ACTIVATION OF THE MEGAKARYOCYTE-SPECIFIC GENE PLATELET BASIC-PROTEIN (PBP) BY THE ETS FAMILY FACTOR PU.1

Platelet basic protein (PBP) is a chemokine family member that is only found in platelets and their precursors megakaryocytes. The PBP gene is physically linked to the gene for another platelet-specific chemoki ne, platelet factor 4. While the. biological basis of platelet factor 4 expression has been pursued by others, the regulatory features contr olling the platelet specific expression of PBP have not been investiga ted, In this article, we examined the molecular basis by which this me gakaryocyte-specific gene is regulated, Transient expression studies o f truncated reporter constructs containing from 4.5 to 0.1 kilobases o f the functional PBP gene 5'-flanking region, demonstrated that the pr oximal 0.1 kilobases of the promoter was sufficient for high levels of expression in human erythroleukemia and CHRF-288 cells, two megakaryo cytic cell lines, He-Never, none of these constructs was expressed abo ve background levels in HeLa and 293 cells, two non-megakaryocytic cel l lines, Further truncation of this promoter suggested that there was an important regulatory element(s) within a pyrimidine-rich tract. Mob ility shift analysis of the pyrimidine-rich tract defined a region bet ween -85 and -64 which bound to a nuclear factor(s), This region conta ins sequences matching the consensus Ets-binding site from -78 to -75 base pairs, In particular, we noted that this site matched a PU,I cons ensus sequence known as a PU box, Mobility shift and supershift studie s with nuclear extracts as well as recombinant PU.1 protein and anti-P U.1 antibody further confirmed that PU.1 was the specific Ets family f actor that bound to this site, Transient expression assays using repor ter constructs which contained point mutations that abrogated PU.1 bin ding also significantly reduced PBP promoter activity in human erythro leukemia and CHRF cells, In addition, while all reporter gene construc ts containing PBP promoters were completely inactive in HeLa cells, tr ansactivation experiments using a PU,I expression construct demonstrat ed that exogenous expression of PU.1 could increase reporter gene expr ession up to 8-fold in these cells, Finally, the role of PU.1 in PBP g ene expression was compared between wild-type and PU.1-null embryonic stem (ES) cells that were differentiated in vitro into cells that rese mbled megakaryocytes both morphologically and immunologically. We foun d that PBP gene expression in the differentiated PU.1(-/-) null ES cel ls (as determined by semi-quantitative reverse transcriptase-polymeras e chain reaction) was more that four times lower than that in the wild -type ES cells, while other platelet-specific genes were expressed equ ally or similarly in the two ES cell lines. Previous reports have show n the PU.1 is expressed in several hematopoietic lineages, including m egakaryocytes. However, the functional role of PU.1 has only been prev iously demonstrated in the myeloid and lymphoid lineages. Therefore, o ur studies are the first to show the biological importance of this nuc lear factor in the regulated expression of a megakaryocyte-specific ge ne.