013 ◽  
Vol 273 (3) ◽  
pp. 635-643 ◽  
Author(s):  
Suriguga ◽  
Xiao-Fei Li ◽  
Yang Li ◽  
Chun-Hong Yu ◽  
Yi-Ran Li ◽  
...  
Keyword(s):  
K562 Cells ◽  
Erythroid Differentiation

Chromatin Conformation and a Distal Regulatory Element Activate the Human Erythroid Ankyrin-1 Promoter.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 803-803
Author(s):  
Ashley N. Owen ◽  
Robert I. Liem ◽  
Andre M. Pilon ◽  
Patrick G. Gallagher ◽  
David M. Bodine
Keyword(s):  
Reporter Gene ◽  
Regulatory Element ◽  
K562 Cells ◽  
Regulatory Elements ◽  
Dnase I ◽  
Luciferase Reporter ◽  
Erythroid Cells ◽  
Luciferase Reporter Gene ◽  
Human And Mouse ◽  
In Erythroid Cells

Abstract Ankyrin forms the bridge between the spectrin/actin network of the erythrocyte membrane skeleton and the red cell membrane by binding to both β-spectrin and band 3. The erythrocyte ankyrin promoter (Ank-1E) is active only in erythroid cells, while two other Ank-1 promoters located 20 kb downstream and 40 kb upstream of Ank-1E are active in the cerebellum and muscle cells respectively. We have been studying the mechanism by which the Ank-1E promoter becomes active in erythroid cells by studying the cis acting regulatory elements and the chromatin structure of the Ank-1 promoter region. We have previously shown that the sequences between −296 and −15 of the Ank-1E promoter are fully sufficient for erythroid specific, copy number dependent uniform expression of reporter genes in transgenic mice. We have also mapped a DNase I Hypersensitive site (5′HS) between −300 and −100 of the human and mouse Ank-1E promoters in human K562 and mouse fetal liver cells. Both the mouse and human 5′HS are capable of preventing the silencing of a β-globin/GFP reporter gene in K562 cells, establishing that they function as barrier elements. Consistent with this observation, the human and mouse 5′HS are hyperacetylated in erythroid cells. The chromatin 10 kb 5′ to the 5′HS is DNase I resistant (associated with inactive chromatin) in human and mouse erythroid and non-erythroid cells. Approximately 6 kb 3′ to 5′HS are two adjacent HS (3′HS1, 3′HS2). Beyond 3′HS2 the chromatin is also DNase I resistant in both human and mouse erythroid and non-erythroid cells. Between 5′HS and 3′HS1 the 6kb region is DNase I sensitive (active) in erythroid cells but not in other cell types. We hypothesized that this 6 kb region contains regulatory elements that activate the Ank-1E promoter. To screen for regulatory elements we isolated overlapping segments of a 10 kb region extending from 2 kb upstream of 5′HS to 2 kb downstream of 3′HS2. We inserted these fragments into a plasmid vector containing the Ank-1E promoter linked to a luciferase reporter gene and transfected these constructs into K562 cells. A single region up regulated Ank-1E/luciferase expression. This region mapped to a 211bp segment that included 3′HS1, but did not include 3′HS2. A fragment containing only 3′HS2 did not up regulate an Ank-1E/luciferase reporter gene, but 3′HS2 was capable of preventing the silencing of a β-globin/Green Fluorescent Protein reporter gene in K562 cells, demonstrating barrier activity. The region around 3′HS1 and 2 was also a site of histone hyperacetylation. The sequence of the 211 bp fragment containing 3′HS1 does not contain consensus sequences for any known erythroid-specific transcription factors, but does contain potential binding sites fro Sp1, AP-1 and E-box binding proteins. Using the Chromatin Conformation Capture assay we demonstrated that 5′HS and 3′HS1 and 2 are in close proximity in K562 chromatin, but are not closely associated in chromatin from other cell types. We propose that an erythroid-specific chromatin loop brings 3′HS1 and 2 into proximity with 5′HS, adjacent to the Ank-1E promoter. This interaction translocates the positive regulatory element in 3′HS1 to the Ank-1E promoter allowing the Ank-1E promoter to become active in erythroid cells.

Role of Cjun in Gγ-Globin Gene Trans-Activation.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1870-1870
Author(s):  
Sirisha Kodeboyina ◽  
Sima Zein ◽  
Moosueng Lee ◽  
Parimaladevi Balamurugan ◽  
Xiao Yao ◽  
...  
Keyword(s):  
Electrophoretic Mobility ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
K562 Cells ◽  
Luciferase Reporter ◽  
Complete Analysis ◽  
Dependent Manner ◽  
Mobility Shift ◽  
Electrophoretic Mobility Shift Assays

Abstract Previous studies from our laboratory demonstrated the role of the G-CRE (Gγ-globin cAMP response element) in drug-mediated fetal hemoglobin induction. The G-CRE located at −1222 to −1229 in the promoter of Gγ-globin gene, contains binding site for trans-factors CREB1, ATF-2 and cJun. We previously demonstrated binding of phosphorylated CREB1 and ATF-2 to this element via p38 MAPK signaling triggered by sodium butyrate (NaB) and trichostatin A (TSA). Electrophoretic mobility shift assays with a probe containing the AC → TG mutation in the G-CRE (TGTGGTCA, m2) abolished trans-factor binding to the G-CRE. Furthermore, Gγ promoter activity was abolished in the PGL3 luciferase reporter vector driven by the Gγ promoter (−1500 to +36) carrying the m2 mutation. (Sangerman et al. Blood108:3590–9, 2006). Subsequent studies in our laboratory were aimed at understanding the role of trans-factor cJun, an AP-1 family member, as a regulator of Gγ-globin expression via the G-CRE site. In K562 cells treated with 2mM NaB or 0.3μM TSA for 48 hrs, cJun phosphorylation increased 2.8-fold and 6.4-fold respectively by western blot analysis. Chromatin immunoprecipitation studies showed 16-fold chromatin enrichment in the −1225 Gγ-globin region compared to IgG control studies indicative of significant cJun binding in vivo at steady state. Electrophoretic mobility shift assays using cJun monoclonal antibody demonstrated a supershifted DNA-protein complex confirming binding of cJun to the G-CRE probe. To gain evidence for a functional role of cJun, we performed enforced expression studies using the pLen-cJun vector. In a concentration dependent manner, over-expression of cJun increased luciferase activity up to 350-fold in the luciferase reporter plasmid controlled by the Gγ-promoter (−1500 to +36). As predicted from binding studies, the m2 mutation in this promoter abolished the cJunmediated trans-activation confirming that the G-CRE is required to mediate effects of cJun. We are currently investigating the ability of cJun to trans-activate the endogenous Gγ-globin gene in K562 cells. To achieve this goal, K562 stable lines were established with the expression vectors pLen-cJun and empty vector. A complete analysis of the stable lines is in progress. Future investigations to identify other components of the functional CREB1/ATF2/cJun enhanceosome complex bound to the G-CRE will be performed using affinity chromatography and mass spectrometry. This information will be used to develop strategies for fetal hemoglobin induction.

MicroRNA-486-5p Targets Foxo1 and Regulates Human Hematopoietic Stem Cell Proliferation and Erythroid Differentiation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3871-3871
Author(s):  
Li-Sheng Wang ◽  
Ling LI ◽  
Liang Li ◽  
Keh-Dong Shiang ◽  
Min Li ◽  
...  
Keyword(s):  
Protein Expression ◽  
Myeloid Cells ◽  
Erythroid Differentiation ◽  
Luciferase Reporter ◽  
Regulatory Function ◽  
Control Vector ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Direct Target

Abstract Abstract 3871 Previous studies have supported a critical role for specific miRNA in regulating hematopoiesis. However the relative abundance and specificity for most miRNAs remains to be investigated, and the role of expressed miRNA in regulating cell fate and function remains poorly understood. Using microRNA microarrays we identified increased expression of miR-486 in chronic myeloid leukemia (CML) compared to normal CD34+ cells. miR-486 is located within the last intron of the Ankyrin-1 gene on chromosome 8 and is reported to be enriched in muscle cells. The expression pattern of miR-486 in hematopoietic cells and its roles in hematopoietic regulation are not known. In CB cells, miR-486 expression level was highest in MEP and was low in HSC. There was 16-fold increased expression of miR-486 during in vitro erythroid differentiation of CB Lin-CD34+CD38– cells, associated with 5-fold increase in Ankyrin-1 gene expression. To explore the role of miR-486 in growth and differentiation of hematopoietic stem and progenitor cells (HSPC), we first expressed hsa-miR-486-5p in CB CD34+ cells using lentiviral vectors. CB CD34+ cells transduced with this vector demonstrated 2–3 fold increased expression of miRNA-486-5p compared to cells transduced with a control vector (Ctrl). CB CD34+ cells expressing miR-486-5p generated modestly increased numbers of cells (1.22 fold) in culture with SCF, IL-3, GM-CSF, G-CSF and EPO for 6 days. Increased numbers of erythroid cells and reduced numbers of myeloid cells were generated in culture (GPA+ cells: Ctrl 58% and miR-486-5p 72.2%; CD33+ cells: Ctrl 30.7% and miR-486-5p 21.9%;, CD11b cells: Ctrl 33.5% and miR-486-5p 21.5%). To further investigate the effect of inhibition of miR-486-5p on growth and differentiation of HSPC, we inhibited miR-486 expression in CB CD34+ cells using a modified miRZip anti-miRNA lentivirus vectors (SBI) expressing anti-miR-486-5p and compared to cells expressing a control scrambled anti-miRNA sequence. Anti-miR-486-5p expressing CB CD34+ cells generated reduced number of cells in growth factor (GF) culture (67.5% inhibition) compared to controls. Greater inhibition of erythroid compared to myeloid cells was seen (GPA+ cells: 62.5% inhibition; CD33+ cells: 37.1% inhibition compared to controls at day 6). Anti-miR-486-5p expressing CB CD34+ cells also demonstrated reduced colony formation (BFU-E: 67% inhibition;, CFU-GM 16% inhibition), and reduced proliferation (43.88% inhibition of proliferation index) compared to controls. Similar results were observed with CB Lin-CD34+CD38- cells transduced with anti-486-5p virus (GPA+ cells: 67% inhibition; CD33+ cells: 30 % inhibition). The number of CD34+ cells was however maintained after culture (117% for miR-486-5p compared to scramble). These results indicate an important role for miR-486-5p in preservation, proliferation and erythroid differentiation of HSC. A search for evolutionarily conserved miR-486-5p targets using Targetscan 5.1 identified Foxo1, a member of the Foxo subfamily of forkhead transcription factors which play negative regulatory roles in hematopoiesis, as the highest ranking target. To demonstrate that Foxo1 is a direct target of miR-486-5p, we generated pMIR-REPORT™ constructs containing two miR-486-5p seed sites (182 and 658) within the Foxo1 3′-UTR. These constructs were cotransfected into HEK293T cells along with a miR-486-5p expression plasmid or empty control vector. Expression of miR-486-5p resulted in a 65% reduction in luciferase activity. Expression of anti-miR-486-5p resulted in increased Foxo1 protein expression in CB CD34+ cells. Expression of miR-486-5p also resulted in 50% decrease of Foxo1 protein expression. Using a Fas-L promoter-luciferase reporter we found that inhibition of miR486-5p increased Foxo1 transactivation activity in HEK293T cells. These results demonstrate that Foxo1 is a direct target of miR-486-5p. We conclude that miR-486-5p expression is modulated during normal hematopoietic differentiation and in leukemic hematopoiesis. Our results indicate a regulatory role for miR-486-5p in the growth hematopoietic stem cells and their erythroid differentiation. We show that miR-486-5p directly inhibits Foxo1 expression, which may potentially play an important role in its hematopoietic regulatory function. Disclosures: No relevant conflicts of interest to declare.

CoCoA/CCAR1 Pair-Mediated Recruitment of Mediator Complex Indicates Novel Pathway for the Function of GATA1 in Erythroid Differentiation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1302-1302
Author(s):  
Chihiro Kaminaga ◽  
Shumpei Mizuta ◽  
Tomoya Minami ◽  
Kasumi Oda ◽  
Haruka Fujita ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Direct Interaction ◽  
K562 Cells ◽  
Erythroid Differentiation ◽  
Luciferase Reporter ◽  
Zinc Finger Domain ◽  
C Terminus ◽  
N Terminus ◽  
Reporter Assays ◽  
Globin Promoter

Abstract Abstract 1302 The mammalian multi-protein complex Mediator, originally identified by ourselves as a nuclear receptor-specific coactivator complex, is a phylogenetically-conserved subcomplex of the RNA polymerase II holoenzyme and serves as an end-point integrator of diverse intracellular signals and transcriptional activators. The 220-kDa Mediator subunit MED1 is a specific coactivator not only for nuclear receptors but for GATA family activators, and serves as a GATA1-specific coactivator that is essential for optimal GATA1-mediated erythropoiesis. In this study, we show a novel nuclear signaling pathway for MED1 action in GATA1-induced transcriptional activation during erythroid differentiation. First, we identified the amino acid residues 681–715 of human MED1 (MED1(aa.681-715)) to be responsible for the direct interaction with GATA1. When MED1 in K562 human erythroleukemic cells was knocked down during hemin-induced erythroid differentiation, the erythroid differentiation was significantly attenuated as assessed by an erythroid differentiation score defined by the number of cells positive for benzidine staining, and the expressions of the GATA1-targeted and erythroid differentiation marker genes, β-globin, γ-globin, PBGD and ALAS-E, were prominently attenuated. However, overexpressions of the N-terminal MED1 truncations without and with nuclear receptor recognition motifs, MED1(aa.1–602) and MED1(aa.1–703), respectively, but neither of which could bind to GATA1 (above), prominently enhanced erythroid differentiation of K562 cells. Luciferase reporter assays by using the human γ-globin promoter and Med1−/− mouse embryonic fibroblasts (MEFs) showed that these N-terminal MED1 truncations rescued GATA1-mediated transactivation, indicating that MED1(a.a.1–602) served as the functional interaction surface for GATA1. Hence, a putative bypass for GATA1-MED1 pathway appears to exist, and is expected to interact with the N-terminus of MED1. As a candidate bypass system, we tested both the recently reported bypass molecule for a nuclear post-activator signaling, CCAR1, and its partner coactivator CoCoA. CCAR1 was reported by others to bypass the estrogen receptor-mediated transactivation by a simultaneous binding of CCAR1 with the estrogen receptor and the N-terminus of MED1. Functionally, serial luciferase reporter assays by using the γ-globin promoter and MEFs demonstrated cooperative transactivation by combinations of GATA1, CCAR1, CoCoA and/or the N-terminus of MED1, but the transactivation mediated by the N-terminus of MED1 was not as prominent as the one mediated by the full-length MED1. An overexperssion of CCAR1 or CoCoA in K562 cells prominently enhanced both the GATA1-mediated erythroid differentiation and the expressions of the GATA1-targeted genes. Next, the mechanisms underlying the CCAR1- and CoCoA-mediated GATA1 functions were analyzed by serial GST-pulldown and mammalian two-hybrid assays, and the following results were obtained. (i) The N-terminus of CCAR1 interacted with the C-terminus of CoCoA. (ii) The N-terminus of MED1 interacted with both the N- and C-termini of CCAR1. (iii) While the N-terminal zinc-finger domain of human GATA1 (GATA1(a.a.204–228)) is known to bind to the well-known GATA1 partner FOG1, intriguingly, the C-terminal zinc-finger domain of GATA1 (GATA1(a.a.258–272)) interacted with all three of the following cofactors; MED1 (MED1(aa.681–715)), CCAR1 (at the C-terminus) and CoCoA (at both the N- and C-termini). The affinity of CoCoA to bind to GATA1 appeared to be a little higher than the other. Thus, the GATA1(a.a.258-272) zinc finger appears to serve as a docking surface for multiple coactivating proteins, where both MED1 and CoCoA/CCAR1 pair can interact, probably in a competitive manner, or perhaps simultaneously. Here, both CoCoA/CCAR1 as a pair and CCAR1 by itself can serve as a bypass. Finally, ChIP assays of hemin-treated K562 cells showed that GATA1, CCAR1/CoCoA and MED1 were all recruited onto the γ-globin promoter during transactivation. Taken together, besides a direct interaction between GATA1 and MED1, the CoCoA/CCAR1 pair appears to relay the GATA1 signal to MED1. The multiple modes of mechanisms for transcription mediated by the GATA1-MED1 axis might contribute to a fine tuning of the GATA1 function, not only during erythropoiesis but also in other GATA1-mediated homeostasis events, within a living animal. Disclosures: No relevant conflicts of interest to declare.

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