Regulation Of Insect Transcription Factor BR-C On The Transcription Of The Shtd Gene

Insect transcription factor Broad Complex(BR-C)is characterized as one of the primary response genes in ecdysone signaling cascade and directly regulates the transcription of downstream target genes involving in the transition of from larvae to pupa and from pupa to adult.It has been clearly demonstrated about the functions of BR-C and the mechanism underlying BR-C-mediated transcriptional regulation.However,few novel targets of BR-C have been identified in the recent years,except for the several known WCP genes and Vg gene.In our previous study,we performed a genome-wide identification of novel target genes of silkworm BR-C based on chromatin immunoprecipitation followed by high-throughput sequencing(ChIP-seq)analysis.Among the potential targets of silkworm BR-C,Shatterred(Shtd)has been shown to be involved in the cell cycle progression.Herein,we cloned the full-length cDNA sequence of silkworm Shtd gene.In addition,we explored the direct transcriptional regulation of BR-C on Shtd transcription by using ChIP-PCR,EMSA and dual luciferase reporter assay.The roles and regulatory mechanism of BR-C and Shtd during the growth and development of wing disc in silkworm and fruit fly were also investigated.The main results are as follows:1.Full-length cDNA cloning and spatio-temporal expression analysis of silkworm Shtd gene Previous ChIP-seq data revealed that silkworm transcription factor BR-C can bind to the promoter of the Shtd gene(BMgn008276),indicating that the Shtd gene is a candidate target of BR-C.We cloned the full-length cDNA of silkworm Shtd,which is6406 bp in length and consists of 16 exons and 15 introns.Domain prediction demonstrated that the amino acid sequence encoded by Bm Shtd gene contains a typical ANAPC1 domain.We used the amino acid sequences of ANAPC1 domain of Shtd proteins from silkworm,fruit fly,red flour beetle,human and other different species to perform a multiple sequence alignment and found that the amino acid sequences of ANAPC1 domains are highly similarity,indicating that the Shtd genes in different species may play conserved physiological functions.Phylogenetic analysis revealed that the silkworm Shtd was firstly grouped with fruit fly Shtd and then grouped with other insects.We used quantitative RT-PCR method to profile the spatio-temporal expression of silkworm Shtd gene.The results showed that on day 3 of the fifth instar(L5D3)of silkworm larvae,the Shtd gene was highly expressed in the several tissues with mitotic cycle,including wing disc,gonads(ovary and testis),and hemocyte,and exhibited a low expression in endocycling tissues of fat body and silk gland.In the wing disc during silkworm larval-pupal transition,Shtd expression was high at early larval stage but was declined at the end of larval stage and pupal stage.Given that wing disc undergoes cell proliferation and cell growth during larval-pupal transition,our result suggested that Shtd may suppress the cell cycle progression in wing disc.2.Direct regulation of silkworm BR-C on the transcription of the Shtd geneInsect BR-C is a zinc-finger transcription factor.We analyzed the regulation of silkworm BR-C on the transcription of the Shtd gene.ChIP-Seq data analysis revealed that the promoter of the Shtd gene has one BR-C ChIP peak.Subsequent ChIP-PCR experiment in either silkworm embryo-derived Bm E cells with BR-C overexpression or wing disc during wandering confirmed that BR-C can directly bind to the identified ChIP peak in the promoter of the Shtd gene.Further electrophoreretic mobility shift assay(EMSA)also in vitro demonstrated the direct binding of BR-C to the promoter of the Shtd gene.We further cloned the promoter of silkworm Shtd gene and analyzed the regulation of BR-C on the activity of the Shtd promoter in Bm E cells by using a dual luciferase reporter assay.The results showed that compared with the empty control,the overexpression of silkworm BR-C significantly decreased the activity of the Shtd promoter.However,the truncated Shtd promoter without BR-C binding site had no response to silkworm BR-C overexpression.Given that developmental expressions of BR-C and Shtd in the wing disc during silkworm larval-pupal transition was contrary,we suggests that silkworm BR-C may negatively regulate the transcription of the Shtd gene by directly binding to its promoter.3.The roles of BR-C and Shtd in the growth and development of silkworm and fruit fly wing discWe performed RNAi experiments to characterize the roles of BR-C and Shtd in the development of silkworm and fruit fly wing disc.The double strand RNA(ds RNA)against silkworm BR-C and Shtd were synthesized.The ds RNA against silkworm BR-C were sequentially injected into silkworm larvae on day 5 and day 7 of the fifth instar,and Shtd ds RNA were sequentially injected on day 1 and day 3 of the fifth instar.At 24 h after injection,the expressions of BR-C and Shtd in silkworm wing disc were significantly decreased,compared with the RFP gene RNAi as the control.The expression of Shtd in the wing disc was reduced following BR-C RNAi,further indicating that BR-C negatively regulates the transcription of silkworm Shtd.In addition,we observed that BR-C RNAi caused a death during silkworm larval-pupal transition.Shtd RNAi had no effect on silkworm pupation but resulted in atrophied wing formation or down-curled wing at the adult stage.Moreover,wing disc-specific knockdown of fruit fly BR-C and Shtd genes driven by either Nubbin-Gal4 or En-Gal4 could also cause pupal death and wing developmental abnormality.We further analyzed whether the RNAi of either BR-C and Shtd affected the cell cycle progression in the wing disc.Ed U staining,which indicates DNA replication,revealed that BR-C RNAi decreased DNA replication in the wing disc of silkworm or fruit fly,but Shtd RNAi promoted DNA replication.Phosphorylated histone H3(pH3)is a mitotic marker.Immunostaining analysis demonstrated that BR-C RNAi and Shtd RNAi decreased and increased the amounts of pH3-positive cells in wing disc of silkworm and fruit fly,respectively.In addition,western blot analysis in silkworm and Drosophila showed that the protein level of both CycE and CycB in both silkworm and fruit fly were decreased following BR-C RNAi,but were increased following Shtd RNAi.Taken together,our data revealed that insect transcription factor BR-C inhibits the transcription of Shtd by direct binding to its promoter and then regulates the cell cycle progression by affecting the protein level of CycE and CycB.