Ets-dependent Regulation of Target Gene Expression during Megakaryopoiesis

Abstract

Megakaryopoiesis is the process by which hematopoietic stem cells in the bone marrow differentiate into mature megakaryocytes. The expression of megakaryocytic genes during megakaryopoiesis is controlled by specific transcription factors. Fli-1 and GATA-1 transcription factors are required for development of megakaryocytes and promoter analysis has defined in vitro functional binding sites for these factors in several megakaryocytic genes, including GPIIb, GPIX, and C-MPL. Herein, we utilize chromatin immunoprecipitation to examine the presence of Ets-1, Fli-1, and GATA-1 on these promoters in vivo. Fli-1 and Ets-1 occupy the promoters of GPIIb, GPIX, and C-MPL genes in both Meg-01 and CMK11-5 cells. Whereas GPIIb is expressed in both Meg-01 and CMK11-5 cells, GPIX and C-MPL are only expressed in the more differentiated CMK11-5 cells. Thus, in vivo occupancy by an Ets factor is not sufficient to promote transcription of some megakaryocytic genes. GATA-1 and Fli-1 are both expressed in CMK11-5 cells and co-occupy the GPIX and C-MPL promoters. Transcription of all three megakaryocytic genes is correlated with the presence of acetylated histone H3 and phosphorylated RNA polymerase II on their promoters. We also show that exogenous expression of GATA-1 in Meg-01 cells leads to the expression of endogenous c-mpl and gpIX mRNA. Whereas GPIIb, GPIX, and C-MPL are direct target genes for Fli-1, both Fli-1 and GATA-1 are required for formation of an active transcriptional complex on the C-MPL and GPIX promoters in vivo. In contrast, GPIIb expression appears to be independent of GATA-1 in Meg-01 cells.Ets family members each contain a conserved winged helixloop-helix DNA binding (ETS) domain that allows recognition of purine-rich DNA sequences with a core GGA(A/T) consensus, designated EBS 1 (Ets binding sequence) (1-3). These transcription factors have critical roles in the transcriptional control of genes important in development, morphogenesis, proliferation, and angiogenesis. Ets factors can function as either positive or negative transcriptional regulators (4, 5) and additional binding of other transcription factors to cis-elements located near EBSs contributes to regulated transcription of specific target genes. Thus, in addition to binding to DNA, Ets transcription factors participate in protein interactions that affect their functions (6, 7).Fli-1, a member of the Ets gene family of transcription factors, performs functions critical for normal development and oncogenesis (for a review, see Ref. 8). Fli-1 is preferentially expressed in cells of hematopoietic lineages and vascular endothelial cells, and has been shown to transcriptionally activate genes, including the stem cell leukemia gene (9), tenascin (10), the stress response gene GADD153 (11), the anti-apoptotic gene Bcl-2 (12) and several megakaryocytic specific genes (see below). Fli-1 also forms ternary complexes through interaction with SRF to bind to SRE elements of fos and Egr-1 promoters (13, 14). Fli-1 protein interaction with other regulatory proteins...

Cells meeting our immunophenotypic criteria of endothelial cells are large platelets

“…Using immunophenotyping and sorting of CMOIC followed by morphology, IHC, and QRT-PCR, we were not able to detect cells with endothelial characteristics among these cells. The positive staining of CMOIC for vWF and Fli-1 was not conclusive with respect to their endothelial origin, as cells from the megakaryocytic lineage express these antigens as well (21,31). Furthermore, nuclear staining and FISH failed to detect cells with a nucleus in this population and finally, electron microscopy revealed CMOIC to be large platelets.…”

p210BCR-ABL reprograms transformed and normal human megakaryocytic progenitor cells into erythroid cells and suppresses FLI-1 transcription

“…We focused our work on FLI-1 as this transcription factor in association with the fact that GATA-1 activates the transcription of several MKrestricted genes such as gpIX (CD42a), gpIba (CD42b), and c-mpl through cooperative DNA binding. [35][36][37] FLI-1 has also the ability to inhibit the erythroid differentiation in several erythroleukemic cell lines. [38][39][40] Altogether, these data strongly suggest that during normal hematopoiesis, FLI-1 not only activates the MK differentiation program but also simultaneously represses the erythroid program.…”

IL‐13 upregulates GPIIb expression in megakaryocytic cell lines via STAT6

“…GPIIb combines with the more broadly expressed b3 (GPIIIa, CD61) subunit to form the GPIIb/IIIa heterodimer receptor that regulates cell adhesion and functions in aggregation of activated platelets by binding fibrinogen and vWF (Jackers et al, 2004;Sevinsky et al, 2004). Because of its unique expression in megakaryocytes and platelets in humans, in this study GPIIb was chosen as a model for IL-13-dependent regulation of lineage-specific marker.…”