Clip-domain serine proteases (CLIPs) play important roles in insect innate immunity and development.Our previous studies indicated that CLIP13,an epidermis-specific gene,was involved in cuticle remodeling during molting and metamorphosis in the silkworm,Bombyx mori.However,the transcriptional regulatory mechanism and regu-latory pathways of CLIP13 remained unclear.In the present study,we investigated CLIP13 expression and the regulation pathway controlled by 20-hydroxyecdysone (20E) in the silkworm.At the transcriptional level,expression of CLIP13 exhibited pronounced spatial and temporal specificity in different regions of the epidermis;homeodomain transcription factors POU-M2,antennapedia (Antp),and abdominal-B (Abd-B) showed similar expres-sion change trends as CLIP13 in the head capsule,thorax,and abdomen,respectively.Furthermore,results of cell transfection assays,electrophoretic mobility shift assays,and chromatin immunoprecipitation demonstrated that POU-M2,Antp,and Abd-B were in-volved in the transcriptional regulation of CLIP13 by directly binding to their cis-response elements in CLIP13 promoter.RNA interference-mediated silencing of POU-M2,Antp,and Abd-B led to a decrease of CLIP13 expression in the head capsule,the epidermis of the 1st to 3rd thoracic segments and the 7th to 10th abdominal segments,respectively.Consistent with CLIP13,20E treatment significantly upregulated expression of POU-M2,Antp,and Abd-B in the silkworm epidermis.Taken together,these data suggest that 20E positively regulates transcription of CLIP13 via homeodomain proteins POU-M2,Antp,and Abd-B in different regions of the silkworm epidermis during metamorphosis,thus af-fecting the molting process.Our findings provide new insight into the functions of home-odomain transcription factors in insect molting.

State Key Laboratory of Silkworm Genome Biology,Southwest University,Chongqing,400716,China;Biological Science Research Center,Southwest University,Chongqing,400716,China;Chongqing Key Laboratory of Sericultural Science,Chongqing Engineering and Technology Research Center for Novel Silk Materials,Southwest University,Chongqing,400716,China;College of Biological Science and Engineering,Shaanxi University of Technology,Hanzhong,Shaanxi Province,723001,China

[1]Huawei Liu;Jingya Heng;Luoling Wang;Youshan Li;Xin Tang;Xuan Huang;Qingyou Xia;Ping Zhao-.Homeodomain proteins POU-M2,antennapedia and abdominal-B are involved in regulation of the segment-specific expression of the clip-domain serine protease gene CLIP13 in the silkworm,Bombyx mori)[J].中国昆虫科学（英文版）,2022(01):111-127

Homeodomain,antennapedia,CLIP13,CLIPs,Antp,odomain

proteins,POU,M2,abdominal,are,involved,regulation,expression,clip,serine,gene,silkworm,Bombyx,mori,Clip,proteases,play,important,roles,insect,innate,immunity,development,Our,previous,studies,indicated,that,epidermis,was,cuticle,remodeling,during,molting,metamorphosis,However,transcriptional,regulatory,mechanism,pathways,remained,unclear,In,present,study,investigated,controlled,hydroxyecdysone,20E,At,level,exhibited,pronounced,spatial,temporal,specificity,different,regions,homeodomain,factors,Abd,showed,similar,change,trends,head,capsule,thorax,abdomen,respectively,Furthermore,results,cell,transfection,assays,electrophoretic,mobility,shift,chromatin,immunoprecipitation,demonstrated,were,directly,binding,their,cis,response,elements,promoter,interference,mediated,silencing,decrease,1st,3rd,thoracic,segments,7th,10th,Consistent,treatment,significantly,upregulated,Taken,together,these,data,suggest,positively,regulates,via,thus,af,fecting,process,findings,provide,new,insight,into,functions

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