Chromosome Conformation Of Silkworm And Its Regulating Mechanism For Fibroin Genes Expression

The phenomenon of insect systhezing silk is very common in our life and it performs different biological significance.Silkworms stand out from a wide range of insects for their excellent silk protein synthesis capability obtaining from evolution and more than 4,000 years' artificial domestication.The silkworm consumes 20 g mulberry leaves in larval stage and produces a 0.5g cocoon.The cocoon shell was consisted of three types of fibroin proteins and three types of sericins.The most enriched fibroin heavy chain(Fib-H)account for 70% of cocoon shell.So one silkworm can systheze 0.35 g Fib-H protein.It is rarely in nature with so high expression level of a gene.The expression of Fib-H gene is regulated by two mechanisms: forming superploidy cells in the posterior silk gland;comprehensive and fine regulation with multiple transcription factors mediated its temporal and spatial expression.But the two factors can not fully explain,whether there are other ways to regulate its expression? Scientist of Japan found out the state of chromosomal was different at the Fib-H gene locus when the gene expressed or not.With the development of technology for genome researching and the understanding of chromosomal structure,we know different chromosomal status is caused by the change of spatial structure of chromosome and involves in the regulation of gene expression.Chromosome conformation capture technique and its derivative technology are commonly ultilzed to study the spatial structure of chromosomes.It is unclear whether the conformation of chromosomes is involved in the regulation of Fib-H gene expression.In the present study,we obtained the whole genome interaction of cell line and silk gland of silkworm utilizing chromosome conformation capture technique(HiC)to analyze the intra-chromosome and inter-chromosome interactions.The chromosome conformation of Fib-H gene region was analyzed from chromosome interaction map and tried to explain the effect of the three-dimensional spatial structure of the genome on the regulation of Fib-H gene expression.Then we performed genome editing of BmlaminA/C for destroying the genome 3D structure to determine the function of conformation for Fib-H gene expression.The results of the study are as follows: 1.Whole genome interaction and three-dimensional model of BmE cell line and silk gland cells in multiple stagesWe detected six samples,BmE cell line(BmE),posterior silk gland(4M-PSG)of 4th molt stage,middle silk gland(5L3-MMSG,5L3-PMSG)and posterior silk gland(5L3-PSG)in the 3rd day of 5th instar,and posterior silk gland of pre-pupal(PP1-PSG)respectively and obtained the whole genome chromosome interaction using the high-throughput chromosome conformation capture technique(HiC).The results showed that interaction frequency between chromosomes(inter-chromosome interation)was weak,whereas the interaction frequency in chromosomes(inter-chromosome interation)were high,indicating that there were chromosome territories in silkworm cells.The trans-interaction map reflects interactions between chromosomes.The interactions between Chr5,Chr13 and Chr22 in BmE cell lines were frequent.It is presumed that the three chromosomes were relatively closer in the nuclei of BmE cells.5L3-PSG had a frequent interaction between Chr11 and Chr14,while in 4M-PSG had no significant frequent interaction between chromosomes.In 5L3-MMSG and 5L3-PMSG,Chr8 and Chr24 were interacted frequently.In PP1-PSG,the interaction between Chr7 and Chr22,Chr11 and Chr28,Ch4 and Chr7 were relatively frequent,suggesting that these chromosomes are closer in space.According to the interaction data,two spatial structure of chromosomes were obtained.BmE cell lines were relatively homogeneous,with high inter-chromosome interaction frequency and silk gland tissue cell had a relatively heterogeneous conformation and high intra-chromosome interaction frequency.2.Compartments and Topologically associated domain(TAD)analysis of silkworm.The main interaction pattern of silkworm cells was intra-chromosome interaction acquired from the whole genome chromosome interaction data.The intra-chromosomal interaction is called as cis-interaction,and displayed as chromosomal matrices.The chromosome interaction matrix of BmE cells and 5 silk gland samples showed that smaller interaction pattern than chromosome territories exist called chromosome compartments.These compartment are cell-type specific and associate with chromatin state like open and closed.The chromosomes are divided into A and B compartments by Principal Component Analysis(PCA).The chromosomal compartment of BmE cells is more obvious and the generally divided the chromosome into 1-6 parts.The compartments of five tissue samples were more complex which needed to combine the first and the second eigenvector to determine,and the differences among the chromosomes were very large.Some chromosomes had only one chromosomal compartment,indicating that the whole chromosomes were distributed in one compartment.The chromosomal compartment between the silk gland tissue samples is not large,indicating that the chromosomal compartment provides the necessary environment for the experiment of cell process function,but it is not a sufficient condition for functional realization.Although the silk gland cells from different part,their function are much similar.If the difference between the two organizations is very large,the chromosomal compartment will also be a corresponding change.Since the chromosomal compartment is not a decisive interaction structure,then the smaller level of interaction is TAD.Interaction frequency is very high when sites within a TAD,but the outside region even very close on linear,the interaction frequency was low.The TAD size of BmE cell lines was 100 kb-1Mb,while the tissue samples contained larger TADs ranged from 0.1kb-2Mb.TAD provides a microenvironment for interacting genes to ensure that gene expression regulation is more accurate.The smaller TAD is responsible for fundamental gene expression,whereas the large TAD is formed to achieve gene regulatory expression.3.Three chromosomes of 3rd day of 5th instar owing special chromosomal structure were Chr11,Chr24 and Chr25.When we analyzed the whole genome interaction,Chr11,Chr24 and Chr25 of 5L3-PSG were found to have a specific chromosomal interaction pattern: a region divided the chromosomes into two relatively independent chromosome regions.Sites in two regions interacted frequently while interaction frequency between the two regions was very low.The region Chr11: 4,100,000-4,300,000 divided the Chr11 into two parts,and no coding gene was reported in this region.The interval region of Chr24 was Chr24: 14,600,000-15,000,000 which was a gap in the silkworm genome.The specific locus in Chr25 was Chr25: 11,700,000-11,900,000 which contained only a Fib-H gene.We found out three genes encoding sericin were located in Chr11,Sericin1 was located in the smaller chromosomal interaction region,while Sericin2 and sericin3 were located in the larger half part.For Chr24 have no silk protein encoding genes,we are not studing in depth.Sericin and silk fibroin accounting for 75% of the total cocoon shell,whether the specific pattern of interaction within the chromosome is involved in the regulation of the high expression of the silk protein genes is still unknown.In order to identify the relationship between the structure of chromosomes and the high expression of silk protein,we analyzed the chromosomes interacting with the other four silk gland tissues(4M-PSG,5L3-MMSG,5L3-PMSG,PP1-PSG).It was shown that Chr11 showed a special chromosome structure in all samples,whereas Chr25 did not.The interactions between 5L3-MMSG and other chromosomes of 5L3-PMSG was much similar,and the interaction of other chromosomes between 4M-PSG and 5L3-PSG was similar.Although PP1-PSG was the posterior silk gland material,the chromosomal structure was much different with the other four samples due to the end up of the silk protein expression which indicating the consistency of the structure and function of chromosomes.Chromosome interaction pattern of Chr25 is much different in five samples,suggesting that there was some relationship with the expression of Fib-H gene.When we analyzed the expression activity of Fib-H gene in five samples,the Fib-H gene was actively experssed in 5L3-PSG,temporarily inactive in 4M-PSG and permanently inactive in PP1-PSG,5L3-MMSG,5L3-PMSG.Therefore,the appearance of specific chromosomal conformation and Fib-H gene expression pattern were temporally and spatially specific.This result was associated with the previous study of Waga finding,and there were significant differences in DNase I sensitivity at the 5' upstream,enhancer-promoter region and the second exon regions of Fib-H gene region[1].In contrast with Chr25,Chr11 showed a specific chromosome interaction pattern in all samples,but there was a slight difference interaction pattern at the Sericin1,Sericin2 and Sericin3 genes loci in 5L3-MMSG and 5L3-PMSG.Therefore,the specific chromosome interaction pattern of Chr11 does not have tissue-specificity,but the chromosomal interaction between Sericins genes is tissue-specific.4.The function of 3D genome structure in silk gland was explored by CRISPR / Cas9.It is not known whether the special chromosome interaction pattern of Chr25 plays a role in regulating the expression of Fib-H gene in the silk glands of the 3rd day of 5th instar.We use genome editing strategy changing the chromosome conformation to study its effect on Fib-H gene expression.Nucleus lamins play an important role in maintaining the morphology of nuclei and involve in attachment of heterochromatin to the inner layer of the nuclear membrane.The loss of the lamins will lead to the disorder of the chromosomal distribution in the nucleus and change the genome conformation in the nucleus.The lamin prtein is highly conserved in many species.We referred the sequence of predicted laminC-like from the full-length cDNA library of silkworm in NCBI,and validated the open reading frame(ORF).It was identical to the sequence laminC-like gene and named as BmlaminA/C.The total length of BmlaminA/C gene was 15111 bp,including 10 exons and 9 introns.The mRNA was 3262 bp and encoded a BmLaminA/C protein containing 620 amino acids which contained two conserved domains.Quantitative PCR was used to detect the tissue expression profile of Bmlamin A/C gene.The results showed that the BmlaminA/C gene was expressed in the head,fat body,malpighian tubules,midgut,silk gland and gonadat 3rd day of 5th instar.With the development of gene editing technology,our laboratory has used the target gene editing tools ZFN,TALEN and CRSIPR / Cas9 technology to achieve gene editing in silkworm.The methods above are efficient for genone editing of individual by embryonic injection of mRNA or plasmid of transient system.The transgenic individuals were screened from the offspring of G0 reared from injected embryo.However,when being used to investigate embryonic lethal gene or developmental regulatory genes,this method has larger limitations.The BmlaminA/C gene encods important structural protein-nuclear lamin.Systematically knockout of it could affect the development and growth of silkworm and was hard to study the effect of chromosome structure on silk protein synthesis.Therefore,it is necessary to construct tissue-specific gene editing system.In this study,we constructed a target site screening system to select highly efficient gene targets.Subsequently we performed transgenic injection to obtain two transgenic lines: tissue-specific Cas9 expression and ubiquitously expressed target gRNA,respectively.The transgenic marker gene was used to screen tissue-specific gene editing individuals.Based on the strategy,we obtained two transgenic lines targeting BmlaminA/C gene specificly in posterior silk gland.The results showed the target site of BmlaminA/C gene was mutated absolutely(100%)at genome level,the mRNA was down 43.7%-74.3% and the protein expression was decreased by more than 83%-91%.In transgenic lines,the ability of silk protein synthesis was significantly affected in two lines.The cocoon layer rate decreased significantly,and one of the strains had no silks which forming a nude pupa.There was no significant change in the number of silk gland cells in the transgenic individuals,but the results of staining showed that the glandular cells were disordered,the silk glands became shorter,the gland lumen became larger and empty,which was similar to the phenotype of transgenic individuals of Fib-H gene knockout.The results of fluorescence quantitative PCR showed that the major silk fibroin gene and transcriptional regulatory genes were down-regulated in the posterior silk glands.We presumed the conformation of chromosomes affects the expression of silk protein genes in the posterior silk glands.Transcriptomics analysis data showed,the Fib-H gene expression was decreased 10% of total TPM in the transgenic lines.So the the changes of genome conformation affected Fib-H gene a lot.The data indicated the 3-D structure of chromosome affected Fib-H gene expression.Above all,we used HiC chromosome conformation capture method to obtain the whole genome chromosome interaction map of silkworm cells and tissues,and found out the chromosomal conformation was involved in the regulation of silk protein expression.The specific chromosome interaction pattern of Chr25 appeared when Fib-H actively expressed.We disordered chromosome structure of silk gland cells with gene editing of lamin protein,and the expression of silk protein was significantly reduced.This suggestedthat the conformation of chromosomes may be a new factor in the regulation of high expression of silk protein.