Linked Life Data

9334192

Source:http://linkedlifedata.com/resource/pubmed/id/9334192

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
42
pubmed:dateCreated
1997-11-17
pubmed:abstractText
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 chemokine, platelet factor 4. While the biological basis of platelet factor 4 expression has been pursued by others, the regulatory features controlling the platelet-specific expression of PBP have not been investigated. In this article, we examined the molecular basis by which this megakaryocyte-specific gene is regulated. Transient expression studies of truncated reporter constructs containing from 4.5 to 0.1 kilobases of the functional PBP gene 5'-flanking region, demonstrated that the proximal 0.1 kilobases of the promoter was sufficient for high levels of expression in human erythroleukemia and CHRF-288 cells, two megakaryocytic cell lines. However, none of these constructs was expressed above background levels in HeLa and 293 cells, two non-megakaryocytic cell lines. Further truncation of this promoter suggested that there was an important regulatory element(s) within a pyrimidine-rich tract. Mobility shift analysis of the pyrimidine-rich tract defined a region between -85 and -64 which bound to a nuclear factor(s). This region contains sequences matching the consensus Ets-binding site from -78 to -75 base pairs. In particular, we noted that this site matched a PU.1 consensus sequence known as a PU box. Mobility shift and supershift studies with nuclear extracts as well as recombinant PU.1 protein and anti-PU.1 antibody further confirmed that PU.1 was the specific Ets family factor that bound to this site. Transient expression assays using reporter constructs which contained point mutations that abrogated PU.1 binding also significantly reduced PBP promoter activity in human erythroleukemia and CHRF cells. In addition, while all reporter gene constructs containing PBP promoters were completely inactive in HeLa cells, transactivation experiments using a PU.1 expression construct demonstrated that exogenous expression of PU.1 could increase reporter gene expression up to 8-fold in these cells. Finally, the role of PU.1 in PBP gene expression was compared between wild-type and PU.1-null embryonic stem (ES) cells that were differentiated in vitro into cells that resembled megakaryocytes both morphologically and immunologically. We found that PBP gene expression in the differentiated PU.1(-/-) null ES cells (as determined by semi-quantitative reverse transcriptase-polymerase chain reaction) was more than four times lower than that in the wild-type ES cells, while other platelet-specific genes were expressed equally or similarly in the two ES cell lines. Previous reports have shown that PU.1 is expressed in several hematopoietic lineages, including megakaryocytes. However, the functional role of PU.1 has only been previously demonstrated in the myeloid and lymphoid lineages. Therefore, our studies are the first to show the biological importance of this nuclear factor in the regulated expression of a megakaryocyte-specific gene.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Oct
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
17
pubmed:volume
272
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
26236-46
pubmed:dateRevised
2007-11-15
pubmed:meshHeading
pubmed:year
1997
pubmed:articleTitle
Activation of the megakaryocyte-specific gene platelet basic protein (PBP) by the Ets family factor PU.1.
pubmed:affiliation
Graduate Group in, the University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.