The GATA Site-Dependent Hemogen Promoter Is Transcriptionally Regulated by GATA1 in Hematopoietic and Leukemia Cells
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Leukemia (2006) 20, 417–425 & 2006 Nature Publishing Group All rights reserved 0887-6924/06 $30.00 www.nature.com/leu ORIGINAL ARTICLE The GATA site-dependent hemogen promoter is transcriptionally regulated by GATA1 in hematopoietic and leukemia cells LV Yang1,9, J Wan2,9,YGe3,4,,ZFu2, SY Kim5, Y Fujiwara6, JW Taub3,7, LH Matherly,3,4, J Eliason8 and L Li1,2 1Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI, USA; 2Department of Internal Medicine, Wayne State University School of Medicine, Detroit, MI, USA; 3Experimental and Clinical Therapeutics Program, Barbara Ann Karmanos Cancer Institute, Detroit, MI, USA; 4Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI, USA; 5Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, USA; 6Children’s Hospital, Harvard Medical School, Boston, MA, USA; 7Children’s Hospital of Michigan, Detroit, MI, USA and 8Asterand Inc., Detroit, MI, USA Hemgn (a gene symbol for hemogen in mouse, EDAG in human pancreas, tonsil, colon and peripheral blood mononuclear and RP59 in rat) encodes a nuclear protein that is highly cells.2 EDAG is highly expressed in acute myeloid leukemia expressed in hematopoietic tissues and acute leukemia. To characterize its regulatory mechanisms, we examined the (AML) and acute lymphoblastic leukemia (ALL) and has been activities of a Hemgn promoter containing 2975 bp of 50 flanking demonstrated to be involved in erythroleukemia differentiation, sequence and 196 bp of 50 untranslated region (50 UTR) survival and apoptosis and cell transformation of NIH3T3 sequence both in vitro and in vivo: this promoter is preferen- cells.4,5 ALL these studies suggest that overexpression of EDAG tially activated in a hematopoietic cell line, not in nonhemato- in acute leukemia may play an important role in leukemogensis. poietic cell lines, and is sufficient to drive the transcription of a More recently, assocaition of overexpression of EDAG and no lacZ transgene in hematopoietic tissues in transgenic mice. Mutagenesis analyses showed that the 50 UTR including two remission in de novo AML has been reported: this result suggests highly conserved GATA boxes is critical for the promoter that EDAG may play a modulator role in AML and could be a 6 activity. GATA1, not GATA2, binds to the GATA binding sites new target in the treatment of AML. and transactivates the Hemgn promoter in a dose-dependent A number of hematopoietic-specific transcription factors are manner. Furthermore, the expression of human hemogen found to regulate the molecular pathways of hematopoiesis (EDAG) transcripts were closely correlated with levels of through direct binding to regulatory elements of key hemato- GATA1 transcripts in primary acute myeloid leukemia speci- 7 mens. This study suggests that the Hemgn promoter contains poietic genes. For instance, GATA proteins bind to the critical regulatory elements for its transcription in hematopoie- consensus sequence WGATAR in the promoter regions of a 8 tic tissues and Hemgn is a direct target of GATA1 in leukemia variety of erythroid genes. Members of the GATA family are cells. generally categorized as hematopoietic GATA factors and Leukemia (2006) 20, 417–425. doi:10.1038/sj.leu.2404105; cardiovascular GATA factors.9 Among the former, GATA1 and published online 9 February 2006 GATA2 play critical roles in hematopoiesis, particularly Keywords: hemogen; EDAG; GATA1; promoter; AML; leukemia erythropoiesis, whereas GATA3 is primarily involved in T-cell development.10 To gain further insights into the regulatory mechanism(s) of Hemgn, we isolated and characterized a Hemgn promoter. In Introduction this report, we show that the Hemgn promoter exhibits hematopoietic-specific activities both in vitro and in vivo. Hemgn is highly expressed in the hematopoietic system.1–3 The Mutagenesis, gel shifting and chromatin immunoprecipitation developmental expression pattern of Hemgn marks its hemato- (ChIP) analyses were used to demonstrate the transcriptionally poietic ontogeny. In early embryogenesis, the expression of important roles of the GATA boxes and their binding protein, Hemgn is detected in blood islands of the yolk sac and in GATA1, in the Hemgn 50 untranslated region (50 UTR). Finally, primitive blood.1 In later development, the expression is we report that expression of GATA1 and EDAG appear to be sequentially localized in active hematopoietic sites, such as coregulated in primary AML cells. fetal liver and bone marrow.1,3 Among hematopoietic lineages, Hemgn is predominantly expressed in erythroid and megakaryo- cytic precursor cells; it is also expressed in hematopoietic Materials and methods stem cells and early progenitors.1,3 However, it is absent in 1,3 matured lymphocytes. Just like Hemgn, the human hemogen Hemgn promoter isolation and sequence analysis orthologue (EDAG) exhibits specific expression in human A BAC clones (# 567P14) containing the full-length Hemgn gene hemotopoietic tissues and cells, including adult bone marrow 1 was screened from a mouse BAC genomic library CITB-CJ7-B and fetal liver. However, no EDAG transcripts are detected in (Research Genetics, Catalogue # 96021) (for details, see adult liver, heart, brain, skeletal muscle, kidney, spleen, Supplementary Information). A Hemgn promoter spanning 3171 bp (positions À2975 to þ 196) was amplified with pfu Correspondence: Dr L Li, 421 E. Canfield Ave. #1107, Detroit, Turbo DNA polymerase (Stratagene, CA, USA) using the BAC as Michigan 48201, USA. a template. The Hemgn promoter was confirmed by DNA E-mail: [email protected] 9These authors contribute equally to the paper. sequencing. The sequence homology comparison of human and Received 20 November 2005; accepted 29 November 2005; mouse Hemgn genes was performed using VISTA (http:// published online 9 February 2006 dcode.org) (Figure 1b).11 The sequences of mouse and human Transcriptional control of the hemogen promoter by GATA1 LV Yang et al 418 Figure 1 Sequence analyses of mouse and human hemogen promoters. (a) Schematic diagram of exon and intron distribution of hemogen. Please note that 1 h and 1t refer to the first exons of hematopoietic and testicular isoforms, respectively. (b) VISTA plot of DNA sequence conservation between mouse and human hemogen. The homology analysis is set at 70% identity within a 20-bp window: the blue and yellow peaks indicate coding sequence and UTRs of the hemogen cDNA, respectively; the red and pink peaks indicate evolutionarily conserved sequence in the promoter and intronal regions, respectively. (c) The alignment of proximal promoters and the first exons of mouse (GenBank accession#: DQ204723) and human hemogen. The conserved nucleotides are marked with *. The TATA box is labeled in red color; GATA boxes in blue color; and the translation initiation codon ATG in green color. The GATA boxes are boxed, and the sites for AML-1, Evi-1 and Ets are indicated by lines (Please refer the html file on the web for the colored Figure 1). proximal promoters and the first exons were aligned using CMS, an acute myeloid leukemia cell line (established from a ClustalW (http://www2.ebi.ac.uk/clustalw). The MatInspector 2-year-old girl with acute megakaryocytic leukemia (AMkL))13 program (http://www.gene-regulation.com/) was used to identify was a gift from Dr A Fuse (National Institute of Infectious putative hematopoietic-specific transcription factor binding sites Diseases, Tokyo, Japan). The AMkL cell line, Meg-01, was in the sequence (Figure 1c). obtained from the American Type Culture Collection (Manassas, VA, USA).14 Single colonies of CMS cells with stably transfected pcDNA3-GATA115 were screened for GATA1 expression by real Cell culture, transfections, and reporter assays time RT–PCR (for details see Supplementary Information). 10T1/2 (a mouse fibroblast cell line), PAC1 (a rat smooth muscle cell line), COS-7 (a monkey kidney cell line), C2C12 (a mouse skeletal muscle cell line) and K562 cells (a human erythroleu- Construction of Hemgn promoter deletion and mutant kemia cell line) were co-transfected with indicated plasmids and constructs 200 ng of pRL-SV40 Renilla Luciferase reporter (as an internal Deletion mutants of the Hemgn promoter were cloned into the control). The culture condition and transfection conditions are luciferase reporter vector pXP1.16 The constructs were verified detailed in Supplementary Information. Cells were harvested by sequencing. The primers for amplifying the corresponding 24 h after transfection and luciferase activities were measured Hemgn promoter and its deletion mutants were: 0 using Dual-Luciferaset reporter assay system (Promega, Madi- pHemgnÀ2975 þ 196:5-CGC GGA TCC CAC ATC AGA GAC son, WI, USA). ACC TTG CC and 50-CCG CTC GAG GGT ATT GGC TTT GAC 0 Drosophila Mel-2 cells (D. Mel-2, a Schneider S2 insect cell TTC AC; pHemgnÀ831 þ 196:5-CGC GGA TCC TTG AAC TAG line) from Invitrogen (Carlsbad, CA, USA) were co-transfected GGT GGC TCT GG and 50-CCG CTC GAG GGT ATT GGC TTT 0 with 1 mg of the Hemgn-luciferase reporter gene construct GAC TTC AC; pHemgnÀ404 þ 196:5-CGC GGA TCC AAC AGC (pHemgn-2975 þ 196luc) and GATA1 (50–400 ng pPacGA- CTA CCT AGG AAG AG and 50-CCG CTC GAG GGT ATT GGC 12 0 TA1) using Fugenet 6 reagent (Roche Diagnostics Corpora- TTT GAC TTC AC; pHemgnÀ2975 þ 12:5-CGC GGA TCC CAC tion, IN, USA). Cells were harvested after 24 h for luciferase ATC AGA GAC ACC TTG CC and 50-CCG CTC GAG ACA CTG assays using the Single Luciferase Assay System (Promega). CAC AGG TGT GAG GG. Luciferase activities were normalized to total cell protein, The core GATA binding sequences in the plasmid measured by the Bio-Rad protein assay system. pHemgnÀ2975 þ 196luc construct were mutated to cATA, a Leukemia Transcriptional control of the hemogen promoter by GATA1 LV Yang et al 419 change reported to abolish GATA binding,17–19 using the Real time RT-PCR Quickchanget site-directed mutagenesis kit (Stratagene, La Total RNAs were extracted from mock-transfected, and GATA1 0 Jolla, CA, USA).