Research On Regulatory Mechanism Of HSPCs Expansion By TANGO6 And HSPCs Homeostasis Maintenance Related Drug Screen

Part Ⅰ:TANGO6 controls HSPCs proliferation via COPI vesicle mediated nuclear import of RPB2Hematopoietic stem/progenitor cells（HSPCs）have the capacity to generate all kinds of mature blood lineages by differentiation and construct the whole blood system.During this developmental process,aberrant HSPCs may lead to a series of diseases,such as leukemia,lupus erythematosus,anemia and rheumatoid arthritis.Therefore,HSPCs homeostasis maintenance is critical for organism health.Meanwhile,at present,HSPCs transplantation has become a major stem cell-based curative therapy in the treatment of malignant hematological disease,which are derived from bone marrow,umbilical cord blood and peripheral blood.Above all,the ability to expand sufficient HSPCs ex vivo prior to transplantation has great clinical significance.Therefore,to expore the regulatory mechanism on HSPCs expansion has become a hot pot in life science and medicine all the time.In mice,primitive hematopoiesis produces early erythrocytes and myeloid cells in yolk sac.With embryogenesis,definitive hematopoiesis generates hematopoietic stem cells（HSCs）in aorta-gonad-mesonephros（AGM）region by endothelial to hematopoietic transition（EHT）.Then,HSPCs migrate to fetal liver as well as to thymus for lymphoid cells commitment,and finally colonize at bone marrow to maintain lifelong hematopoiesis.During this process,fetal liver is considered as an important organ for HSPCs expansion.Previous studies have shown that growth factor,Notch signaling and epigenetic modification play important roles in HSPCs proliferation.However,HSPCs regulatory network remain to unclear,especially for novel factors identification.Zebrafish has become an ideal model organism to investigate hematopoiesis,due to its transparency,high reproduction rate and conserved regulatory mechanism.The current studies provide that,similar with mice,zebrafish hematopoiesis contains two waves,primitive hematopoiesis and definitive hematopoiesis.About 26 to 28 hours of post fertilization（hpf）,HSCs emergences at ventral wall of dorsal aorta（VDA）via EHT process.Then,HSPCs migrate to caudal hematopoietic tissue（CHT）region,which is similar with fetal liver of mammals,conduct expansion and differentiation.Finally,these cells colonize at thymus and kidney to maintain perpetual hematopoiesis.Hence,CHT region is suitable for analyzing HSPCs expansion network in zebrafish.In order to explore the key factors involved in HSPCs expansion,we carried out large scale forward genetic screen,and identified cq72 mutant.In this mutant,HSPCs emergence was normal,but the number of HSPCs decreased from 3.5 dpf in CHT region,meanwhile,the HSPCs proliferation level was also decreased significantly.Consistently,other mature blood lineages development was defective.By map-based cloning,we found a point mutation（G to T）in tango6（transport and golgi organization 6）at exon6,which led to early termination of Tango6 translation in 392 amino acid（Glu）.By detecting tango6 expression pattern,fluorescence in situ hybridization（FISH）,transgenetic line rescue and HSPCs transplantation experiments,we demonstrated that Tango6intrinsically regulates HSPCs expansion in zebrafish.Simultaneously,we also constructed the other two mutant alleles,tango6^Δ16/Δ16（delete 16 bp at exon4）and tango6^Δ5/Δ5（delete 5 bp at exon12）.Due to the two alleles phenotype are identified with tango6^cq72,we primarily utilized cq72 mutants for further study.Additionally,we successfully constructed Tg（coro1a:tango6）transgenic lines,the results indicated that overexpress tango6 promotes HSPCs population enlarged,in contrast with tango6mutants.These data demonstrate that tango6 is critical for HSPCs homeostasis maintenance and expansion in zebrafish.Studies about tango6 were extremely limited,only one paper reported that in Drosophila S2 cells,Tango6 localized at Golgi apparatus,its deficiency led to Golgi fragmentation.But its biological functions,especially for HSPCs expansion is still unclear.On account of no suitable primary antibodies against to Tango6 in zebrafish,we further studied TANGO6 function in He La and U251 cells.Above all,we knock-out tango6 in U251 cells by CRISPR/Cas9 and knock-down this gene in He La cells by specific si RNAs,and find the aberrant proliferation of U251 and He La cells upon TANGO6 ablation,which was identical to the observations of HSPCs in tango6 mutant zebrafish,indicating the conserved function of TANGO6.Surprisingly,in He La cells,we found a large part of the monitored cells（～80%）manifested the cytoplasmic appearance of TANGO6⁺signals,and around 20%of cells showed nuclear localization of TANGO6⁺puncta.Consistently,we discovered the distribution of TANGO6 is closely related to cell cycle progression.Then,we carefully characterized the position of TANGO6 in cytoplasm using high-resolution imaging of immunofluorescent staining（Airyscan and STED）and isolated different organelles,TANGO6⁺signals were predominantly detected in COPI vesicles（mediate Golgi apparatus to ER transport）rather than COPII vesicles（control ER to Golgi apparatus transport）.Additionally,both N and C terminus of TANGO6 were localized at cell cytoplasm,and cytoplasmic TANGO6 largely tethers to the COPI vesicles via 2-pass transmembrane（TM）domains.Meanwhile,impaired COPI vesicles lead to TANGO6⁺signals diffused into the whole cytoplasm,but TANGO6 plays a limited role in the configuration of COPI vesicles and Golgi complex integrity.Previous studies have demonstrated that membrane proteins nuclear import need nuclear import signal（NLS）,importins and ER assistance.As for TANGO6,we found a putative NLS sequence（232 aa to 262 aa）,and Arg（arginine）at 252 site was critical for TANGO6 nuclear import by different site-directed mutagenesis experiments.Consistently,we identified KPNB1（a kind of importinβ）interaction with TANGO6,and its deficiency gave rise to TANGO6 nuclear import defective.Furthermore,TANGO6nuclear import was also blocked when we broke ER integrity through the application of thapsigargin（TA）to induce ER stress,indicated that its nuclear import also required ER participation.The above results implied that TANGO6 pushed cell cycles by shuttling key factors between the cell cytoplasm and nucleus.To this end,we conducted mass spectrum analysis to identify candidate molecules.The results revealed that RPB2,the second largest subunit of RNA polymerase II complex,presented substantial interaction with TANGO6.To verify mass spectrum result,co-immunoprecipitation（CO-IP）,immunostaining and sucrose density gradient centrifugation were performed,the results indicated that TANGO6 interacted with RPB2,but no interaction with RPB1（the largest subunit of RNA polymerase II complex）,was observed.Indeed,when we knock-down or knock-out TANGO6,led to RPB2 accumulated at cell cytoplasm,demonstrated that TANGO6 was critical for RPB2 nuclear import.Meanwhile,when we broke COPI vesicle integrity by knocking-down COPA（a key component of COPI vesicle）,it also caused RPB2 accumulated at cell cytoplasm.Consistently,RPB2 deficiency also inhibited cell proliferation,which was identical with TANGO6 defective.These data indicated that TANGO6 regulates cell proliferation by directing RPB2 nuclear import through COPI mediated transport.To further validate the conserved regulatory mechanism of the TANGO6-RPB2 axis in zebrafish hematopoiesis,firstly,we detected polr2b expression pattern and triple staining（polr2b,tango6 and Runx1-GFP）,the results indicated that polr2b was highly enriched at HSPCs expansion stage,which was identical with tango6.Then,by CO-IP experiment,we found Tango6 similarly interacted with Rpb2 in zebrafish.Meanwhile,Rpb2 protein level was decreased significantly in tango6^cq72 mutant nucleus.These results suggest Tango6 function is conserved in zebrafish.Furthermore,in order to validate Rpb2 is critical for HSPCs homeostasis maintenance,we conducted knock-down（morpholino）or knock-out（CRISPR/Cas9）polr2b,the data showed that Rpb2 deficiency led to the number of HSPCs and proliferation level decreased significantly,which is similar with tango6 mutants.Based on this,we fused the IWR1 NLS sequence into polr2b N terminus.Interestingly,supplying NLS-polr2b elegantly enlarged the population of cmyb⁺cells in tango6 mutants.These results suggest that in zebrafish,especially for HSPCs expansion,Tango6 regulates Rpb2 nuclear import to ensure cell cycle progression.Finally,we focused on mice models.Whole-mount in situ hybridization（WISH）results indicated that Tango6 was ubiquitously expressed in whole embryos during E9.5to E14.5.We initially created Tango6 conventional knock-out mice,unfortunately,no homozygous mutant offspring were identified,even at E9.5.It indicated that Tango6 was essential for early embryonic development.Therefore,we generated a Tango6 conditional knock-out allele with lox P sites flanking exon2.We bred Tango6^f/f with Vav-i Cre mice to obtain Vav-i Cre/Tango6^f/f（conditional knock-out,c KO）mice.Then,we narrowed the lethal time window of c KO mice at E16.5-E17.5.Furthermore,we analyzed Lin^-Sca-1⁺c-Kit⁺（LSK）cells population in fetal liver at E15.5,to mark HSPCs population,the result indicated that HSPCs reduced obviously in c KO mice.Meanwhile,in fetal liver,RPB2protein level was decreased and accumulated at cell cytoplasm in c KO mice.These data indicated that TANGO6-RPB2 axis is functionally conserved in mice.In summary,we identified tango6^cq72 mutant zebrafish by forward genetic screen,its deficiency led to HSPCs proliferation level decreased significantly,and therefore,restrained other mature blood lineages development.Then,we investigated regulatory mechanism of TANGO6 on cell proliferation in cell lines.By mass spectrum analysis,we identified RPB2,as a strong candidate,interaction with TANGO6.Further study demonstrated that RPB2 nuclear import required TANGO6 assistance,when TANGO6deficiency,led to RPB2 accumulated at cell cytoplasm and reduced in cell nucleus.Therefore,we supplied NLS fused polr2b m RNA to tango6^cq72 mutants,it could mitigate HSPCs defective phenotype in cq72 mutants.Finally,by analyzing HSPCs population in Tango6 c KO mice,we found the number of HSPCs was also reduced in c KO mice.Taken together,we discover TANGO6,as a novel factor,is critical for HSPCs expansion in zebrafish and mice,uncover TANGO6-RPB2 axis function in regulating cell proliferation,and provide new insight to HSPCs expansion regulatory network.Part Ⅱ:An FDA-approved drug screening for compounds that facilitate hematopoietic stem and progenitor cells（HSPCs）expansion in zebrafishAs we mentioned at previous section,HSPCs abnormal development bring out many hematological diseases,and the major therapy to treat these sicknesses is HSPCs transplantation.Therefore,the ability to expand sufficient HSPCs prior to transplantation has great clinical significance.Our research emphasis is in identifying small molecule drugs to be used as efficient agonist for HSPCs expansion.Over the past two decades,a panel of experiments have been conducted on drug exploration in zebrafish.For instance,dm PGE2 and trifluoperazine have been applicated at clinical phase II or phase I stage.Base on this,we successfully established a screening system in zebrafish by adopting an FDA-approved drug library to identify candidates that promoted HSPCs expansion.To date,we have screened 171 drugs of 7 categories,including antibacterial,antineoplastic,glucocorticoid,NSAIDS,vitamins,antidepressant,and antipsychotic drugs.By quantifying cmyb⁺signal areas in CHT region,we found 21 drugs that contributed to HSPCs expansion,32 drugs administration caused HSPCs diminishment and 118 drugs treatment elicit no effect on HSPCs amplification.Among these 21 drugs,due to advantages of easy delivery and limited side effects,we focused on 6 vitamins（biotin,α-tocopheryl acetate,ergocalciferol,panthenol,ascorbic acid and retinol）.Consistently,Ed U incorporation assay indicated that the 6vitamins significantly promoted HSPCs expansion.Furthermore,we found only ergocalciferol and panthenol administration could mitigate ikzf1 mutants HSPCs expansion defective phenotype.Meanwhile,fluorescence-activated cell sorting（FACS）analysis indicated that the proportion of CD41-GFP^low population（HSPCs）in whole CHT cells increased obviously after treating with ergocalciferol and panthenol,supported that the two drugs drastic effects on HSPCs expansion.Additionally,we uncovered ergocalciferol and panthenol facilitate HSPCs expansion in a dosage-dependent manner,however,their analogs failed to present the similar effects.In summary,by preliminary screening of FDA-approved 171 drugs,we identified21 drugs contributed to HSPCs expansion.Among these drugs,we focused on vitamin drugs,especially ergocalciferol and panthenol,due to their administration could mitigate ikzf1 mutants HSPCs expansion defective phenotype,furthermore,their effects on HSPCs promotion in a dosage dependent manner.Collectively,our study highlights the significance of small molecule drugs on HSPCs homeostasis maintenance,and uncovers the clinical application potential of ergocalciferol and panthenol.