The leukemia associated nuclear corepressor ETO homologue genes MTG16 and MTGR1 are regulated differently in hematopoietic cells
© Ajore et al; licensee BioMed Central Ltd. 2012
Received: 26 November 2011
Accepted: 23 March 2012
Published: 23 March 2012
MTG16, MTGR1 and ETO are nuclear transcriptional corepressors of the human ETO protein family. MTG16 is implicated in hematopoietic development and in controlling erythropoiesis/megakaryopoiesis. Furthermore, ETO homologue genes are 3'participants in leukemia fusions generated by chromosomal translocations responsible of hematopoietic dysregulation. We tried to identify structural and functional promoter elements of MTG16 and MTGR1 genes in order to find associations between their regulation and hematopoiesis.
5' deletion examinations and luciferase reporter gene studies indicated that a 492 bp sequence upstream of the transcription start site is essential for transcriptional activity by the MTG16 promoter. The TATA- and CCAAT-less promoter with a GC box close to the start site showed strong reporter activity when examined in erythroid/megakaryocytic cells. Mutation of an evolutionary conserved GATA -301 consensus binding site repressed promoter function. Furthermore, results from in vitro antibody-enhanced electrophoretic mobility shift assay and in vivo chromatin immunoprecipitation indicated binding of GATA-1 to the GATA -301 site. A role of GATA-1 was also supported by transfection of small interfering RNA, which diminished MTG16 expression. Furthermore, expression of the transcription factor HERP2, which represses GATA-1, produced strong inhibition of the MTG16 promoter reporter consistent with a role of GATA-1 in transcriptional activation. The TATA-less and CCAAT-less MTGR1 promoter retained most of the transcriptional activity within a -308 to -207 bp region with a GC-box-rich sequence containing multiple SP1 binding sites reminiscent of a housekeeping gene with constitutive expression. However, mutations of individual SP1 binding sites did not repress promoter function; multiple active SP1 binding sites may be required to safeguard constitutive MTGR1 transcriptional activity. The observed repression of MTG16/MTGR1 promoters by the leukemia associated AML1-ETO fusion gene may have a role in hematopoietic dysfunction of leukemia.
An evolutionary conserved GATA binding site is critical in transcriptional regulation of the MTG16 promoter. In contrast, the MTGR1 gene depends on a GC-box-rich sequence for transcriptional regulation and possible ubiquitous expression. Our results demonstrate that the ETO homologue promoters are regulated differently consistent with hematopoietic cell-type- specific expression and function.
The highly conserved ETO (Eight-Twenty-One) corepressor gene family traced to Drosophila ETO homologue Nervy contains the myeloid translocation gene (MTG) 16 (MTG16) ( gene name CBFA2T3) or murine ETO-2 (gene name cbfa2t3), MTG8 ( gene name CBFA2T1, RUNXT1, ETO) and MTG-related protein 1 (MTGR1) ( gene name CBFA2T2). The ETO homologues do not bind directly to DNA but rather function as protein scaffolds and bring about gene repression indirectly through an interplay with multiple transcriptional regulatory proteins [2, 3]. MTG16 has been shown to interact with canonical transcription factors such as PLZF, BCL6, TAL1/SCL, Gfi1and Heb [4–9], and MTGR1 with TAL1/SCL . The ETO homologues also recruite chromatin regulating proteins such as nuclear corepressors [3, 11, 12] and histone deacetylases (HDACs) that catalyze chromatin modifications, resulting in transcriptional repression. Importantly, ETO homologue genes are involved in chromosomal translocations of acute leukemia, MTG16 in the generation of the AML1- MTG16 fusion gene of t(16;21)  in patients with therapy-induced leukemia, ETO in the generation of the AML1-ETO fusion gene of t(8;21) [14, 15] and MTGR1 in the generation of AML1-MTGR1 fusion gene of t(20;21) . The leukemic fusion proteins so encoded disrupt normal function of transcriptional regulators, to promote dysregulation of hematopoietic cell differentiation .
Expression of the MTG16 (murine ETO-2) corepressor is confined to early hematopoiesis whereas MTGR1 is present throughout hematopoietic maturation . Thus, ETO-2 increases during differentiation of murine embryonic stem cells into hematopoietic cells  suggesting a role in the development of the blood cell compartment. This is consistent with MTG16 being the most highly expressed ETO homologue gene in the stem/progenitor cell compartment . Furthermore, MTG16/ETO-2 has a role in controlling erythropoiesis and megakaryopoiesis. In this context, MTG16/ETO-2 is incorporated in Ldb1 (LIM domain- binding protein 1) and TAL1 (T-cell acute lymphocytic leukemia protein 1) containing transcription factor complexes [5, 8, 20, 21], mediating transcriptional suppression. As a heteromer with MTG16, also MTGR1 complexes with TAL1 in erythroid cells, enhancing transcriptional repression . The ETO-2 recruitment within nuclear complexes may determine the onset of terminal erythroid/megakaryocytic differentiation , while the downmodulation of MTG16/ETO-2 at late stages of erythropoiesis suggests that decreased transcriptional repression is necessary for late, terminal erythroid differentiation to occur. For example, in erythroid MEL cells, induction of differentiation is accompanied by dissociation of nuclear protein complexes concomitant with a decrease in the relative level of ETO-2 . Moreover, shedding the negative regulator ETO-2  transforms the Ldb complex into a positive regulator of final erythroid differentiation, supporting the notion that multi-protein complexes may keep erythroid target genes suppressed until corepressor ETO-2 is eliminated at final differentiation . Similarly, in megakaryopoiesis the ETO-2 expression is restricted to immature cells consonant with a function in repression of genes that should be only terminally expressed . Finally, a physiological hematopoietic role for MTG16 has been proven by results from targeted disruption of murine MTG16 (ETO-2). Although no complete block in hematopoietic differentiation was discovered by ETO-2 knockout, a requirement was demonstrated for ETO-2 in fate decisions, proliferation and stress- dependent hematopoiesis. As a corroboration of functional differences between ETO homologues, targeted disruption of MTGR1 showed no phenotypic consequences for hematopoiesis .
Endogenous cell-type-specific hematopoietic expression of the ETO homologues  suggests differences in gene regulation. The ETO promoter is regulated by the GATA transcription factor in erythroid/megakaryocytic cells . We now examined functional promoter elements upstream of the coding sequences for MTG16 and MTGR1. A critical role in transcriptional regulation was shown for an evolutionary conserved GATA binding site of the MTG16 promoter. In contrast, MTGR1 has a strong TATA-less promoter with multiple GC box-rich regions of possible importance for constitutive expression. Our results show that the ETO homologue promoters are regulated differently, consistent with cell-type- specific expression.
Identification of transcriptional start site and sequence analysis of proximal promoters of MTG16 and MTGR1
However, a conserved GC box close to the transcriptional start site (Figure 1A) may regulate transcriptional initiation. Sequence comparisons showed the GATA binding site at -301 to be conserved between eight species (Figure 1B).
Functional evaluation of MTG16 promoter
A GATA consensus binding site is critical for MTG16 promoter function in erythroid/megakaryocytic cells
The GATA-301 site is necessary but not sufficient for transactivation of the MTG16 promoter as some deletion constructs (-359, -339) that retain the GATA -301 site do not retain transcriptional activity (Figure 4). However, all mutation experiments were done with the longer -668 construct and its promoter activity was blocked completely by mutation of the GATA-301 site. An element upstream of the GATA-301 site that cooperates with GATA bound to -301 might explain our results. However, no such element was identified as disruption of all upstream potential binding sites that were identified e. g. the SP1 -425, ETS1 -491, ETS1 -419, SP1 -316, and GATA -356 sites did not affect the reporter signal (data not shown). Disruption of the MZF1 -191 sites downstream of the GATA-301 site did not affect the luciferase signal either (data not shown). Mutation of the downstream MZF1 - 251 increased the signal except in HEL cells (data not shown), suggesting that this site possibly mediates binding to some transcriptional repressor.
GATA-1 binds in vitro/in vivo to the -301 consensus site in the MTG16 promoter
In addition to the specific shift, another band is clearly visible in the EMSA (Figure 6). This band-shift is unspecific since it is unaffected or only weakly affected by the competitor probe. However, it obviously contains GATA-1 protein as it is lost by supershifting with anti GATA-1 (Figure 6).
The SP1 -425 and the MZF1 -251 binding sites of the MTG16 promoter were also examined by EMSA. No supershifting was observed by appropriate antibodies suggesting lack of binding of these transcription factors to the respective consensus binding sites (data not shown).
Taken together, the results from EMSA/supershift assays demonstrate GATA-1 binding in vitro to the GATA -301 binding site and results from ChIP assays demonstrate binding in vivo of both GATA-1 and GATA-2 to the putative MTG16 promoter. This result further supports a functional role at least for GATA-1 in activation of the MTG16 promoter, consistent with the results of the mutagenesis studies shown in Figure 5.
Overexpression of GATA-1
Repression of GATA-1 by siRNA and inhibition of GATA-1 transcriptional activity with HERP2 leads to decreased MTG16 promoter activity
Functional evaluation of the MTGR1 promoter
To define the promoter in more detail, a series of constructs was generated by sequential deletions from the 5'end of the -989 +52 bp region after which transcriptional activity was analyzed in HEL cells (Figure 11). Deletion to -499 did not affect transcriptional activity, but further deletion reduced the luciferase reporter expression. Thus the sequence from -499 to +52 bp retained full transcriptional activity and therefore is likely to contain the core proximal MTGR1 promoter. Further sequential 5' deletions of the -499 to +52 bp region resulted in a step by step decreasing luciferase reporter signal (Figure 11) suggesting cooperation between factors binding to different regions of the promoter.
Site-directed mutagenesis was used to examine whether potential transcription factor binding sites contributed to promoter transactivation. The -308 to +52 bp region retains approximately two thirds of the transcriptional activity of the fully active promoter as was shown in Figure 11. This region is GC box-rich with 9 SP1 consensus binding sites (Figure 2) located immediately upstream of the transcription start site at -146 bp consistent with the housekeeping gene character of MTGR1 w ith ubiquitous expression. However, disruption of individual SP1 sites; SP1-285, SP1-243, SP1-229, SP1-225, SP1-213, SP1-206, SP1-192, SP1-178, SP1-170; did not affect reporter gene activity relative to intact MTGR1 promoter (data not shown), suggesting that SP1 is not important for promoter activity. However, it is possible that multiple active SP1 binding creates a redundancy in SP1 binding and elimination of several SP1 binding sites is needed to reveal their functional relevance. Disruption of the -335 ETS1, the -276 ETS1 and the -301 YY1 sites did also not affect promoter activity (data not shown). Overall, our results indicate that a GC-box-rich sequence with multiple SP1 sites is important for transcriptional regulation of MTGR1 promoter.
AML1-ETO represses MTG16 and MTGR1 gene reporters
The negative transcriptional regulator ETO was originally discovered as the 3' participant in the leukemia chromosomal translocation t(8;21) . Now, ETO and its homologues MTG16 and MTGR1 are known to be involved in transcriptional regulation of genome-wide targets. To explain cell-type-specificity of ETO homologue expression and function  exploration of promoter regulation is warranted. The human ETO proximal promoter is regulated by the GATA-1 transcription factor in an erythroid/megakaryocytic-specific manner . Here, we continued to identify essential cis-acting elements and trans-acting factors also of the promoters of MTG16 and MTGR1 within human hematopoietic cells.
MTG16 promoter regulation
TATA and CAAT-like promoter motifs facilitate interactions with general transcription factors and are thus involved in initiation of transcription. The proximal MTG16 promoter investigated lacks these motifs. GC-boxes are known to regulate the utilization of start sites in TATA-less promoters [32–35]. Therefore, the conserved GC-box situated close to start site (Figure 1A) may be involved in transcriptional initiation. Among hematopoietic cell lines, the promoter activity was strongest in the erythroid HEL/TF-1, erythroid/megakaryocytic UT-7 and megakaryocytic MEG-01 cells consistent with a role of MTG16 in erythropoiesis/megakaryopoiesis [5, 8, 20, 21]. Deletion analysis of the proximal MTG16 promoter defined a 492 bp region upstream of the transcription start to be required for maximal promoter activity. This region includes a potential GATA binding site (-301), which is strongly conserved between species. Disruption of the GATA -301 binding site, repressed transactivation of the MTG16 promoter. The importance of this cis-acting binding site was confirmed by results from examination in vitro with EMSA, which revealed specific GATA-1 binding. Further, in vivo binding of both GATA-1 and GATA-2 to this region of the promoter was confirmed by ChIP/PCR analysis. Taken together these results demonstrate binding of the GATA-1 transcription factor to the MTG16 promoter and strongly support a role for GATA-1 in transcriptional regulation of the MTG16 gene in hematopoietic cells.
MTGR1 promoter regulation
A 499 bp region upstream of the translation start site of MTGR1 was defined as sufficient for maximal activity in erythroid HEL cells. Upon analysis using different cell lines, we found that the promoter is active in most of them, which is in agreement with ubiquitous presence of native MTGR1 gene product. Furthermore, the lack of classical TATA or CCAAT boxes, the presence of multiple SP1 sites and a CpG island covering the transcriptional initiation site of the MTGR1 promoter (Figure 2) are similar to characteristics of a housekeeping gene , expected to be generally active in a non-tissue specific manner. Few potential binding sites for known transcription factors were found in the -499 promoter, rather a GC-box-rich sequence stretch with multiple SP1 binding sites is a prominent part of the promoter. Importantly, the GC-box-rich region was responsible for most of the transcriptional activity. However, we were not able to inhibit promoter activity by the elimination of individual SP1 binding sites. Possibly, individual SP1 binding sites may be equally involved in guaranteeing constitutive MTGR1 expression. Further 5'deletion of the -499 promoter region showed gradually decreasing promoter activity observed upon successive deletions, indicating transcriptional cooperation between promoter sequences consistent with cooperating SP1- binding sites.
ETO homologues in hematopoiesis
The regulation of both the ETO and the MTG16 promoter (this work) by GATA-1 is consistent with a role of human ETO/MTG16-mediated gene suppression at a phase of erythropoiesis/megakaryopoiesis when GATA-1 levels are high [37, 38]. However, despite shared trans-activation, MTG16 and ETO are differently expressed in human hematopoiesis ; MTG16 is downmodulated at late stages of erythropoiesis whereas ETO is expressed transiently in (human) erythropoiesis. Therefore, cell-type-specific expression of these ETO homologues may be governed also by trans-activating factors other than GATA. Although both MTG16 and ETO are expressed in human erythroid cells  yet the role of ETO in murine erythropoiesis is not yet supported .
GATA-1 and GATA-2 are expressed reciprocally during development of the erythropoietic lineage, GATA-2 decrease is followed by GATA-1 increase . GATA-2 seems to be essential for the development of early and definitive erythropoiesis/megakaryopoiesis and GATA-1 for terminal erythroid/megakaryocytic maturation [26–28]. The present results are consistent with upregulation of MTG16 as a result of rising GATA-1 levels. Furthermore, results from genome-wide analyses  are consistent with early GATA-2 induction of murine ETO-2 during erythropoiesis. These analyses also showed that most occupied sites are located away from promoters indicating that GATA factors may be involved in long- range control by occupancy at non-promoter regions.
Our finding of in vitro binding of GATA-1 but lack of GATA-2 binding to the GATA -301 binding site raises questions about the relative importance of GATA-1/GATA-2 in MTG16 promoter regulation. GATA-2 is reported to contribute in hematopoietic stem cell and progenitor survival  and GATA-1 in erythroid cell survival [42, 43]. Furthermore, genetic complementation studies show that GATA-1 can replace GATA-2 [44, 45]. A lack of binding in EMSA experiments suggests that GATA-2 may not be a primary transcription factor to initiate nucleation of the transcription complex at the MTG16 promoter. Possibly, GATA-2 has a weaker binding affinity than GATA-1. However, our finding in ChIP assay of the binding of both GATA-1 and GATA-2 to MTG16 promoter suggests that GATA-1 and GATA-2 cooperate. This is supported by results from in vivo studies by ChIP-seq with human K562 erythroleukemia cells  showing chromatin occupancy by both GATA-1 and GATA-2 at the conserved GATA motif corresponding to the -301 site of the MTG16 promoter. In addition, a role of GATA-2 in stimulating murine ETO-2 expression was also suggested . Thus, GATA-2 was found to occupy a conserved GATA binding site in the murine erythroid G1E cell, which lack GATA-1 expression; the occupied murine site corresponds to the human -301 GATA binding site of MTG16 promoter. Overall, available data suggest a role of both GATA-1 and GATA-2 in MTG16 promoter activation.
Murine MTG16 (ETO2) is involved in erythroid progenitor expansion , which is coupled to inhibition of differentiation. This is also the case in megakaryopoiesis where ETO2 represses inappropriate early expression of terminal genes  coupled to restrained differentiation of immature megakaryocytes. MTG16 and ETO show reciprocal expression during in vitro-induced human erythropoiesis; MTG16 decreases and ETO increases during the peak of erythropoiesis . As both promoters are regulated by GATA-1, specific negative autoregulation of MTG16 may explain the difference. This is consonant with a role for MTG16 in repressing genes associated with terminal erythroid differentiation thereby inhibiting differentiation and a role for ETO in repressing genes associated with self-renewal and proliferation thereby supporting erythroid differentiation.
Suppression of MTG16/MTGR1 promoter by AML1-ETO
The leukemic AML1-ETO fusion protein [14, 15] binds to the promoter region of a number of genes resulting in transcriptional suppression . However, some genes are affected by AML1-ETO in an indirect manner and do not bind the fusion protein directly [30, 46]. The observed suppression of the MTG16 and the MTGR1 promoter by AML1-ETO is likely to be indirect as binding sites for AML1 were not detectable on the promoters. Furthermore, a potential mechanism for the MTG16 promoter inhibition by AML1-ETO would be suppression of GATA-1 through impaired acetylation . Suppression of MTG16 is consistent with the low MTG16 promoter reporter signal observed in AML1-ETO-positive Kasumi cells, which showed very low MTG16 transcripts (Figure 3), whereas MTGR1 transcripts were high in this cell line (Figure 12). AML1-ETO-mediated suppression of the ETO promoter has also been reported  and suggested to facilitate the AML1-ETO- induced block of erythroid lineage commitment [47, 48]. AML1-ETO-mediated repression of MTG16 may have a similar effect because of blocking MTG16/ETO2-mediated erythroid progenitor expansion . The repression of all ETO homologue genes by AML1-ETO may have a general role in inhibition of hematopoietic differentiation in t(8;21) leukemia.
This work shows differences in promoter regulation between MTG16 and MTGR1 that may explain differences in hematopoietic expression of these genes. Evolutionary conserved GATA binding sites are critical in transcriptional regulation of both MTG16 (this work) and ETO promoters. The MTGR1 promoter has a GC box-rich sequence with multiple SP1 binding sites, which may secure transcriptional regulation and ubiquitous expression of this gene.
Myelomonocytic U-937, erythroid HEL and TF-1, megakaryocytic MEG-01 cells were grown in RPMI-1640 medium supplemented with 10% Fetal Bovine Serum (FBS) (Gibco BRL, Life Technologies, Rockville, MD, USA); TF-1 cells also received 20 ng/ml GM-CSF (R&D Systems, MN, USA). The erythroid/megakaryocytic UT-7 cells were grown in MEM Alpha modification medium with L-Glutamine and Nucleosides (PAA Laboratories GmBh, Austria) supplemented with 20% FBS and 30 ng/ml GM-CSF. The acute myeloid leukemia Kasumi-1, Burkitt's lymphoma Raji, and promyelocytic HL-60 cells were supplemented with 20% FBS. Monkey kidney COS cells were grown in DMEM medium with 10% FBS supplemented with high glucose (4.5 g/L) and L-glutamine.
Identification of the transcription start site of the MTG16 gene
The 5'-end (transcription start) of the mRNA was identified by 5'-RACE carried out with mRNA prepared from HEL cells using Oligotex Direct mRNA mini Kit (Qiagen, Hilden, Germany), using the First choice RNA Ligase mediated (RLM)-RACE kit (Ambion Inc., TX, USA). Nested PCR of RACE reactions was performed with adapter primer 5'-CGCGGATCCGAACACTGCGTTTGCTGGCTTTGATG-3' and nested gene specific primer 5'-GCCTTAGCTTTCCTGTCCACTGG-3'. RACE- products were cloned into pGEM-T Easy Vector system (Promega Corporation, WI, USA) and sequenced.
Amplification of MTG16 promoter region
A 1208 bp region upstream of the translation start (from -1264 to -57 bp) was amplified from human genomic DNA by PCR. The forward and reverse primers used were 5'-TGAGGCGGTACCACCTCCAGCCATGGCATC -3' and 5'-TGCCTAGATCTACCTCCTGCAGCCTTGAGG -3', respectively. Regions corresponding to -905 to -57 and -820 to -57 bp were amplified from the 1208 bp fragment by nested PCR with forward primers 5'- TATGGTACC GTGGGGGAGCCCTGCTGTCTCCACA -3' (KpnI restriction site underlined),5'- AGGCGGGAGGTACC TTGAGGACAGGTCAGG -3' (KpnI restriction site underlined), and a common reverse primer 5'-TGCCTAGATCT ACCTCCTGCAGCCTTGAGG -3' (BglII restriction site underlined).
Sequential 5' deletions of the -820 to -57 bp promoter region were generated by PCR from the cloned genomic DNA as template to generate -668 to -57, the -512 to -57, the -359 to -57 bp, the -339 to -57, the -299 to -57 and the -219 to -57 regions. Forward primers were 5'-TA AGGTACC CTGGGGAAAGCTCGGCTGAC-3', 5'-TATGGTACC CCTGCCCTGGGGCCGAGG -3', 5'-CCAGGTACC TCCTGTCAGGGAAGTGGCGG-3', 5'-TATGGTACCGGGCGCAGCTGCAGGCC
3', 5'- TATGGTACC TCACGGGGACACAGCTGGC-3' and 5'-TATGGTACC ATG AGCCCCAGGGCTCCCACCC -3' with KpnI restricton sites underlined. The common reverse primer is the same as used for amplification of the -1208 to -57 bp region. All sequences were verified by sequencing.
Amplification of MTGR1 promoter region
A 5' flanking region of 1965 bp from -1913 to +52 bp was amplified from human genomic DNA by PCR. The forward and reverse primers used were 5'-TATTGGTACCGCAACACCATGTCTGGCTAA -3' (KpnI restriction site underlined) and 5'- CTACAGATCTGCATTCACGCCCCACTTAC -3' (BglII restriction site underlined), respectively. Regions corresponding to -989 to +52, -656 to +52 and -499 to +52 bp were amplified from the 1965 bp fragment by nested PCR with forward primers 5'-TTGCGGTACCCTTCAAACTCCTGACCTCGTGA -3' (KpnI restriction site underlined), 5'- TTGCGGTACCCCCGGCCAACAGTTATTAAA -3' (KpnI restriction site underlined), 5'- TTGCGGTACCTGTAATCCCTCCCCCTAGCACT -3' (KpnI restriction site underlined), and a common reverse primer 5'-CTACAGATCTGCATTCACGCCCCACTTAC -3' (BglII restriction site underlined).
Sequential 5' deletions of the -499 to +52 bp promoter region were generated by PCR from the cloned genomic DNA as template to generate -308 to +52, the -257 to +52, the -207 to +52 bp and the -145 to +52 regions. Forward primers were 5'-TTGCGGTACCGTGCTTAATGGGAGCTGGTCT.-3', 5'-TTGCGGTACCGCGGCGAGAGCGCGCGGCGGGGGCG -3', 5'-TTGCGGTACCGCCGGGGGCGGGGCGGGGCGCGGCC -3' and 5'-TTGCGGTACCGTGGTGGTGTCTGGTTAGCTC-3', with KpnI restricton sites underlined. The common reverse primer is the same as used for amplification of the -499 to +52 bp region. All sequences were verified by sequencing.
Site-directed mutagenesis of transcription factor-binding sites
Oligonucleotide primers including the desired mutations were synthesized and used in two-step spliced overhang extension PCR. The following potential MTG16 promoter transcription factor binding sites were mutated: ETS1/PU.1 sites at positions -491, 5'-AGGA AA-3' changed to 5'- ATTC AA-3'; SP1 sites at positions -425, 5'-GGGA GG-3' changed to 5'-GTTC GG-3'; ETS1/SPI1 sites at positions -419, 5'-AGGA AGT-3' changed to 5' - ATTC AGT-3' TFAP2A sites at positions -389, 5'-GCCCGA GGG-3' changed to 5'-GCCTTC GGG-3'; GATA/YY1 sites at positions -356, 5'-GATGG T-3' changed to 5'-GACTT T-3'; SP1 sites at positions -316, 5'-CTCCGGC CCC-3' changed to 5'-CTCCTTA CCC-3'; GATA sites at positions -301, 5'-TATC A-3' changed to 5'-TGGT A-3' and MZF1 sites at positions -251 & -191, 5'-TCCC CA-3' changed to 5'-TAAA CA-3'.
The following potential MTGR1 promoter transcription factor binding sites were mutated: ETS1 sites at positions -335, 5'-CTTCC G-3' changed to 5'-CGTAT G-3'; YY1 sites at positions -301, 5'-CCCA TT-3'changed to 5'-CTTC TT-3'; SP1 sites at positions -285, 5'-CC CG GC CCCA-3' changed to 5'- CA CT GT CCCA -3'; ETS1 sites at positions -276, 5'-CT TCC G-3' changed to 5'- CG TAT G -3' SP1 sites at positions -243, 5'-CCCCC GCC GC-3' changed to 5'- CCCTT GCA GC -3'; SP1 sites at positions -229, 5'-CCCC GCC CGC-3' changed to 5'- CCTT GCA CGC -3'; SP1 sites at positions -225, 5'-CCCC GCC CCG-3' changed to 5'- CCTT GCA CCG -3'; SP1 sites at positions -213, 5'-CCCC GCC CCC-3' changed to 5'- CCTT GCA CCC -3'; SP1 sites at positions -206, 5'-CCCC GCC CCG-3' changed to 5'- CCTT GCA CCG -3'; SP1 sites at positions -192, 5'-CGCC GCC GCC-3' changed to 5'- CGTT GCA GCC -3'; SP1 sites at positions -178, 5'-CGCC GCC GCC-3' changed to 5'- CGTT GCA GCC -3' and SP1 sites at positions -170, 5'-CGCC GCC GCC-3' changed to 5'- CGTT GCA GCC -3'. After subcloning into promoterless pGL3/Basic reporter plasmid, all mutations were verified by sequencing.
Luciferase reporter assays of MTG16 and MTGR1 promoter
MTG16 promoter was cloned into promoterless pGL3/Basic reporter plasmid using firefly luciferase as reporter to generate pGL3 -905-57, pGL3 -820-57, pGL3 -668-57, pGL3 - 512-57, pGL3 -359-57, pGL3 -339-57, pGL3 -299-57 and pGL3 -219-57 reporter constructs. Mutants were cloned into the same reporter plasmid. Transient transfections of hematopoietic cell lines were performed by electroporation as previously described by . The pGL3/SV40-promoter vector served as positive control and promoterless pGL3/Basic vector as negative control. Renilla luciferase, pRL/SV40 vector (Promega Corporation, WI, USA) was used as internal control for transfection efficiency. Thirtyfive μg pGL3 DNA were used for HL-60 target cells and 15 μg for HEL, TF-1, U937, UT-7, MEG-01, Raji and Kasumi-1 target cells. COS cells were transfected with 1.5 μg of DNA by use of Polyfect (Qiagen, Hilden, Germany). Twentyfour hours after transfection, cells were lysed and subjected to luciferase dermination using the Dual luciferase reporter assay kit (Promega Corporation, WI, USA). Firefly and Renilla luminiscense were quantified (Run Promega Protocol, DLR-0-INJ) in a GLOMAX 20/20 Luminometer (Promega Corporation, WI, USA). Firefly was normalized to Renilla luciferase as internal control for transfection efficiency. Results are given as adjusted Relative Luciferase Units (AdjRLU) normalized to pGL3/promoter (set to 1). Three to five independent transfections were performed in each case and each sample was measured in triplicate.
Similarly, MTGR1 pGL3 -1913 + 52, pGL3 -989 + 52, pGL3 -656 + 52, pGL3 -499 + 52, pGL3 -308 + 52, pGL3 -257 + 52, pGL3 -207 + 52 and pGL3 -145 + 52 reporters were generated, transfected into cells and assayed as described above.
Transfection of GATA-1 small interfering RNA
Twentyfour-well Falcon plates (Becton Dickinson, New Jersey 07417, USA) were coated with 1 mg/ml retronectin (Takara Bio inc, Verviers, Belgium) in PBS for two h at room temperature. Excess retronectine was removed and wells were blocked with 2% BSA in PBS for 30 min. After removal of the supernatant, 50,000 HEL cells were added per well in 600 μl RPMI medium containing 10% FBS and 20 nM phorbol 12-myristate 13-acetate (PMA) to generate adherent cells by incubation at 37°C for 24 h. After washing with PBS transfection was carried out with 5 μM siRNA using the N-TER transfection reagent (Sigma-Aldrich Corp. St. Louis, MO USA) in RPMI without FBS. After 4 h, 300 μl of RPMI with 20% FBS was gently added and cells were incubated for 48 h. Eventually, gently rinsed with PBS, tripsinized and examined by Western blotting. The GATA-1 siRNA sequence was 5'-AGUUGAGGCAGGGUAGAGC-3' (Oligo SASI_Hs02_00333089, Sigma).
Inhibition of GATA-1 transcriptional activity by HERP2
The transcription factor HERP2 represses transcriptional activation by GATA-1  by physical interaction. To get support for a role of GATA-1 in gene activation of MTG16, HEL, TF-1, UT-7, and MEG-01cells were co-transfected with 10 μg MTG16 pGL3 -668 luciferase reporter and 10 μg HERP2 plasmid (kindly provided by Dr N Goldfarb, Charlottesville, Virginia) and the reporter signal was measured after 24 h as described above for luciferase reporter assays. The results were normalized to the luciferase activity of pGL3 -668 luciferase reporter.
Electrophoretic Mobility Shift Assay (EMSA) of MTG16 promoter
EMSA was performed as described previously . The potential GATA-1 sites at -301 was examined. The probe sequence was biotin-5-CCCGGCATTATCACGGGGACAC -3'. Nuclear extracts from HEL, TF-1, UT-7 and MEG-01 cells were prepared as described by Andrews and Faller . One to two μl polyclonal anti-GATA-1 (Active Motif, Carlsbad, CA, USA), monoclonal anti-GATA-2 (Santa Cruz Biotechnology Inc., CA, USA, sc-9008) or polyclonal anti-CD63 (sc-7080, Santa Cruz Biotechnology Inc., CA, USA) antibodies were added to the reaction mixtures.
Chromatin Immunoprecipitation (ChIP) assay of MTG16 promoter
ChIP was performed as described previously . For IP, 4 μl of polyclonal anti-GATA-1 (Active Motif, Carlsbad, CA, USA) or 0.8 μg monoclonal anti-GATA-2 antibodies (R&D Systems, MN, USA) were used. Forward and reverse primers for GATA sites were 5'-CAGATGGTTCCTGTCAGGGAAGTGGCG-3' and 5'-AGGTCTCCCTGCAGCCTGCGGGTGAG-3'. Control forward and reverse primers were: 5'-TAACACAGAGTACCCAGCCACTGTGC-3 ' and 5'-TGGTTGCACGGACAGAAGCCCCT-3'. Three different chromatin preparations were used for each IP.
Quantitative real-time PCR
Real-time PCR was performed as described previously . Transcript levels were calculated from a standard curve based on the Ct values of the samples,. Relative quantification based on the ΔCt method  was used. Normalization: ΔCt = Ct (sample) - Ct (HEL corresponding DNA concentration). Relative quantification = 2 - ΔCt .
Immunoprecipitation (IP) and Western blotting
IP and Western blotting were performed as described previously . The following antibodies were used: polyclonal anti-GATA-1 (Active Motif, Carlsbad, CA, USA) and polyclonal anti-GATA-2 (R&D Systems, MN, USA).
The cDNA sequences were analyzed by use of the NCBI Blast program http://www.ncbi.nlm.nih.gov/BLAST/. Conserved regions were searched by multiple alignment to genomic sequences using ClustalW http://www.ebi.ac.uk/Tools/clustalw2/index.html.
Potential transcription factor binding sites were examined with the MatInspector http://www.genomatix.de//matinspector.html and the Jaspar database (Jaspar.genereg.net).
The statistical significance between two samples was determined by unpaired t-test in Prism Programme. Triple and single asterisk represents P < 0.0001 and P < 0.05, respectively.
This work was supported by the Swedish Cancer Foundation and the Swedish Childhood Cancer Foundation. We are grateful to Ann-Maj Persson for kind help and expert guidance.
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