Novel function of PITH domain-containing 1 as an activator of internal ribosomal entry site to enhance RUNX1 expression and promote megakaryocyte differentiation.

Abstract

Altered gene expression coincides with leukemia development and may affect distinct features of leukemic cells. PITHD1 was significantly downregulated in leukemia and upregulated upon PMA induction in K562 cells undergoing megakaryocyte differentiation. We aimed to study the function of PITHD1 in megakaryocyte differentiation. K562 cells and fetal liver cells were used for either overexpression or downregulation of PITHD1 by retroviral or lentiviral transduction. FACS was used to detect the expression of CD41 and CD42 to measure megakaryocyte differentiation in these cells. Western blot and quantitative RT-PCR were used to measure gene expression. Dual luciferase assay was used to detect promoter or internal ribosomal entry site (IRES) activity. Ectopic expression of PITHD1 promoted megakaryocyte differentiation and increased RUNX1 expression while PITHD1 knockdown showed an opposite phenotype. Furthermore, PITHD1 efficiently induced endogenous RUNX1 expression and restored megakaryocyte differentiation suppressed by a dominant negative form of RUNX1. PITHD1 regulated RUNX1 expression at least through two distinct mechanisms: increasing transcription activity of proximal promoter and enhancing translation activity of an IRES element in exon 3. Finally, we confirmed the function of PITHD1 in regulating RUNX1 expression and megakaryopoiesis in mouse fetal liver cells. PITHD1 was a novel activator of IRES and enhanced RUNX1 expression that subsequently promoted megakaryocyte differentiation. Our findings shed light on understanding the mechanisms underlying megakaryopoiesis or leukemogenesis.