GATA-1-Specific Coactivator MED1/TRAP220, Transcriptional Mediator Subunit, Mediates Erythroid Differentiation in K562 Erythroleukemia Cells.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2259-2259
Author(s):  
Tomoya Minami ◽  
Osamu Horie ◽  
Kana Inoue ◽  
Sawako Toji ◽  
Norinaga Urahama ◽  
...  

Abstract The multi-protein complex TRAP/Mediator is a subcomplex of RNA polymerase II holoenzyme and acts as the end-point integrator of a variety of activators and intracellular signalings. The Mediator activates transcription in concert with a group of recruited general transcription initiation factors. Among the circa 25 subunits, the MED1/TRAP220 subunit was originally demonstrated by us as a key component that acts as a ligand-dependent nuclear receptor-specific coactivator, and proved to play an important role in retinoic acid-dependent granulocytic and 1,25-dihydroxyvitamin D3-dependent monocytic differentiation of both normal hematopoietic precursor and acute promyelocytic leukemia cells, via retinoic acid receptor (RAR) and vitamin D receptor (VDR), respectively. MED1 was recently re-identified as a specific coactivator for GATA family activators and found to function in the process of GATA-1-mediated normal erythroblast differentiation. In this study, we analyzed the role of MED1 in erythroid differentiation of malignant erythroblastic (leukemic) cells. We used K562 human erythroleukemia cells as a model since GATA-1-mediated erythroid differentiation of K562 is efficiently induced with hemin. When K562 cells were exposed to 50nM hemin for 5 days, 44% of cells became positive for benzidine staining and the expression of the erythroid lineage-specific (and GATA-1-targeted) genes such as β-globin, γ-globin, porphobilinogen deaminase (PBGD) and 5-aminolevulinate synthase (ALAS-E) were strongly induced. The Mediator subunits including MED1 were also induced strongly together with these erythroid-specific genes in this process. Since the precedent study has demonstrated that Mediator subunits are induced during nuclear receptor-mediated myelomonopoiesis, and since genes whose products have physiological significance are often induced, Mediator might be employed in GATA-1-mediated erythroid differentiation of erythroleukemia cells as well. Then we focused on the GATA-1-specific coactivator MED1 among the Mediator subunits. In order to analyze the role of MED1, we first reduced the expression of endogenous MED1 in K562 cells with the MED1 knockdown small hairpin RNA (shRNA) expression vector. When the transiently knocked down cells (approximately 25% of the control) were treated with hemin, the number of benzidine-positive cells were half of the control in 3 days, indicating that hemin-induced erythroid differentiation was strongly inhibited. Further, the expression of direct target genes for GATA-1 such as β-globin, γ-globin, PBGD and ALAS-E were significantly reduced by approximately 70% in knockdown cells. Moreover, when the knockdown cells were cultured for 3 days without hemin, the basal expression levels of the erythroid lineage-specific genes such as β-globin, γ-globin and PBGD were significantly reduced by approximately 30%. We next asked if overexpressed MED1 might enhance erythroid differentiation. Since the MED1 domain near the N-terminus (a.a. 622 to 701) interacts with GATA-1 and that the N-terminal domain that contains two LxxLL nuclear receptor recognition motifs (NR box; aa. 592 to 703) is sufficient for in vitro and most in vivo transcription events, we tested the N-terminal truncation of MED1, N-MED1 (aa. 1 to 703), for overexpression. When N-MED1 was stably expressed in K562 cells after selection with G418, MED1 expression was boosted at least 2.2 folds of the control transfectant cells. When these cells were treated with hemin, benzidine-positive differentiated cells were 3-fold reduced and the expression of the erythroid differentiation marker genes (above) were approximately 2-fold increased in N-MED1 cells after one day. However, after 3 days, the number of benzidine-positive cells and the expression of erythroid marker genes in N-MED1 cells were saturated and caught up by the control cells. Thus, it appears that overexpressed MED1 enhances the erythroid differentiation in K562 cells. Together with the finding that GATA-1 is physically associated with MED1 and that MED1 is co-occupied with GATA-1 in the enhancer region, these results indicate that MED1 functions as a GATA-1-specific coactivator in erythroid differentiation of not only normal erythroblasts but erythroleukemic cells. MED1 also appears to be a key coactivator for two distinct lineages of normal and malignant hematopoiesis, namely myelomonopoiesis and erythropoiesis.

2019 ◽  
Vol 39 (13) ◽  
Author(s):  
David McClellan ◽  
Mattie J. Casey ◽  
Diana Bareyan ◽  
Helena Lucente ◽  
Christopher Ours ◽  
...  

ABSTRACTGrowth factor independence 1B (GFI1B) coordinates assembly of transcriptional repressor complexes comprised of corepressors and histone-modifying enzymes to control gene expression programs governing lineage allocation in hematopoiesis. Enforced expression of GFI1B in K562 erythroleukemia cells favors erythroid over megakaryocytic differentiation, providing a platform to define molecular determinants of binary fate decisions triggered by GFI1B. We deployed proteome-wide proximity labeling to identify factors whose inclusion in GFI1B complexes depends upon GFI1B’s obligate effector, lysine-specific demethylase 1 (LSD1). We show that GFI1B preferentially recruits core and putative elements of the BRAF-histone deacetylase (HDAC) (BHC) chromatin-remodeling complex (LSD1, RCOR1, HMG20A, HMG20B, HDAC1, HDAC2, PHF21A, GSE1, ZMYM2, and ZNF217) in an LSD1-dependent manner to control acquisition of erythroid traits by K562 cells. Among these elements, depletion of both HMG20A and HMG20B or of GSE1 blocks GFI1B-mediated erythroid differentiation, phenocopying impaired differentiation brought on by LSD1 depletion or disruption of GFI1B-LSD1 binding. These findings demonstrate the central role of the GFI1B-LSD1 interaction as a determinant of BHC complex recruitment to enable cell fate decisions driven by GFI1B.


Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1311-1311
Author(s):  
Etsuko Matsubara ◽  
Ikuya Sakai ◽  
Jun Yamanouchi ◽  
Taichi Azuma ◽  
Hiroshi Narumi ◽  
...  

Abstract Early hematopoietic zinc finger protein (EHZF), the human homolog to mouse Evi3, is a transcription factor with 30 zinc fingers and a highly conserved N-terminal FOG repression motif. The EHZF mRNA expression is abundant in early hematopoietic progenitors and declines during differentiation. In most of the leukemic cells from acute myelogenous leukemia patients, significant levels of EHZF mRNA are detected. These findings suggest that EHZF play an important role in hematopoietic differentiation. In the present study we investigated whether siRNA-mediated depletion of EHZF affected the erythroid differentiation of K562 cells. Three kind of EHZF siRNA were designed and their expression vectors were constructed. After transient expression of these siRNAs by electroporation, hemoglobinization of K562 cells were analyzed by Benzidine staining. In all of these siRNAs introduced cells, the levels of hemoglobinizations were abundant compared to control cells, which suggested that EHZF might influence the erythroid differentiation of K562 cells. To further analyze the effects of EHZF siRNA in erythroid differentiation, we established K562 clones in which EHZF was depleted by siRNA. EHZF siRNA expression vectors were introduced into K562 cells by electroporation and the expression of EHZF mRNA were examined by RT-PCR in G418 resistant clones. The depressions of EHZF mRNA in the clones were confirmed by Northern blotting. We isolated several EHZF depleted clones in each kind of EHZF siRNA introduced cells. Most of the EHZF depleted clones showed marked hemoglobinization by Benzidine staining compared to control clones, and surface expressions of glycophorin A were also increased in these clones. These results confirmed the relevant role of EHZF in erythroid differentiation of K562 cells. Comparison of mRNA expressions between an EHZF depleted clone and a control clone using DNA array showed increase expressions of hemoglobin alpha, beta, delta mRNA in EHZF depleted clone compared to control clone. Several transcription factors which are involved in erythroid differentiation also showed the difference mRNA levels between two clones. Studies are underway to elucidate the mechanisms underlying the role of EHZF in erythroid differentiation, the relationship to other transcription factors (GATA-1, GFi-1B, FOG-1 etc) which involved in erythroid differentiation.


1990 ◽  
Vol 10 (3) ◽  
pp. 1209-1216 ◽  
Author(s):  
J Wu ◽  
G J Grindlay ◽  
P Bushel ◽  
L Mendelsohn ◽  
M Allan

The human epsilon-globin gene has a number of alternative transcription initiation sites which correspond with regions of DNase I hypersensitivity upstream of the canonical cap site. Transcripts originating from the promoters located -4.3/-4.5 and -1.48 kilobase pairs (kbp) and -900 and -200 base pairs (bp) upstream of the major epsilon-globin cap site can, at certain stages of erythroid differentiation, extend through the gene and are polyadenylated. The 350-bp PolIII transcripts, originating within the Alu repetitive element -2.2 kbp upstream of the cap site, extend in the opposite direction from the gene, are nonpolyadenylated, nucleus confined, and are detectable only in mature K562 cells or mature embryonic red blood cells where the epsilon-globin major cap site is maximally transcribed. Fragments containing the promoters located between -4.5 and -4.3 kbp upstream of the gene down regulate transcription from the epsilon-globin gene 20- to 30-fold in a transient expression assay in which both erythroid and nonerythroid cell lines were used. This occurs only when the direction of transcription from the -4.3/-4.5-kbp promoters is towards the gene, and we hypothesize that down regulation is caused by transcriptional interference. Fragments containing the Alu repetitive element -2.2 kbp upstream of the gene can overcome down regulation of the epsilon-globin gene by the -4.5-kbp element when interposed in the direct orientation between this element and the epsilon-globin gene.


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

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2727-2727
Author(s):  
Mitsuhiro Ito ◽  
Norinaga Urahama ◽  
Akiko Sada ◽  
Kimikazu Yakushijin ◽  
Katsuya Yamamoto ◽  
...  

Abstract The TRAP/Mediator complex, the metazoan counterpart of the yeast Mediator complex, is master transcriptional regulatory complex composed of approximately 30 subunits. It was originally isolated as a thyroid hormone receptor (TR)-associated protein (TRAP) complex that mediates TR-activated transcription from DNA templates in vitro and probably acts in vivo after the action of other receptor-interacting coactivators involved in chromatin remodeling. The TRAP220/MED1 subunit of the TRAP/Mediator complex is proposed to act on a variety of major and specific biological events, including growth, differentiation and homeostasis, through physical interaction with nuclear receptors. The vitamin D receptor (VDR) and retinoic acid receptor (RAR), coupled with retinoid X receptor (RXR), are nuclear receptors which have important roles for monopoiesis and granulopoiesis, respectively. In this study, we present the functional role of TRAP220/MED1 in nuclear receptor-mediated monopoiesis and granulopoiesis. Since TRAP220 knockout (Trap220-/-) mice were mortalities during the early embryonic period before definitive hematopoiesis within the hepatic primordia becomes dominant, the function of TRAP220/MED1 in adult hematopoiesis was mostly unknown. However, these embryos appeared to have normal composition of nucleated erythroid cells. Therefore, the E9.0 yolk sac-derived hematopoietic precursor cells were used to differentiate into definitive hematopoietic colony forming units within the methylcellulose blood cell culture. The number of monocytic colonies (CFU-M) was significantly lower in knockouts than in wild type controls, while the numbers of other types of colonies (CFU-GEMM, CFU-GM, CFU-G and CFU-E) were comparable. Hence, TRAP220/MED1 appeared to be indispensable for optimal monocytic differentiation. Next, the HL-60 acute promyelocytic leukemia cells were used to elucidate directly and mechanistically the roles of TRAP220/MED1 in RAR- and VDR-dependent differentiation of the hematopoietic precursor cells into granulocytic and monocytic lineage cells. The expression of the TRAP220/MED1 subunit as well as other TRAP/Mediator subunits was induced when the cells were treated with their ligands, all-trans retinoic acid and 1,25-dihydroxyvitamin D3. Flow cytometric analyses showed that HL-60 cells, wherein TRAP220/MED1 was down-regulated, did not differentiate efficiently into monocytes and granulocytes by stimulation with 1,25-dihydroxyvitamin D3 and all-trans retinoic acid, correspondingly. The expression of direct target genes of VDR or RAR, as well as the differentiation marker genes, was low in the knockdown cells by stimulation with these ligands. In contrast, 12-O-tetradecanoylphorbol-13-acetate (TPA)- and dimethylsulphoxide (DMSO)-mediated monocytic and myeloid differentiation, which bypasses nuclear receptor-mediated signaling pathways, was not affected in knockdown cells. Collectively, these results indicated an indispensable role of TRAP220/MED1 in the optimal VDR- and RAR-mediated myelomonocytic differentiation processes in mammalian hematopoiesis.


2001 ◽  
Vol 264 (2) ◽  
pp. 193-200 ◽  
Author(s):  
Wilhelm Woessmann ◽  
Nahid F. Mivechi

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3443-3443
Author(s):  
AI Inoue ◽  
Tohru Fujiwara ◽  
Yoko Okitsu ◽  
Noriko Fukuhara ◽  
Yasushi Onishi ◽  
...  

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.


Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4730-4730
Author(s):  
Qian Xiong ◽  
Zhaojun Zhang ◽  
Hongzhu Qu ◽  
Xiuyan Ruan ◽  
Hai Wang ◽  
...  

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.


2011 ◽  
Vol 227 (3) ◽  
pp. 1123-1129 ◽  
Author(s):  
Wen-Hui Tsai ◽  
Chung-Hung Shih ◽  
Hung-Yi Wu ◽  
Hong-Yu Chien ◽  
Yee-Chen Chiang ◽  
...  

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