热带爪蛙合子基因组激活时期增强子鉴定及基因编辑研究

多细胞生物的细胞异质性源于基因的时空差异化表达，基因的表达主要由顺式作用元件和反式作用因子共同调控。动物胚胎发育早期，在合子基因组激活之前，胚胎发育完全依赖于母源物质。合子基因组激活是胚胎发育过程中基因表达从无到有的过程，这时基因的激活依赖于增强子与反式作用因子的调控，合子基因组激活是研究体内基因表达调控机制的理想模型，鉴定合子基因组激活时期全基因组增强子并探索合子基因组激活过程中相关的基因调控机制将为探索先天性疾病的发病机制提供帮助。然而，受到试验材料的限制，使用已广泛应用于全基因组鉴定增强子的STARR-seq技术鉴定胚胎时期全基因组增强子至今未有报道。热带爪蛙大量的胚胎数量且与人类遗传距离相对较近，这为鉴定合子基因组激活时期全基因组增强子提供理想的动物模型。

为了系统性鉴定合子基因组激活时期增强子，本研究采用自主开发了基于特异性RNA捕获的PcStarr-seq技术用于热带爪蛙合子基因组激活时期全基因组增强子鉴定，并分析了鉴定增强子在基因组上的分布。发现鉴定的增强子在启动子区域富集，增强子的长度越长其活性越强，强度越强的增强子对应临近基因的表达量也越强。在启动子区域的增强子被称为E-promoter，本研究发现E-promoter所对应基因主要富集到多个生物体的基础代谢活动中，说明E-promoter对生物体的基本存活至关重要。

本研究进一步对热带爪蛙合子基因组激活时期鉴定增强子的表观遗传特征进行分析。基于刀豆素A磁珠捕获细胞核的染色质开放性检测方法（CANTAC-seq技术），本研究构建了热带爪蛙ZGA时期染色质开放性图谱，发现染色质开放性区域在启动子区域富集，位于开放性区域的增强子活性强于非开放性区域的增强子，开放性区域增强子与激活性组蛋白修饰以及相关蛋白高度富集，与某些抑制性组蛋白修饰及相关蛋白微弱富集。本研究通过组蛋白修饰类型将染色质开放性区域增强子分为11类，其中4类增强子富集形态发生素的效应因子，且存在1类双功能增强子（富集H3K4me1和H3K27me3），发现增强子活性与富集母源因子的强度呈正相关。同时中心区域富集活性组蛋白修饰的增强子活性低，两端富集活性组蛋白修饰的增强子活性高。此外，本研究在开放区域增强子中筛选出343个超级增强子，这些超级增强子富集多个胚胎发育过程中，提示超级增强子参与热带爪蛙ZGA时期重要基因转录调控。

物理距离的靠近是增强子与靶基因互作基本模式，本研究通过全基因组互作图谱筛选增强子的靶基因，并分析了它们的调控关系。本研究使用Hi-C技术绘制了热带爪蛙早期胚胎全基因组互作图谱，发现染色质高级结构TAD随着合子基因组激活而不断建立，通过杂交胚胎获得热带爪蛙合子基因组激活时期新生RNA表达谱。通过增强子从全基因组互作网络中筛选出靶基因信息，发现增强子数目与靶基因表达呈正相关，增强子的活性可能随着靶基因数目增加而被稀释。通过基因集表达水平与增强子活性、增强子-启动子交互强度以及组蛋白修饰类型进行幂函数拟合，本研究发现基因表达水平与增强子活性、增强子-启动子交互强度存在非线性关系。

基因编辑方法是验证体内增强子活性的重要工具，然而目前在热带爪蛙中仅报道SpCas9可高效进行基因编辑，这限制了验证体内增强子活性时编辑位点的选择。因此，本研究探索了可用于热带爪蛙基因编辑的技术，通过检测目前已报道的8种CRISPR核酸酶，发现SaCas9和KKH SaCas9、LbCas12a/crRNA在热带爪蛙中有高效的基因编辑效率，KKH SaCas9和LbCas12a/crRNA可以在热带爪蛙中实现增强子DNA片段删除。这些高效的编辑酶都将为在热带爪蛙中进行增强子体内验证提供助力。

综上所述，本论文首次在热带爪蛙早期胚胎中建立了改进的STARR-seq功能性高通量增强子技术—PcStarr-seq技术，开发了热带爪蛙早期胚胎开放染色质检测的CANTAC-seq技术，绘制了热带爪蛙早期胚胎全基因组互作图谱，提出了基因表达与增强子活性、增强子-启动子交互强度的非线性模型，拓宽了热带爪蛙基因组编辑核酸酶的选择。这些方法及数据为在热带爪蛙中研究基因组功能奠定了坚实的基础。

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 (PcStarr-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 E-promoter. 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 SpCas9 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 SaCas9, KKH SaCas9, and LbCas12a/crRNA RNP complexes were highly effective in G0 embryos. I also found that multi-gRNA of KKH SaCas9, and paired crRNAs of LbCas12a/crRNA 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 PcStarr-seq method and identified whole-genome 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.

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