p19^INK4d Modulates Human Terminal Erythroid Differentiation By Post-Transcriptionally Regulating GATA1 Expression

Erythropoiesis is a process during which hematopoietic stem cell (HSCs) are first committed to erythroid progenitors, which subsequently undergo terminal erythroid differentiation to produce mature red blood cells. During terminal erythroid differentiation, proerythroblasts undergo 4-5 mitoses to sequentially generate basophilic erythroblasts, polychromatic erythroblasts and orthochromatic erythroblasts that expel their nuclei to produce enucleated reticulocytes. Terminal erythropoiesis is a tightly regulated process. The most well studied regulatory mechanisms include EPO/EPOR mediated signal transduction and transcription factors among which GATA1 plays critical role. Terminal erythroid differentiation is also tightly coordinated with cell cycle exit, which is regulated by cyclins, cyclin-dependent kinases and cyclin-dependent kinase inhibitors (CDKI), yet their roles in erythropoiesis remain largely undefined. Our RNA-seq of human terminal erythroid differentiation shows that of seven CDKI members, only three of them, p18^INK4c, p19^INK4d and p27^KIP1, are abundantly expressed in erythroid cells and their expressions are significantly upregulated in late stage erythroblasts, which were further confirmed by western blotting analysis. In contrast to demonstrated roles of p18^INK4c and p27^KIP1 in terminal erythroid differentiation, the function of p19^INK4d this process has not been studied. To explore the role of p19^INK4d during human erythropoiesis, we employed a shRNA-mediated knockdown approach in CD34⁺ cells and found that p19^INK4d knockdown delayed erythroid differentiation, inhibited cell growth, led to increased apoptosis and generation of abnormally nucleated late stage erythroblasts. Unexpectedly, p19^INK4d knockdown did not affect cell cycle. Rather it led to decreased GATA1 protein levels. Importantly, the differentiation and nucleus defects were rescued by ectopic expression of GATA1. As GATA1 protein is protected by nuclear HSP70, to explore the mechanism for the decreased GATA1 protein levels, we examined the effects of p19^INK4d knockdown on HSP70 and found p19^INK4d knockdown led to decreased nuclear localization of HSP70 due to reduced ERK activation. Further biochemical analysis revealed that p19^INK4d directly binds to Ras kinase inhibitor PEBP1 and that p19^INK4d knockdown increased the expression of PEBP1 which in turn led to reduced ERK activation. These results demonstrate that p19^INK4d maintains GATA1 protein levels through PEBP1-pERK-HSP70-GATA1 pathway. Our findings identify previously unknown and unexpected roles for p19^INK4d in human terminal erythroid differentiation via a novel pathway.