Regulation Of DNase Hypersensitive Sequence For Specific Expression Of Coagulation Factor Ⅷ In Endothe Lial Cell

BackgroundCoagulation factor Ⅷ(FⅧ)is an important regulator of blood coagulation and hemostasis.FⅧ circulates as a complex with von Willebrand factor protein(vWF)and serves as a pro-cofactor for activated Factor IX in converting Factor X into activated Factor X.The FⅧ gene(F8)is located at the end of the long arm of the X chromosome.F8 defect can reduce or abolish the expression of FⅧ activity and cause hemophilia A,while increased FⅧ activity is linked to the susceptibility to the thrombosis.Therefore,studying the underlying mechanisms of F8 gene expression will be beneficial to elucidate the role of FⅧ in physiological hemostasis and the formation of coagulopathy disease.A study has determined that FⅧ is synthesized by specific blood vascular endothelial cells and lymphatic vascular endothelial cells using microarray and single-cell RNA-seq-based profiling.However,the molecular mechanisms underlying its endothelial cell subset-specific expression remains to be explored.In this study,we used phylogenetic analysis to identify an evolutionarily conserved segment of about 500 bases located in the middle of the first intron.This fragment is co-resident with a DNase-hypersensitive sequences(DHS)specifically manifested in FⅧ-expressing endothelial cells,but not in other endothelial cells and other cell types.These findings led me to hypothesize that this conserved region plays a role in F8 transcription.Through a series of in vitro biochemical experiments and in vivo gene knockout of its DNA fragments,the data as summarized in this thesis show that this conserved intronic DNA fragment plays a crucial role in governing endothelial cell subset-specific transcription of F8.This study reveals an unprecedented mechanism for F8 gene regulation,thus advancing the fundamental knowledge of FⅧ biology as well as providing important insight into clinical pathologic regulation of FⅧ.ObjectivesThe study aims to investigate the transcriptional mechanisms of F8 specific in endothelial cell.Methods1.F8 genome analysis.Queried the F8 genomic sequences of several mammals in the UCSC genome browser,compared the sequences with Bio Edit,explored the DNA regulatory elements exist in the conserved sequences with Encode database,and analyzed their function.2.Effect of DNase-hypersensitive sequences on the expression of luciferase.a.Cloned DNase-hypersensitive sequences by PCR in vitro.b.Inserted it into the upstream of the luciferase reporter gene promoter in a forward and reverse manner,respectively.c.Transfected it into cells by liposome transfection.d.Collected and tested cells for luciferase activity after 48 hours.3.Effect of DNase-hypersensitive sequences on F8 mRNA expression in cells.DHS-KO-bEnd3 cells and bEnd3 cells were cultured in 6-cm dishes until the confluence reached 90%.Collect cells for RNA extraction.F8 mRNA expression was analyzed by real-time PCR.4.Effect of DNase-hypersensitive sequences on FⅧ:C activity in cells.The same number of DHS-KO-bEnd3 cells and bEnd3 cells were cultured and kept in the same proliferation rate.Culture supernatant was collected and tested for the FⅧ:C activity using a FⅧ assay kit.5.Effect of DNase-hypersensitive sequences on F8 mRNA expression in tissues.Liver,spleen,kidney,lung,and lymph nodes from WT and DHS-KO male mice were isolated.RNA was extracted after tissue homogenization and tested F8 mRNA expression in different tissues by real-time PCR.6.Effect of DNase-hypersensitive sequences on FⅧ:C activity in plasma.Blood was drew from the inferior vena cava of WT and DHS-KO male mice.During the process,3.8%sodium citrate was used for anticoagulation.Blood was centrifuge at 3000g for 15min and the upper plasma was collected for FⅧ:C activity test.Results1.Discovery of DNase-hypersensitive sequences.We identified an evolutionarily conserved region at the middle of the first intron of the F8 gene.The conserved region is 509bp in length and contains a cluster of transcription factor binding site,such as GATA box,E-box-GATA composite motif and MEF2.Meanwhile,this region is co-resident with the DNase-hypersensitive sequences,which is highly active in FⅧ-expressing cells,suggesting that these sequences may related with F8 transcription specific in endothelial cells.2.DNase-hypersensitive sequences enhance the expression of downstream gene.We constructed the luciferase reporter gene system with DNase-hypersensitive sequences and transfected it into cells that express FⅧ.We found that the activity of luciferase significantly increased,indicating that DHS may enhance the expression of downstream gene like enhancer.3.Deletion of DNase hypersensitive sequences significantly reduces the expression of F8 mRNA in cells.Quantitative detection of F8 mRNA in bEnd3 cells and DHS-KO bEnd3 cells revealed that the deletion of DNase-hypersensitive sequences significantly reduced F8 mRNA expression(P<0.0001),implying that DHS affects F8 transcription.4.Deletion of DNase-hypersensitive sequences reduces FⅧ:C activity in cell.Cell culture medium of bEnd3 and DHS-KO bEnd3 were collected and tested for FⅧ:C activity.Results showed that FⅧ:C activity was reduced significantly(P<0.0001)after DHS deletion.5.Deletion of DNase-hypersensitive sequences significantly reduces F8 mRNA expression in tissues.Liver,kidney,spleen,lung and lymph nodes from WT and DHS-KO male mice were isolated,and RNA was extracted.Real-time PCR showed that the expression of F8 mRNA was significantly reduced in the knockout mice in all tissues,especially in the liver(P<0.001)and lymph nodes(P<0.0001).6.Deletion of DNase-hypersensitive sequences reduces plasma FⅧ:C activity in mice.Plasma from WT and DHS-KO mice was collected and tested for FⅧ:C activity.Results showed that when DNase-hypersensitive sequences were knocked out,the plasma FⅧ:C activity was significantly reduced(P<0.05),suggesting that DHS affects FⅧ activity even in complex environments such as in vivo condition.Conclusions(1)The DNase-hypersensitive sequences in F8 function like enhancer,which can significantly enhance the expression of downstream genes;(2)Deletion of DNase-hypersensitive sequences significantly reduces F8 mRNA expression and FⅧ:C activity in cells;(3)Knockout of the DNase-hypersensitive sequences in vivo reduces F8 mRNA expression in tissues and FⅧ:C activity in plasma.