Three-Dimensional Folding Dynamics Analysis Of The Xenopus Tropicalis Genome During Embryogenesis

The three-dimensional genomics research technology represented by highthroughput chromatin conformation capture technology(Hi-C)has opened the door to the study of the relationship between chromatin structure and function.Structures such as chromatin topology-associated domain(TAD),chromatin loop and compartment were often proved to be related to the regulation of gene expression.The structure of TAD and loop in the animal genome is considered to be conservative in evolution.Current studies believe that TAD structure is mainly composed of CCCTCbinding factor(CTCF),cohesin complex,chromatin remodeling complex(ISWI)and other factors involved in the establishment.In view of this,studies of how these highlevel chromatin structures are established in embryos at the beginning of life,and whether they are affected by the loss of transcription or related factors,can help people understand the changes in chromatin conformation during embryonic development and their impact on embryo development,cell fate determination and other mechanisms for a deeper level.In order to explore the dynamic changes in the three-dimensional structure of chromatin during embryonic development,we use Xenopus tropicalis as the research model to study the establishment process of TAD,loop,and compartment structures in the early embryonic development.Other treatments including transcriptional repression,knock-down of CTCF,Rad21 and SNF2 H were also processed to further reveal the dependencies between the establishment of chromatin high-level structure and related environments or factors during embryonic development.At the beginning of this work,we found that there were many assembly errors in the original Xenopus tropicalis v9.1 reference genome.In order to solve this problem,we carried out de novo assembly of high-quality reference genome,using Pac Bio sequencing data for contig assembly,and Hi-C interaction data for subsequent assisted assembly.In order to solve the problem of TAD identification and compartment identification in the early embryonic development of Xenopus tropicalis,we propose a TAD identification strategy integrating three methods and a compartment identification method by tracing back to the unstable compartment state of early embryos from the late embryo compartment state.Subsequently,we determined the establishment process of TAD structure in Xenopus embryos,which first began to establish after ZGA initiation,and gradually consolidated with embryonic development.This is coincided with the upregulation of CTCF and Rad21 expression during embryonic development.The establishment of the compartment structure also conforms to the characteristics of the establishment of the TAD structure.However,this process is accompanied by relatively frequent A/B compartment transformations in the early stages of embryonic development,and then gradually stabilizes.This work also determined by two forms of transcriptional repression experiments that,similar to the results in mice and Drosophila,the establishment of TAD structure in early Xenopus tropicalis embryos is independent of transcriptional processes,whereas studies have shown that TAD establishment in human embryos is transcribtion required.This indicates that although the formation mechanism of chromatin TAD structure is conserved in animals,the process of its de novo establishment still differs in different species.We used morpholino knockdown experiments for CTCF and Rad21 proteins to verify the dependence of TAD on CTCF and cohesin in the de novo establishment of early embryonic development.In addition,this paper also found that the de novo establishment of the Xenopus TAD structure also requires the participation of ISWI,and compared with the deletion of CTCF and Rad21 proteins,the impact of SNF2 H knock down on the chromatin structure can not be completely rescued by overexpression experiments.This work also found that there is an unbalanced interaction mode in the TAD structure,which is positively correlated with the binding of CTCF and cohesin,and the sides with stronger interaction have higher gene expression.The K-means clustering of the Directionality Index(DI)can well distinguish the TADs of this unbalanced interaction.Finally,this paper conducted a comparative study of the chromatin interaction maps of adult tissue cells such as brain,liver and sperm.We found that chromatin structures such as TADs and compartment are not conserved in different tissue cells of Xenopus tropicalis.Compartment and TAD structures in brain cells were stronger than those in embryonic and liver cells,while corresponding structures in liver were weaker than those in immature embryos.These results indicate that the chromatin structure of Xenopus adult tissues is not conserved.This suggests the great significance for further research and analysis of the relationship between differences of chromatin structure and cell function.