| Human endogenous retroviral elements(HERVs)are derived from the integration of endogenous retroviruses into the germ line during primate evolution.The long terminal repeat(LTR)elements of endogenous retroviruses can hijack the host’s transcription factors and transcriptional complex to generate protoviral RNA,providing HERVs with potential cis-regulatory ability.As species evolve,the endogenous retrovirus sequence accumulates mutations,truncations,and loss of function in the viral protein,leading to the loss of the ability for horizontal transmission within the genome.Homologous recombination of the genome results in the loss of the entire viral structure gene,leaving only a solo LTR element,which is then referred to as an endogenous retrovirus element.These HERV elements have highly similar sequences and contain binding sites for host transcription factors.In some cases,the host can use these elements as temporary cis-regulatory elements to regulate its own gene expression.Endogenous retroviral elements have long been considered as "junk DNA" and genome parasites due to their ability to horizontally transmit within the genome,leading to increased instability.However,in recent years,researchers have discovered that the human endogenous retroviral element LTR5-HERVK is highly expressed during normal human embryonic development and has anti-viral function.It can even remodel the epigenetic state of local chromatin,indicating that endogenous retroviral elements have important biological significance,although the sequences are roughly similar,sequence differences resulting from accumulated mutations or truncations during evolution have hindered the studies on endogenous retroviral elements.Most reports have been limited to certain subclass and the systematic investigation is lacked.When compared to somatic cells,the chromatin of embryonic stem cells is more loosely packed and endogenous retroviral elements are more active.This makes them a good model for studying the role of endogenous retroviral elements.In this study,we used Ch IP-seq data of all histone modifications,transcription factors,and DNaseseq data from the human embryonic stem cell line H1 in the ENCODE database to represent the epigenetic status of chromatin.We then re-analyzed the high-throughput sequencing data by optimizing the multiple alignment sequence pipeline.Our research revealed that 163 out of 584 human endogenous retroviral elements were enriched with at least one histone modification.Through principal component analysis,we identified two of the most representative endogenous retroviral elements.The promoter-like MER57E3 elements was significantly enriched with H3K4me3 and H3K27 ac,and the MER57E3 elements of the promoter region had higher integrity and conservation than the intergenic MER57E3 elements.The enhancer-like LTR5 elements was significantly enriched in H3K4me1 and H3K27 ac.In addition to bioinformatic analysis,we also performed functional experiments on two endogenous retroviral elements.For the MER57E3 elements,our research revealed that the transcription factor TEF of the PAR b ZIP family can bind directly to the MER57E3 elements through transcription factor motif enrichment analysis.Furthermore,we confirmed that TEF can activate the MER57E3 elements directly,as shown through dual luciferase reporter assays.Additionally,our research conducted knockout/CRISPRi experiments on the MER57E3 elements and performed overexpression experiments of the TEF protein on the MER57E3 elements.These experiments provided evidence that the MER57E3 elements can regulate the expression of adjacent genes in human embryonic stem cells.Regarding the LTR5 elements,in order to target the majority of LTR5 elements,our research first designed sg RNA on the most conservative sequence of LTR5 elements at different sites,and reduced the length of the sg RNA to16 nt.We then constructed a lentivirus vector by tandem,where the lentivirus delivery vector expressed sg RNA that interacted with d Cas9-KRAB protein.As a result,we successfully suppressed the activity of LTR5 elements,and the transcript of the protovirus sequence HERVK,which is associated with LTR5 elements,was also significantly suppressed.Through RNA-seq analysis,we found that LTR5 elements can regulate gene expression whether they are located upstream,intron,or downstream of a host gene.Additionally,our research revealed that LTR5 elements are more active in early pluripotent stage cell lines with high expression of upstream transcription factors such as Naive embryonic stem cells and extended pluripotent stem cells.Furthermore,we transformed Primed embryonic stem cells into extended pluripotent stem cells and found that the transcriptional activity of LTR5 elements regulatory genes was significantly improved.The cis-regulatory effect of LTR5 elements was also found to be stronger when the element is closer to the host gene.To summarize,our research has generated the most comprehensive enrichment landscape of histone modifications and transcription factors of endogenous retroviral elements in human embryonic stem cells through bioinformatics analysis.In addition,we have improved the model of endogenous retroviral elements regulating host gene expression through a series of functional experiments. |
