Characterization Of Upstream Sequence Regulating The Wool Follicle Cortex Specific Expression Of Sheep KAP11.1 Gene

In the formation of hair follicles,the hair matrix cells can differentiate into cells of the inner root sheath and hair shaft,and the hair shaft finally become the hair.The hair shaft consists of medulla,cortex and cuticle,the cortex is an important part of hair shaft.Most of keratin associated proteins(KAPs)specifically express in cortex,the content of KAPs and interaction between KAPs and keratin influence the physicochemical property of hair,and finally influence the quality of hair.In our previous study,founding that the sheep KAP11.1 expressesspecifically in cortex,and after the transgenic mice model was used to confirm that the sheep KAP11.1 gene upstream 5952-bp sequence could drive expression of exogenous gene specifically in cortex.However,the mechanism of KAP11.1 gene expressing specifically in cortex is still unclear.In this study,bioinformatics prediction,dual-luciferase reporter gene detection,sites-directed mutagenesis,RNA interference,EMSA,immunofluorescence and other methods were used to identify the sheep KAP11.1 gene upstream regulatory sequence,and the KAP11.1-Cre transgenic vector expressing Cre recombinase was constructed.The main results as follows:(I)The study of sheep KAP11.1 gene upstream regulatory sequence 1.The sheep KAP11.1 gene 5’-upstream sequence contains crucial regulatory regionFirstly,applying deletion analysis for the sheep KAP11.1 gene 5’-upstream sequence(the A of ATG is +1)and obtaining seven constructs,named F5(-2576 ~ +1),F6(-2009 ~ +1),F7(-1594 ~ +1),F8(-1026 ~ +1),F9(-569 ~ +1),F10(-251 ~ +1),F11(-148 ~ +1).The constructs were subcloned into dual-luciferase reporter vector,and transfected into HEK 293 T cells.The results demonstrate that-251 ~-148 bp region of F10 construct contains crucial regulatory region influencing the expression of sheep KAP11.1 gene.Bioinformatics prediction shows that three transcription factors(Hoxc13,Tst-1/Oct6 and Ets1)related to hair follicle development binding sites are located at-251 ~-148 bp region.Secondly,RNA interference and sites-directed mutagenesis were applied to the three transcription factors and its binding sites to confirm that the three transcription factors binding sites regulate the expression of sheep KAP11.1 gene.2.The interaction between sheep KAP11.1 geneupstream-251 ~-148 bp region and candidate transcription factorsThe probes were designed according to Hoxc13,Tst-1/Oct6 and Ets1 binding sites in-251 ~-148 bp region.Electrophoretic Mobility Shift Assay was used to detect the interaction between the three transcription factors and binding sites,the results demonstrate that binding sites of Hoxc13,Tst-1/Oct6 and Ets1 interact with nuclear proteins,and DNA-protein complex was formed.3.The sheep KAP11.1 geneupstream-251 ~-148 bp region maybe a importanttranscription regulatory regionFirstly,the skin of sheep and KM mice were obtained for slices,immunofluorescence detection shows that Hoxc13,Tst-1/Oct6 and Ets1 were detected in hair follicle cortex of sheep and mice.Secondly,the pKAP11.1-sKAP13.1-2A-ZSG mice(named Plus mice)was prepared,which can express KAP13.1 and ZSG at the regulatory of sheep KAP11.1 gene 5’-upstream sequence.In this study,the skin of 9-day(anagen),15-day(late anagen),18-day(catagen),21-day(telogen)Plus mice were obtained for slices,immunofluorescencedetection shows that Hoxc13,Tst-1/Oct6 and Ets1 express in hair follicle cortex of Plus mice in anagen and late anagen,and not in catagen and telogen.The expression pattern of Hoxc13 and Ets1 are same as KAP13.1 in Plus mice.Thirdly,the sheep KAP11.1 gene 5’-upstream sequence,which Ets1 binding sites in-251 ~-148 bp region was mutated,was subcloned into dual-luciferase reporter vector and transfected into cells.Finding that the luciferase activity of KAP11.1 gene 5952-bp upstream sequence is obviously decreased,demonstrates that-251 ~-148 bp region maybe a transcription regulatory region of sheep KAP11.1 gene upstream sequence.(II)The construction of KAP11.1-Cre transgenic vector expressing Cre recombinaseThe sheep KAP11.1 gene 5952-bp upstream sequence that can drive expression of exogenous gene specifically in hair follicle cortex was utilized to construct the KAP11.1-Cre vector expressing Cre recombinase,and the KAP11.1-Cre vector was overexpressed in three kinds of cells(HEK 293 T,HHK 21,PK 15).The Cre recombinase activity was detected in vitro,and the KAP11.1-Cre vector was used to preparation of transgenic mice.