Identification Of Xenopus Tropicalis Enhancers During Zygotic Genome Activation And Expanding Genome Editing Tools In Xenopus Tropicalis

The identity of various cells in multicellular organisms is determined by precise spatial and temporal gene expression,which is mainly regulated by cis-regulatory elements and trans-acting factors.The very early stages of embryonic development in animals are completely dependent on the maternal materials before zygotic genome activation(ZGA).ZGA is the de novo gene transcription of the zygotic genome that is initially transcriptionally silent after fertilization.ZGA is regulated by enhancers and trans-acting factors and is an ideal model to study the regulatory mechanisms of gene expression in vivo.Systematic identification of genome-wide active enhancers during ZGA and mechanistic analysis of ZGA initiation would contribute to the understanding of the pathogenesis of congenital diseases.Unfortunately,to date,due to limited embryonic materials,it is hardly possible to use the Self-Transcribing Active Regulatory Region Sequencing(STARR-seq)method,a massively parallel reporter assay to identify transcriptional enhancers directly based on their activity in entire genomes initially developed for Drosophila cell lines,for high-throughput functional screening of enhancer activities during ZGA in any model organisms.With large number of embryos and relatively close genetic distance to humans,Xenopus tropicalis provide an ideal animal model for identifying genome-wide enhancers during ZGA.To identify genome-wide enhancers during ZGA of Xenopus tropicalis,I developed an improved version of the STARR-seq,designated as the Probe-capturing STARR-seq(Pc Starr-seq),which can specifically capture RNAs transcribed from injected reporter constructs,and analyzed the genomic distribution of the identified enhancers.The obtained data show that the identified enhancers are enriched in the promoter regions.Longer enhancers have stronger activities,which is correlated with higher expression levels of adjacent endogenous genes.The enhancer in the promoter region is called Epromoter.The genes corresponding to E-promoters identified in this study are mainly enriched in the basal metabolism,suggesting that E-promoters are essential for survival of organisms.Next,I analyzed the epigenetic characteristics of the identified enhancers during Xenopus tropicalis ZGA.Using the Concanavalin A beads-based Nucleus capture followed by Tn5-mediated Accessible Chromatin assay with sequencing(CANTAC-seq)newly developed for Xenopus tropicalis early stage embryos,I found that open chromatins were enriched in promoter regions during ZGA.Enhancers located in open chromatins were more active than those in non-open chromatins and were highly enriched with activating histone marks.the open chromatin enhancers were categorized into 11 groups by histone marks.Among them,4 groups were enriched in morphogen effectors and 1 group showed bivalent character(enriched with both H3K4me1 and H3K27me3).In general,the enhancer activity was positively correlated with the enrichment of maternal factors.The enhancers with active histone marks enriched in the central region have lower activities,while with active histone marks enriched in the flanking margins have higher activities.In addition,343 super-enhancers were screened from the open region enhancers.These super-enhancers related genes were enriched in multiple embryonic development biological processes suggested that super-enhancers are involved in the transcriptional regulation of important genes during the ZGA of Xenopus tropicalis.Physical proximity is the basic interaction mode between enhancers and target genes.Using the Hi-C data,I screened the target genes of enhancers through the genome-wide interaction mapping and analyzed the relatedness between the enhancer and its target gene.The topologically associating domain chromatin(TAD)structure was continuously established during Xenopus tropicalis ZGA.RNA-seq analysis with hybrid embryos derived from Xenopus laevis eggs fertilized by Xenopus tropicalis sperm detected de novo RNA expression profiles of Xenopus tropicalis genome during hybrid ZGA.With target genes of enhancers screened out from the at genome-wide chromatin contacts,I found that enhancer quantity is positively correlated with target gene expression levels and the activity of enhancers might be diluted with the increase of target genes.Through power function fitting analysis,a nonlinear model was revealed that can explain the relatedness among gene expression level,enhancer activities,enhancer-promoter interaction strength,and histone marks.Genome editing is a powerful tool for validating enhancer function in vivo.Currently,only Sp Cas9 is widely used in Xenopus tropicalis,which limits the selection of editing sites when validating enhancer activity in vivo.In this study,I evaluated the genome editing efficiency of 8 different Cas nucleases in Xenopus tropicalis and found that Sa Cas9,KKH Sa Cas9,and Lb Cas12a/cr RNA RNP complexes were highly effective in G0 embryos.I also found that multi-g RNA of KKH Sa Cas9,and paired cr RNAs of Lb Cas12a/cr RNA RNP complexes can efficiently induce enhancer DNA fragment deletion in Xenopus tropicalis embryos.These Cas nucleases will facilitate the in vivo enhancer validation of Xenopus tropicalis.In summary,in this study,I established the Pc Starr-seq method and identified wholegenome active enhancers during Xenopus tropicalis ZGA.Integrating the chromatin accessibility data,histone mark data,and Hi-C data,I proposed a nonlinear model to explain the relatedness among gene expression,enhancer activity,and enhancer-promoter interaction strength.Last but not least,I expended the genome editing scope of CRISPR/Cas in Xenopus tropicalis embryos.These data and methods provide a solid foundation for further studying genome function in Xenopus tropicalis.