Expression Of Runx1 And Its Function In Regulating Transcription Activity Of Wnt5a During Mouse Hematopoiesis

Runt-related transcription factor 1( RUNX1) is an essential regulator both in embryonic and adult hematopoiesis. In adult, RUNX1 regulates transcription activity of several genes which play important role in hematopoietic stem cells (HSCs) self-renew and differentiation. RUNX1 deficiency in bone marrow showed inhibition of megakaryocytic maturation and defective T and B lymphocyte development. Mutation or chromosomal rearrangements of Runx1 is correlated with acute myeloid leukemia (AML). Runx1 is essential for generation of definitive HSCs from haemangioblast during mouse ontogeny. Knockout Runx1 leads to the lost of intra-aortic clusters in mouse aorta-gonad-mesonephres (AGM) region and the complete block of generation definitive hematopoietic progenitors. Existed researches indicate that Runx1 generate two alternative splice forms, Ex5 andΔEx5, in different developmental stages. The ability ofΔEX5 to activiate the target genes is much lower because of the absence of protein sequence translated from exon5. In vertebrates, the transcription of Runx1 is also under tight ctronl of two alternative promoters, the distal promoter 1 and proximal promoter 2. Runx1b and Runx1c are spatio-termporal differently expressed during developmental hematopoiesis, but their functions and the precise mechanism still remains rudimentary. We firstly detected the spatio-termporal expression of mouse Runx1 alternative splice forms , then study the effects of RUNX1 on hematopoiesis and its microenvironments.To analyze the spatio-temporal expression of Ex5 andΔEx5, RT-PCR was performed with various blood tissues from embryonic day 7.5(E7.5) to 11.5.ΔEx5 is constantly transcripted both in AGM and YS between E7.5-11.5; Increasing expression in AGM while decreasing expression in YS of Ex5 were detected during this developmental course. The results indicated that the function of RUNX1 was mainly carried out by EX5 during mouse embryonic hematopoiesis. (The results indicated that the primitive and defintive hematopoiesis during mouse gestation maybe balanced by the expression of EX5.) The expression of Runx1b and Runx1c during mouse hematopoiesis were also detected with different hematopoietic tissues including adult bone marrow (BM) and fetal liver (FL), placenta ( PL) at E11.5. Results of RT-PCR suggested Runx1b mRNA is domiant in both AGM , while in FL the transcription of Runx1c get the run upon Runx1b. There is no significant difference between the two isoforms in YS an PL. Runx1b transcription in adherent BM stromal cells exceeded that of Runx1c, and the vice verse in the non-adherent hematopoietic cells. Mesenchymal stem cells (MSCs), major constituents of hematopoietic microenvironment, were then purified and were found to highly express Runx1b. It can be concluded from the above data that RUNX1c may affect the proliferation and differentiation of HSCs both in FL and adult hematopoietic cells, while RUNX1b directly controls embryonic hematopoiesis in AGM and regulate hematopioesis indirectly by modifying adult hematopoietic microenvironments.The possible indirect regulatory effects of Runx1 on hematopoiesis was then detected in MSCs .The essential functions of MSCs as HSCs niche cells in BM are to secret cytokines and differentiate into progenies indispensable for the hemostastis of hematopoiesis. RUNX1 regulates hematopoiesis through activating downstream genes, but how it regulates MSCs is unknown. Using the promoter analysis tools, we find Wnt5a and Runx2 promoters have several RUNX1 binding sites. Then chromatin immunoprecipitation was used to investigate the direct in vivo interaction between RUNX1 protein and the two genes. The result came out that RUNX1 can bind to the promoter of Wnt5a but not that of Runx2. Then RUNX1 overexpression model in mouse BM MSCs was established to detect the regulation of RUNX1 on the transcription activity of Wnt5a. The result shows that the expression of Wnt5a was enhanced with the increase of Runx1. It is concluded that the Wnt5a is one of the target genes of RUNX1 in mouse BM MSCs and its transcription is activated by RUNX1.Wnt5a, a member of Wnt gene family (wingless-type MMTV integration site family), plays an important role in WNT/Ca+ pathway, and was reported to stimulate the proliferation of HSCs. Our experiments showed that the cytokine WNT5A inhibits adipogenic differentiation of MSCs. Expression of Adipsin which is specific for adipogenesis decreased after WNT5A were added to adipogenic differentiation culture system. Its effect on colony forming potency of BL-CFC was then checked, BL-CFC was convinced to represent hemangioblast in vivo in early hematopoiesis and gives rise to blast colonies with both endothelial and hematopoietic cells. The number of blast colonies was dramastically increased when WNT5A was added into BL-CFC culture system, which indicates that WNT5A secreted by MSCs may support early hematopoiesis.Conclusively, the expression of Runx1 alternative splice forms spatio-temporally varied during mouse hematopoiesis. In BM MSCs RUNX1 regulates the transcription of Wnt5a, which can inhibit adipogenic differentiation of MSCs and enhance blast colony forming potency of BL-CFC in vitro.