Unraveling Three-Dimensional Chromatin Structural Dynamics And Gene Regulatory Mechanisms During Spermatogonial Differentiation

Spermatogonial stem cells(SSCs)have bi-directional potential for self-renewal and differentiation,maintaining the stability of the stem cell pool and the continuous production of sperm in mammals.Differentiating spermatogonia has more heterochromatin in the nucleus,along with significant alterations in cell cycle progression,metabolic patterns,and proliferative capacity.The previous reports have demonstrated that the chromatin undergoes reorganization during spermatogenesis,which occurs in both diploid spermatogonia,tetraploid spermatocytes and haploid spermatids.Although the changes have occurred in differentiating spermatogonia,whether this process was accompanied by changes in chromatin structure was unclear.The changes in chromatin structure have emerged as important factors in the regulation of gene expression.The technique of high-throughput chromosome conformation capture(Hi-C)allow us to study the regulation of gene expression by chromatin changes during spermatogenesis at the three-dimensional genomic level.Therefore,in this study,Hi-C,RNA-seq and Ch IP-seq were used to explore whether significant chromatin structural changes occur during the differentiation of porcine spermatogonia and the effects of these changes on gene expression.The main results are as follows:(1)Undifferentiated spermatogonia(SSEA4+ spermatogonia)were sorted by flow cytometric sorting,and differentiating spermatogonia were enriched by STA-PUT.The purity of these cells was identified by cell morphology and immunofluorescence staining.The results manifested that the purity of all types of germ cells sorted was above 95%.(2)In this study,mini MDS was used to construct the 3D conformation model.The results revealed that the level of inter-chromosomal interaction and intermingling was decreased,and the distance of inter-chromatin was increased.In addition,the values of intra-chromosomal entropy were increased during spermatogonia differentiation,and the interactions of intrachromatin were more disordered.The inter-chromosomal interactions tended to form larger DNA loop.The localization patterns of centromere and telomere changed with differentiation,gradually shifting from centromere outward to telomere outward patterns.(3)The proportion of compartment A and B was largely unchanged during spermatogonial differentiation.The degree of chromatin compartmentalization and the intensity of the compartment in differentiating spermatogonia significantly decreased.The analysis showed that 4.55% of the genome regions occurred compartment switching,and 4.83%of the genome regions increased compartment strength.The genes which contained within these regions exhibited significantly altered expression levels during spermatogonia differentiation.In addition,the degree of compartmentalization and the intensity of the compartments in X chromosome decreased significantly after differentiation and tended to form two super interacting units.Moreover,the X chromosome was largely absent after differentiation,which explains the possible reason for the low expression of X chromosome genes.(4)TADs constituted the majority of the genome,with a decrease of the TAD number but increase of the mean TAD size during spermatogonial differentiation.The calculating of the D-score,DI and IS values showed a sharply decreased of intra TAD interactions and the TAD boundaries insulation during spermatogonia differentiation.Also,the enrichment of transcriptional start sites and housekeeping genes was decreased after differentiation.The effect of TADs on gene expression was mainly based on their ratio of internal compartments,the genes in A TAD were higher than B TAD.The calculating of the correlation of DI values at the TAD boundaries in the two stages showed that there were 1,482 specific TAD boundaries in undifferentiated spermatogonia and 926 specific TAD boundaries in differentiating spermatogonia.Genes enriched at stage specific TAD boundaries were associated with spermatogonial differentiation.Finally,the TAD cliques were identified by TAD long-range interaction,with a higher proportion of component B in the TAD clique upon differentiation.(5)PSYCHIC was used to identify the promoter-enhancer interactions during spermatogonia differentiation.The analysis found that the regulation of enhancers has an additive effect.The gene promoter with more regulatory enhancer had higher expression levels.The transform analysis of this effect to the RP values showed that the genes with higher expression levels had higher RP values.(6)By analyzing the enrichment of H3K4me3 and H3K27 ac during spermatogonia differentiation,the promoters were divided into active and inactive,and the enhancers were grouped into regular enhancers(REs)and super enhancers(SEs).In addition,the SEs were further divided into hierarchical and non-hierarchical structure based on the spatial interaction strength.The hierarchical structure contained hub enhancers and non-hub enhancers.Among them,hub enhancers had the highest RP value and regulated genes had the highest expression.Comparison of the genes with different RP values identified the self-renew genes FGF9 and DND1,which were regulated by hub enhancers,and the differentiation genes DMC1,EZH2,and CATSPER2,which were regulated by hub enhancers.In sum,this study found a large alteration in chromatin conformation during spermatogonial differentiation,which prepared for meiosis synapsis and homologous recombination.Overall,interchromosomal interactions decreased while the intrachromosomal interactions became more disorder.As for the different hierarchical structures,the degree of compartmentalization and the intensity of the compartment have decreased.Also,the TAD interaction strength and boundary insulation scores have decreased.The effect of this structural change on gene expression was mainly based on compartment A/B.In addition,the hub enhancer elements were involved in the regulation of self-renewal and differentiation genes through promoter-enhancer interactions.The present study has for the first time reported the process of transcriptional regulation by promoter-enhancer interactions during spermatogonial differentiation,and provides a desirable reference catalog for diagnosis of the regulatory mechanism of male spermatogenesis.