Platelet Protein Kinase C-θ Deficiency With Human RUNX1 Mutation
PRKCQ Is a Transcriptional Target of RUNX1
Objective—Mutations in the hematopoietic transcription factor RUNX1 cause thrombocytopenia and impaired platelet function. In a patient with a heterozygous mutation in RUNX1, we have described decreased platelet pleckstrin phosphorylation and protein kinase C-θ (PKC-θ, gene PRKCQ) associated with thrombocytopenia, impaired platelet aggregation, and dense granule secretion. Little is known regarding regulation of PKC-θ in megakaryocytes/platelets. We have addressed the hypothesis that PRKCQ is a direct transcriptional target of RUNX1.
Methods and Results—In a chromatin immunoprecipitation assay using megakaryocytic cells, there was RUNX1 binding in vivo to PRKCQ promoter region −1225 to −1056 bp containing a RUNX1 consensus site ACCGCA at −1088 to −1069 bp; an electrophoretic mobility shift assay showed RUNX1 binding to the specific site. In RUNX1 overexpression studies, PKC-θ protein expression and promoter activity were enhanced; mutation of RUNX1 site showed decreased activity even with RUNX1 overexpression. Lastly, PRKCQ promoter activity and PKC-θ protein were decreased by short interfering RNA knockdown of RUNX1.
Conclusion—Our results provide the first evidence that PRKCQ is regulated at the transcriptional level by RUNX1 in megakaryocytic cells and a mechanism for PKC-θ deficiency associated with RUNX1 haplodeficiency.
- Received December 17, 2010.
- Accepted January 7, 2011.
- © 2011 American Heart Association, Inc.