Epigenetics Mechanism Underlying Photoperiod-Induced Seasonal Activation Of The Reproductive Axis In Quail

Seasonal breeding is an evolutionary strategy birds adopt to the natural environment and maximize the survival of their offspring in the long-term evolution process.The mechanism of timely initiation of the reproductive axis is the most challenging scientific problem in the study of seasonal reproduction.Photoperiod regulates the seasonal reproduction of animals through the reproductive axis,in which the hypothalamus is the regulatory center that senses light signals and transforms them into endocrine signals.Quail is an important model for studying photoperiodic regulation and seasonal reproduction.Using quail as a model,Triiodothyronine(T3)in Medial Basal Hypothalamus(MBH)is the dominant signal controlling the initiation of the reproductive axis.However,how T3 affects the initiation of the downstream reproductive axis,its related neural pathways,action cells,and molecular targets are still unclear.Numerous studies have shown that photoperiodic adaptation produces phenotypes associated with active changes in epigenetic marks.Cytological evidence also shows that the process of reproductive axis initiation is highly dependent on the reversible dynamic changes of chromatin structure,openness,and modification in cells,which undoubtedly opens a new breakthrough for the analysis of the seasonal reproductive axis initiation mechanism.In this experiment,sexually mature male quail(Coturnix japonica)were reared under a short day(SD)(6L:18D)program for 4 weeks and then transferred to long day(LD)(20L:4D),samples were collected from the MBH area of the hypothalamus at 0,3,7 and 28 days after the light change(SD28,LD3,LD7,and LD28)for open chromatin sequencing,and the transcriptome data of SD28 and LD28 were retrieved from the public database.The dynamic changes of the transcriptome during the initiation of the reproductive axis were analyzed.The genome-wide binding profiles of key transcription factors during reproductive axis initiation were verified by DNA affinity purification and sequencing.The experiment results show that:(1)During the initiation of the reproductive axis,the gene expression levels in the MBH area of quail hypothalamus changed dynamically.In the comparison group of SD28 and LD7,125 Differential expression genes(DEGs)were identified,including 41 upregulated genes and 84 down-regulated genes.In the comparison group of SD28 and LD28,169 differentially expressed genes were identified,including 117 up-regulated genes and52 down-regulated genes.There were 36 genes in the two comparison groups,including key genes of deiodinase system(TSHB,DIO2 and DIO3)and key genes of reproductive function(GHRH).89 genes showed time-specific differences in LD7 and 133 genes showed time-specific differences in LD28,suggesting their potential functions in photoperiodic response and initiation of the reproductive axis.(2)The results of chromatin openness data analysis showed that most of the open chromatin in the hypothalamic region was concentrated in the promoter region,intronic region and distal intergenic region,and there was no large-scale remodeling of the open chromatin during the photoperiod change.Differential analysis showed that there were only two differentially open regions at LD7 and LD28 time points,which indicated that the changes in chromatin accessibility status during photoperiod changes in the hypothalamus of Japanese quail tended to be stable after7 days.All the differential accessibility regions were divided into four clusters K1,K2,K3 and K4 using the K-means clustering method.After sequence statistics of each cluster,it was found that K1 cluster was mainly enriched in RFX,NKX,SOX family transcription factors and X-box.NKX family and MEF family members were significantly enriched in K2 cluster.The Nuclear Receptor(NR)superfamily(GRE,PGR,ARE,THRA,THRB,PR,AR-Halfsite,etc.)and BZIP family(Jun-AP1,HLF,NFIL3)were significantly enriched in K3 cluster.Significant enrichment of NR family transcription factor members was also found in the K4 cluster,including LXRE,RORGT,THRA,THRB.The results of the footprint analysis of the differential open regions indicate that the transcription factors THRA and THRB tend to be stable at the later stage of the photoperiod change.Secondly,transcription factors AR,NR3C1 and NR3C2 all had significant footprint binding activities in different groups,indicating that these transcription factors also played a certain role in the whole process of responding to photoperiod changes.(3)The joint analysis results of chromatin accessibility and gene expression showed that all genes were divided into seven types according to the expression changes and open chromatin changes: atac Down＿rna Down,atac Down＿rna Up,atac No Sig＿rna No Sig,atac No Sig＿rna Sig,atac Sig＿rna No Sig,atac Up＿rna Down,atac Up＿rna Up.The results showed that most genes did not show a large number of chromatin accessibility changes in the hypothalamus under the stimulation of photoperiod changes.There was no significant correlation between open chromatin changes and the expression changes of related genes at SD28 and LD7 time points(Pearson correlation,r =-0.006,p = 0.48).However,the correlation between open chromatin changes and the expression changes of related genes at SD28 and LD28 time points was significant(Pearson correlation,r = 0.041,p = 3.302e-06).In addition,a number of genes were identified in the association analysis,such as KLF9,ZNF366,DRD3,HRH1,ALS2 CL,ETNPPL,CSRNP1,ADAD1,SCNN1 D,etc.The expression changes of these genes may be directly regulated by the epigenetic state.(4)DAP-seq technology was used to perform binding verification of THRB transcription factors and screen downstream target genes.It was found that THRB protein tended to bind to non-promoter regions in quail genome to play A regulatory role,and the binding site of THRB to target genes was confirmed as [T/C/A][G/A/C][A/G]GG[T/A]CA by statistical analysis.Combined with transcriptome analysis,we identified TRPA1,RASD2,PGR,TRPA1,SHROOM1,TRPM6,FRMPD1,LOC107307440 as potential targets of THRB.Based on the results of multi-omics analysis and published literature,we constructed a regulatory model of hypothalamic response to photoperiodic changes during the initiation of the reproductive axis.In conclusion,the combined analysis of ATAC-seq,RNA-seq and DAP-seq preliminarily revealed the differentially expressed genes,time-specific expressed genes,changes in chromatin openness and the relationship between transcription factors and THRB protein targeting regulation during the initiation of the male quail reproductive axis.Based on the results and literature reports,a model of hypothalamic regulation of seasonal reproduction was proposed.In conclusion,our results demonstrate the advantages of multi-omics analysis of complex traits and also provide new ideas and perspectives for expanding the theoretical system of photoperiod regulation of seasonal reproduction in animals.