Preliminary Study On The Regulation Of Human RUNX1 Gene On Hematopoiesis

Background:Transcription factor is the main regulator of the hematopoiesis process,in which RUNX1 is an important regulatory gene for hematopoiesis,especially definitive hematopoiesis,and in the mouse model completely knockout RUNX1 its embryos would die of ischemia at 12.5 days.The full length of RUNX1 gene is 216 kb with 10 exons,which has a complex gene regulation mode,has distal promoter P1 and proximal promoter P2.P1 initiates RUNX1c transcription,and P2 starts RUNX1a and RUNX1b transcription,and the most common protein products are RUNXla,RUNX1b and RUNX1c.Except for the difference in the N-terminal 32 amino acid sequences,remaining sequences of RUNXlb/c are identical and contain an N-terminal DNA binding domain(binding to a specific DNA sequence)and a C-terminal transcriptional regulatory domain(interacting with other proteins)while RUNX1a has only the N-terminal DNA binding domain and lacks the C-terminal transcriptional regulatory domain.RUNXla/b are widely expressed during hematopoiesis while RUNX1c is expressed only in hematopoietic stem/progenitor cells,which is a landmark molecule.Antagonistic effect exists between RUNX1a/b.RUNX1a can promote hematopoiesis while RUNXlb maintains hematopoiesis at a normal level,inhibit the hyperplasia of hematopoietic cell,which is generally considered as a tumor suppressor.It is of great significance to explore how RUNX1 regulates the generation of hematopoietic cells from hESC.Objective:We investigate the effect of RUNXl on hematopoiesis using hESC/AGM-S3 co-cultures system and eukaryotic inducible over-expression technology,and further explore the related molecular mechanisms.Methods:In this study,in vitro co-culture system(hESC/AGM-S3)and eukaryotic inducible overexpression technology(PB-tet-on-OE based on PiggyBac)were used to investigate the effect of RUNX1 on hematopoiesis.In hESC/AGM-S3 co-cultures,we tested functional roles of various splicing transcription of RUNX1 in the early development stage of hematopoiesis by a timed Doxycycline(DOX)inducing mode.Using cellular biological methods(Fluorescence Activated Cell Sorting(FACS),immunofluorescence staining method(IF),colony assay et al.)and molecular biological methods(Western blot,qRT-PCR et.al.),we try to elucidate the detail role of RUNX1b in hESC-derived early hematopoiesis.Results:1.Establish the inducible overexpression of RUNXla/b/c hESC cell line,which based on H1 hESC,and the overexpression system,and whose pluripotency and the ability of differentiation to the three germ layers are normal.2.Inducible overexpression of RUNX1b from D0-D2 blocks the generation of CD34+cells,with loss of the consequential CD34+CD43+ and CD34+CD45+ populations.3.Inducible overexpression of RUNXlc from D0-D2 exerted similar blockage to early hematopoiesis as RUNXlbwhile inducible overexpression of RUNXla has no obvious effect on hematopoiesis in early stage and could promote hematopoiesis in latestage.4.Hematopoiesis-related genes are down-regulated by inducible overexpression of RUNX1b at early stage during hematopoiesis.5.Hematopoiesis blockage by RUNX1b can be partially rescued by RepSox,an inhibitor of TGF-? signaling pathway.6.The effect of RepSox and RUNX1b on hematopoiesis may be related to the change of the cell cycle status.Conclusion:The inducible overexpression of RUNX1b in human embryonic stem cells strongly blocks the product of CD34+ cells,with loss of the hematopoietic stem/progenitor cells,and down-regulates the expression of multiple important hematopoiesis-related genes.Such blockage effect occurred only at the very early stage before the generation of hemogenic endothelium,and disappeared after D6.It was partially rescued by RepSox,a specific inhibitor of TGF-? signaling pathway.The effect of RepSox and RUNX1b on hematopoiesis may be related to change the cell cycle status.