PML4 Participates In Erythroid Differentiation And Maturation By Enhancing GATA-1 Transcriptional Activity

Hematopoiesis is cascade of cell differentiations starts from a handful hematopoietic progenitor cells. This process has been widely studied as a model for molecular mechanisms of cell differentiation. Each step of hematopoiesis is stringently regulated by a spectrum of inter- and intra- cellular signals which ultimately target transcriptional regulators and determine cell differentiation. Depletion or abnormality of hematopoietic transcriptional factors always leads to hematopoiesis defect or even hematopoietic diseases. Thus, precise regulation to the expression and function of these transcriptional factors constitute critical aspect in hematopoiesis.Erythrocytes present the most abundant cells population generated during hematopoiesis. Transcriptional regulation of erythropoiesis has been extensively studied. Erythropoiesis is an ordered process accompanied by dynamic gene expression patterns. Up to now, multiple transcriptional factors and signal pathways have been revealed to participate in erythropoiesis. During erythropoiesis, erythroid genes gradually open up and non-erythroid genes go silence to reach erythroid specific phenotype.GATA-1 is the master transcriptional factor during erythroid differentiation and maturation. Almost all the erythroid specific genes are target genes of GATA-1 which functions either as an activator or a repressor. GATA-1 interacts with other transcriptional factors and cofactors to coordinate different regulatory events. Therefore, identification of novel protein partners of GATA-1 is beneficial to further understanding GATA-1 function and transcriptional regulation during erythropoiesis.Promyelocyte leukemia protein PML, a tumor suppressor, is mainly found in the form of PML nuclear body. Such proteinaceous body structure has been shown critical for functional implications of PML. Through recruiting or releasing associating protein factors, PML participates in multiple cellular processes such as cell senescence and apoptosis, cell proliferation suppression, genome stability maintenance and anti-virus function. A role of PML in hemotopoiesis has also been reported, including hematopoietic progenitor cell and granulocytic differentiation. However, functions of PML in erythropoiesis have not been clearly demonstrated.Considering that gene expression mode correlates with gene functions, we started from detecting PML expression in erythroid cells of distinct developmental stages and differentiation status. Realtime RT-PCR showed marginal PML expression in 8.5d to 10.5d yolk sac, the expression flare up from 11.5d to 14.5d in fetal liver. The pattern is consistent with that of GATA-1 during embryonic and fetal erythropoiesis. During induced erythroid differentiation of G1E-ER4 (GATA-1 rescued cell line derived from GATA-1 knockout mES cells), MEL and K562 cell lines, PML was found expressed consistently. Similar erythroid differentiation of human CD34+ cells induced by EPO confirmed the similarity between expression pattern of PML and GATA-1. Subsequently, direct interaction of PML and GATA-1 was discovered using co-IP and GST-pulldown methods. Immunofluorescence experiment demonstrated that PML recruited GATA-1 to PML nuclear bodies. We then constructed truncated constructs of PML and GATA-1, and found that C-Zinc Finger of GATA-1 and coiled-coil domain of PML mediated interaction between the two proteins.To detect whether interaction of PML and GATA-1 is of functional importance, we performed luciferase reporter assay and found that PML enhanced transcriptional activity of GATA-1 and interaction of the two proteins was indispensable for influence of PML on GATA-1 activity. To further show how PML influence GATA-1 activity, we performed a series of luciferase reporter assay, IP and ChIP analyses. We showed that PML enhanced GATA-1 DNA binding in vivo. Furthermore, PML was found to increase cooperation of GATA-1 and its cofactor p300, which in turn leads to increased acetylation of GATA-1 by p300 and increased recruitment of CBP/p300 at GATA-1 binding sites in vivo. Effect of PML expression on the differentiation and maturation of erythrocyte and GATA-1 dependency of the process were then investigated:PML potentiation in K562 by IFNa was accompanied with enhanced globin genes expression. PML overexpressed K562 was found suppressed at G1 phase, along with activated erythroid differentiation markers like globin genes and GPA. The observation reminds that of GATA-1 overexpressed K562 cell. Knockdown of PML in K562 made opposite effects. Similarly, when PML was overexpressed in G1E-ER4 cells, globin gene transcription were enhanced, and hemoglobin production was shown enhanced with benzidine staining, indicating that erythroid maturation was promoted by PML. PML was also overexpressed and knocked down in human primary erythroid progenitor cells, the effects on globin genes expression were after that in cell lines. In addition, both GATA-1 deficient G1E cell and uninduced G1E-ER4 cell showed irresponsive to PML overexpression in erythroid gene expression, suggesting that promotion of erythroid maturation by PML was GATA-1 dependent.In summary, our studies revealed that PML could function as a novel regulator of GATA-1. Recruitment of GATA-1 to PML nuclear bodies, promotes cooperation of GATA-1 and CBP/p300, and hence enhances GATA-1 acetylation and DNA binding in vivo. This together increases transcription of GATA-1 target genes, exemplified by the globin genes, AHSP and GPA genes, and eventually facilitates terminal erythroid differentiation through modulation of GATA-1 activity.