Identification of Essential GATA and Ets Binding Motifs within the Promoter of the Platelet Glycoprotein Ibα Gene

Abstract

Platelet glycoprotein (GP) Ib-IX-V is a multisubunit adhesion receptor that supports platelet attachment to thrombogenic surfaces at sites of vascular injury. The congenital absence of the receptor results in a bleeding disorder associated with "giant" platelets, a condition linking the expression of the complex to platelet morphogenesis. To understand better the expression of the GP Ib-IX-V complex, studies were undertaken to define the essential genetic elements supporting the expression of the ␣-subunit of the complex (GP Ib␣). GP Ib␣ promoter activity was evaluated by transfection of human erythroleukemia cells with reporter plasmids coding for the enzyme, luciferase. Studies were initiated with a fragment extending 2,738 nucleotides 5 to the transcription start site and lead to the identification of 253 nucleotides retaining full promoter activity in human erythroleukemia cells. In cells of nonhematopoietic lineage, human endothelial and HeLa cells, the GP Ib␣ promoter activity was no greater than background levels obtained with promoterless constructs. Gel shift assays and site-directed mutagenesis studies defined essential GATA and Ets binding motifs 93 and 150 nucleotides upstream of the transcription start site, a finding which further substantiates these elements as important determinants of megakaryocytic gene expression. The results define essential cis-acting elements responsible for the expression of GP Ib␣ and provide insights into molecular events coinciding with the release of normal platelets into the bloodstream.Basic information on the differentiation of megakaryocytes and the mechanisms responsible for the release of platelets into the bloodstream has significantly lagged behind basic studies of other cellular components of myeloid tissue. Major reasons for the lag have been the lack of suitable cell lines mimicking the unique properties of megakaryocytes in vitro. Certainly, the recently described c-Mpl ligand is essential for the differentiation of the pluripotent stem cell to a megakaryocyte (1-3), but the late events of the process, specifically those that result in the release of platelets from the mature megakaryocyte are still poorly understood. To this end, the expression of megakaryocytic or platelet-specific antigens is one unique aspect of megakaryocytopoiesis and platelet production that can be examined and the importance of such studies has been validated by the recent work identifying the transcription factor, NF-E2, as an essential genetic factor for platelet production (4).A role for the platelet membrane receptor, glycoprotein (GP) 1 Ib-IX-V, in platelet morphogenesis is suggested by the congenital absence of GP Ib-IX-V, a condition associated with the release of abnormal or "giant" platelets and referred to as the Bernard-Soulier syndrome (5, 6). The platelet GP Ib-IX-V complex is assembled from four distinct gene products, the ␣-and ␤-subunits of GP Ib (GP Ib␣ and GP Ib␤), GP IX, and GP V (7-11). Considerable progress has been made defining the essential role of this recep...

Physical association of the patient-specific GATA1 mutants with RUNX1 in acute megakaryoblastic leukemia accompanying Down syndrome

“…25 To investigate the role of DNA-binding ability of the C-finger in producing the synergic effects of GATA1 and RUNX1, we generated luciferase reporter plasmids containing the GPIba promoter with a mutation in the functional GATA1 site, as described in a previous report. 17 As expected, introduction of the mutation in the GATA1-binding site resulted in markedly reduced transcriptional activity of GATA1 at the GPIba promoter, whereas RUNX1 could still activate the GPIba promoter (Figure 5b). However, in the presence of RUNX1 and CBFb, GATA1, GATA1 FC and GATA1 DC synergistically activated the GPIba reporter with a mutation in the functional GATA1 site, although the reporter activities were reduced by about 50% compared to those of the intact GPIba promoter.…”

“…The GPIba promoter contains a single putative RUNX1 site, which was identified by the TFSEARCH program, and one functional GATA1-binding site (Figure 4a). 17,22 First, to ask whether RUNX1 actually binds to the GPIba promoter in vivo, we performed a ChIP assay. Because it is technically not feasible to use an anti-RUNX1 antibody for this assay, we transiently transfected Xpress-tagged RUNX1 with (Figure 4b lanes 9-12), or without (Figure 4b lanes 5-8) the CBFb expression vector into CMK cells derived from DS-AMKL, and ChIP assays were performed using anti-Xpress antibody.…”

“…17 The GPIba promoter region, containing 253 nucleotides 5 0 to the transcription start site, the nontranslated exon I, the single intron, and the first six base pairs of exon II immediately preceding the initiating methionine codon, was amplified by PCR and subcloned into the Picagene luciferase basic vector (Toyo Ink., Tokyo, Japan). The derived construct PICA GPIba À253 GATA mut was created using PCR, and then the blunt ended PCR products were self-ligated.…”

Ets transcription factors bind and transactivate the core promoter of the von Willebrand factor gene

“…c-Ets-1 and c-Ets-2 transactivate a large number of genes through interactions with a variety of factors (reviewed by Tymms and Kola, 1994), like Fos/Jun , Sp1 (Ge gonne et al, 1993;Block et al, 1996), GATA-1 (Lemarchandel et al, 1993). Essential Ets binding motifs cooperate with GATA elements for the tissue-speci®c expression of megakaryocytic genes such as glycoprotein (GP)IIb (Block et al, 1996;Lemarchandel et al, 1993;Martin et al, 1993), GPIba (Hashimoto and Ware, 1995), platelet factor four (PF4, Ravid et al, 1991), thrombopoietin receptor (MPL, Deveaux et al, 1996). The speci®c Ets proteins involved have not been identi®ed.…”

Thrombopoietin responsiveness reflects the number of doublings undergone by megakaryocyte progenitors

“…It had been established that GATA and cis-acting sequences coregulate the megakaryocyte progenitor expression of Mpl, 33 CD41, 34 and CD42a. 35 TPO responsiveness was recorded both as distributions of TPO thresholds (ie, the TPO concentrations necessary and sufficient to ensure the survival of megakaryocyte progenitors, either as single cells or as colonies) and as the medians of these distributions, termed TPO 50 (ie, the TPO concentration corresponding to the , and positive correlations were obtained between responsiveness and number of previous doublings undergone in vivo (NbDp; middle). As a result, a reciprocal relationship was found between NbDr and NbDp, indicating that a high number of previous doublings were associated with a low number of residual doublings.…”