Mechanisms Of Klf5 In Regulating Selfrenewal And Reprogramming Of The Trophoblast Stem Cells

The placenta is an important organ that connects the fetus to the mother’s body during pregnancy,mainly providing nutrients and hormones,excreting metabolic waste,and producing immune protection,playing an important role in fetal development.Somatic cell nuclear transfer(SCNT)has huge potential for application in the rapid reproduction of excellent livestock and the protection of endangered species,but the birth rate of cloned animals is only about 1%.By correcting abnormal DNA methylation,incomplete the zygotic genome activation(ZGA),abnormal histone modifications,and over-activated imprinting genes in cloned embryos,the birth rate of cloned animals can be increased to about 15%.However,developmental abnormalities in the TE cells of cloned embryos still exist,leading to reduced formation of placental blood vessels and placental hypertrophy caused by expansion of the spongy nourishing layer.The stable isolation of trophoblast stem cells(TSCs)from the early embryo trophectoderm(TE)provides an excellent cellular model for studying complications in placental pregnancy.Klf5 is expressed in mouse TE and participates in regulating the pluripotency of embryonic stem cells(ESCs).However,it is unclear whether Klf5 is involved in the regulation of TSC pluripotency,how it maintains TE development,and whether it plays a role in SCNT.In this study,it was found that Klf5 recruits p300 to affect the enrichment of H3K27 ac in the promoter regions of pluripotency genes,maintaining the chromatin in an open state and thereby regulating the expression of TSCs pluripotency genes by knocking down(KD)Klf5 in TSCs and combining multiomics data.In addition,Klf5 is highly enriched in the reprogramming barrier regions during cloned embryo development.The main research results are as follows:(1)To identify the pluripotent state and differentiation potential of TSCs derived from fertilized embryos and nuclear transfer trophoblast stem cells(nt TSCs)derived from nuclear transfer embryos,the results showed that nt TSCs can maintain a good pluripotent state in the stem cell culture system.nt TSCs can express various placental trophoblast cell markers upon in vitro differentiation,but the chimeric efficiency of nt TSCs in the 18.5-day placenta in vitro is lower than normal TSCs.(2)The chromatin accessibility of cumulus cells(CCs),TSCs,and nt TSCs was compared.The results showed that the chromatin accessibility and transcriptional regulatory network transition in nt TSCs are highly similar to TSCs,but there are still some chromatin regions resistant to reprogramming in nt TSCs.This is mainly manifested by the difficulty in fully reprogramming the distal region and the retention of some genetic imprints from the somatic cell genome.The trophoblast developmentrelated genes,such as Klf5 and Cdx2,are highly enriched in the reprogramming resistance region.(3)Klf5-KD TSCs cannot maintain a flat epithelial-like clone.The expression of pluripotency-related genes is significantly downregulated,while the expression of TE development-related genes is significantly upregulated.The cell proliferation ability is weakened,proving that Klf5 is crucial for TSCs self-renewal.(4)By combining transcriptome sequencing(RNA-seq)data and Klf5 chromatin immunoprecipitation sequencing(Ch IP-seq)data,it was found that Klf5 is mainly enriched in the proximal promoter region of TSCs pluripotency genes.Using RNA-seq and Klf5 Ch IP-seq during TSCs differentiation,495 Klf5-targeted pluripotency genes were screened.Ch IP-seq analysis of chromatin accessibility(Assay for Transposase Accessible Chromatin sequencing,ATAC-seq)was performed on Klf5-KD TSCs,and it was found that the transcription levels of Klf5-targeted pluripotency genes were highly positively correlated with changes in chromatin accessibility.(5)Analysis of changes in histone modifications in Klf5-KD TSCs showed a significant downregulated trend of H3K27 ac protein levels.After Klf5-KD,the enrichment levels of H3K27 ac in the promoter region of Klf5-targeted pluripotency genes were significantly reduced.The acetyltransferase p300 of H3K27 ac has an interaction with Klf5,and the enrichment level of p300 in the promoter region of Klf5-KD TSCs pluripotency genes is significantly lower than the control group.Furthermore,by activating p300,it was found that the enrichment of H3K27 ac in the promoter region of pluripotency genes affected by Klf5-KD can be restored to the level of the control group.(6)Knocking out(KO)Klf5 in embryonic resulted in a significant decrease expression of H3K27 ac in TE cells,which led to almost no expression of TE development-related genes.Proximity Ligation Assay(PLA)showed that Klf5 and p300 have an interaction in the trophectoderm.Klf5-KO embryos cannot form typical blastocyst expansion and cannot obtain a stable TSC cell line in vitro.In summary,Klf5 plays an important role in maintaining TSCs self-renewal and TE development and reprogramming processes.Klf5 is highly enriched in the chromatin regions that are not open during reprogramming.As an activator transcription factor,Klf5 occupies the proximal promoter region of TSCs pluripotency genes and maintains their chromatin in an open state through H3K27 ac.The regulation of H3K27 ac modification by Klf5 is crucial for TSCs establishment and TE normal development.This study further deepens the understanding of the TSCs pluripotency regulatory network,reveals the mechanism of histone modification in TE development,and provides a theoretical basis for improving animal birth rates.