Genome-wide Identification And Functional Characterization Of Novel Targets Of Silkworm BR-C

Insect growth and development are coordinated by ecdysone(20E)and juvenile hormone(JH).The transcription factor BR-C is involved in the 20 E signaling pathway.Previous studies have shown that BR-C is a 20 E primary response gene and plays key roles in larval molting and larval-pupal transition in insect.To date,it is clear how BR-C mediates 20 E signal and how post-translational modification of BR-C affects its transcriptional activity.However,little is known about the identification of downstream target genes directly regulated by the BR-C transcription factor,except for several cuticle protein genes and vitellogenin gene.Therefore,the identification of novel targets of insect BR-C will undoubtedly contribute to elucidate novel roles and signaling network of BR-C transcription factors.In this study,we focused on silkworm BR-C and perform a genome-wide identification of novel targets of silkworm BR-C via chromatin immunoprecipitation sequencing(ChIP-seq)approach.Furthermore,we deciphered the mechanism underlying BR-C regulation on the transcription of novel targets and explored the functions of novel target during silkworm growth and development.The main results are as follows: 1.ChIP-seq-based identification of novel targets of silkworm BR-CWe carried out a ChIP-seq analysis to identify novel targets of silkworm BR-C at genome-wide scale.According to the principle of ChIP-seq approach,we performed BR-C overexpression in silkworm embryo-derived BmE cells.The cells overexpressing the BR-C were fixed and then sonicated to shear genome into DNA fragments of 200-1,000 bp in length.The protein-chromatin complexes were immunoprecipitated using the anti-BR-C antibody and genomic DNA of the complex were collected for high-throughput sequencing.Our results showed that 16,477,790 raw reads and 16,455,296 clean reads were obtained from BR-C immunoprecipitated products,and 16,297,532 raw reads and 16,281,588 clean reads were obtained from input sample as a control without BR-C immunoprecipitation(NCBI accession number PRJNA518741).Compared with input sample,a total of 1,006 unique BR-C ChIP peaks as potential binding sites of BR-C were identified from BR-C immunoprecipitated products.After a mapping with the silkworm genome,we found that about 63% of BR-C ChIP peaks were located in the distal intergenic region,15% in the 3.0 kb promoter region upstream of transcriptional start sites(TSS),18% in the introns,3% in the exons,and only about 2% in the downstream region.Based on MEME Suite program,we further characterized the motifs within the BR-C ChIP peaks and observed that five motifs were most enriched within BR-C ChIP peaks.Tomtom program-based analysis found that these enriched motifs located within either promoter or non-promoter regions could be matched with known motifs that are recognized by other zinc finger transcription factors.Moreover,a genomic alignment revealed that 829 protein-coding genes were associated with BR-C ChIP peaks.GO enrichment analysis showed that these genes could be functionally classified into different categories.Within the class of biological process,high portion of genes were involved in biosynthetic process,catabolic process,metabolic process,ribosome biogenesis,developmental process,autophagy,cell communication,and protein folding.In addition,the categories of catalytic activity,binding,and structural constituent were mostly enriched in the class of molecular function.2.Validation of the candidate targets of silkworm BR-CGiven that transcription factor generally regulates the transcription of its targets by directly binding to the promoter of the targets,we focused on the genes that their promoters contained BR-C ChIP peaks.As a result,133 of 829 genes have BR-C ChIP peaks within their promoters.Considering that the BR-C gene is highly expressed during insect metamorphosis with larval-pupal transition,we further analyzed the transcriptome data of silkworm fat body during metamorphosis that we previously reported and found that 76 of 133 genes having BR-C ChIP peaks in their promoter regions were expressed in fat body during silkworm metamorphosis.Notably,25 of 76 genes,including nuclear hormone receptor HR96(BMgn010782),juvenile hormone esterase(JHE,BMgn000776),and soluble guanylyl cyclase alpha-1 subunit(GC-?1,BMgn009300),exhibited an expression patters similar to the BR-C gene.Further quantitative RT-PCR experiments revealed that in fat body,HR96,GC-?1,and BR-C were highly expressed during prepupa while JHE exhibited a high expression at the beginning of wandering.In wing disc,the expressions of HR96,GC-?1,and JHE showed a similar developmental change with BR-C.These results,together with their relationship with BR-C ChIP peaks,suggested that these three genes were potential targets of silkworm BR-C.We further confirmed the binding of silkworm BR-C to the promoters of its targets and the effects of silkworm BR-C on promoter activities.According to the position and DNA sequence of BR-C ChIP peaks within the promoters of silkworm BR-C targets,we designed specific primers to conduct ChIP-PCR experiment and electrophoretic mobility shift assay(EMSA)and demonstrated that silkworm BR-C can directly bind to the promoter regions of the potential target genes,including HR96,GC-?1,and JHE.We further cloned the promoter sequences of these three genes and performed a dual-luciferase reporter assay in silkworm BmE cells.The results showed that compared with the control overexpressing red fluorescent protein(RFP)gene,the activities of the complete promoters of either HR96 or GC-?1 were significantly promoted following BR-C overexpression,while BR-C overexpression has on effects on the activity of the JHE promote.When the BR-C binding sites in the HR96 and the GC-?1 promoter region were deleted,BR-C has no effect on the transcriptional activity of these truncated promoters.Taken together,our results concluded that silkworm BR-C positively regulates the transcription of HR96 and GC-?1 by a direct binding to the specific sites within their promoter regions.3.Function of the HR96 gene as a target of silkworm BR-CWe performed a transient RNA interference(RNAi)targeting the HR96 gene as silkworm BR-C target to investigate the function of HR96 in silkworm growth and development.According to the principle of RNAi,we synthesized double-stranded RNA(dsRNA)against silkworm BR-C or HR96 and injected into silkworm individuals at the beginning of wandering.At 24 h after injection,the mRNA expression of either BR-C and HR96 in the fat body was significantly decreased after injection with their dsRNA,compared with an injection of dsRNA against RFP gene as a control.Moreover,compared with the control,the RNAi-mediated knockdown of silkworm BR-C significantly decreased HR96 expression,further indicating that the HR96 gene is a target of silkworm BR-C.Further phenotype analysis revealed that compared with the control,the RNAi of either BR-C or HR96 caused larval death and abnormal pupation.Given that HR96 is involved in the regulation of lipid metabolism,we performed Nile Red staining and found that the RNAi of either BR-C or HR96 resulted in a significant decrease in the content of lipid droplets in the fat body,and the lipid droplets became smaller.Taken together,we concluded that silkworm BR-C transcription factor promoted the expression of HR96 by directly binding to the HR96 promoter region and subsequently regulated the lipid metabolism during silkworm metamorphosis with larval-pupal transition.