The Mechanisms Of MRG15 In Cell Fate Determination Of Human Embryonic Stem Cells

Since the successful establishment of a stable in vitro platform for differentiation of human embryonic stem cells（hESCs）,scientists have explored multiple biological mechanisms in the process of differentiation of hESCs into three germ layers and have achieved remarkable results,which greatly enriched our understanding in this area.However,due to the complexity of the differentiation process of hESCs,the regulatory role of epigenetic mechanisms still needs further exploration.In our project,we aimed to find the key regulatory factors in the differentiation process of hESCs.Based on this issue,we took advantage of the in vitro differentiation platform of hESCs into neuroectodermal（h NECs）and the genome-wide CRISPR-Cas9 knockdown library.By analyzing the enrichment level of sg RNAs,we identified a key regulatory gene in the differentiation process of hESCs,the mortality factor on chromosome 4（MORF4）-related gene on chromosome 15（MRG15）.MRG15 was originally identified by Bertram et al.in 1999 and can reverse the immortal phenotype of a subset of cells.Since then,thanks to the continuous efforts of scientists,we have gained a broader and deeper understanding of the functions and regulatory mechanisms of MRG15.Over the years,studies of MRG15 mainly focused on two aspects.MRG15 is a key component of a variety of histone modification complexes and is involved in regulating various biological processes through histone modifications.On the other hand,MRG15interacts with different co-factors to participate in the regulation of biological processes such as DNA damage response and homologous recombination in cells.Based on these two regulatory mechanisms,researchers found that MRG15 plays a key role in regulating stem cell self-renewal in mouse embryonic stem cells（m ESCs）by repressing abnormal gene transcription.Moreover,MRG15 participates in the differentiation process of chicken retinal cells.the expression level of MRG15 homolog MRG-1 in C.elegans plays an important regulatory role in the self-renewal and differentiation of germ cells.MRG15 is essential for maintaining the proliferation of human neural precursor cells.However,less is known about how MRG15 participates in the biological processes during the differentiation of hESCs.Based on this,we aimed to explore the regulatory mechanisms of MRG15 in hESCs.We first successfully obtained MRG15 knockdown hESCs and compared their phenotypes to that of normal hESCs.The pluripotent morphology of hESCs significantly changed upon MRG15 knockdown.In addition,the expression level of pluripotency genes was significantly downregulated upon MRG15 knockdown.Those results indicate that hESCs lost the pluripotency and started to differentiate.Interestingly,we found that MRG15 knockdown hESCs exhibited a completely different differentiation path from normal hESCs.According to the developmental theory of Barresi and Gilbert,the default differentiation fate of embryonic stem cells is neural stem cells.That is,when cultured in spontaneously differentiating media,hESCs eventually differentiate into neural stem cells.This was confirmed by our results of high-throughput transcriptome sequencing（RNA-seq）of day 0 hESCs and hESCs spontaneously differentiated to day 10.hESCs spontaneously differentiated to day 10had significantly up-regulated expression of ectoderm-associated genes,but no expression of meso-/endoderm-associated genes.In contrast,significant upregulation of genes related to mesoderm,endoderm and ectoderm occurred after MRG15knockdown.This indicates that hESCs began to differentiate indiscriminately toward the three germ layers.We subsequently confirmed our results by q RT-PCR and immunofluorescence experiments.To investigate the regulatory mechanism of MRG15 in inducing this differentiation phenotype,we performed high-throughput sequencing experiments of MRG15 chromatin immunoprecipitation（Ch IP-seq）to identify the genomic binding targets of MRG15 in hESCs.We found that MRG15 binds to a subset of differentiation-associated genes.MRG15 knockdown abnormally upregulated a subset of genes that induce the initiation of differentiation of hESCs toward three germ layers.These genes were meant to be highly expressed at the late stage of the spontaneous differentiation process of hESCs.This result suggests that MRG15 is essential in maintaining the specific spatiotemporal expression of key genes during the differentiation of hESCs.Furthermore,we identified a key downstream target gene of MRG15,transcription factor AP-2 alpha（TFAP2A）.We then verified the binding of MRG15 on TFAP2A gene by Ch IP-q PCR and other experiments.Through systematically comparing the differentiation phenotypes of normal hESCs,MRG15 knockdown hESCs and MRG15^KD/TFAP2A^KO double mutant hESCs at day 10,we revealed the key\role of the MRG15/TFAP2A regulatory axis in the differentiation fate decision of hESCs.In addition,we found that the expression levels of a portion of MRG15 target genes involved in cell proliferation and DNA damage repair were not significantly changed upon MRG15 knockdown.The MRG15 enrichment and gene expression levels on those target genes were significantly higher than those of MRG15 target genes involved in the regulation of differentiation.To investigate the epigenetic regulatory mechanism of these results,we categorized MRG15 binding sites into three groups:target genes that significantly up-regulated upon MRG15 knockdown（up-regulate）,target genes that significantly down-regulated upon MRG15 knockdown（down-regulate）and target genes that not differentially expressed upon MRG15 knockdown（none-DEGs）.A systematic analysis of histone modifications and Pol II recruitment was performed according to the three groups.We found that the none-DEGs group was in an active epigenetic environment（stronger enrichment of histone modifications H3K4me3 and H3K9ac and enhanced Pol II signaling）.In contrast,the up-regulate group wad in a repressive or bivalent epigenetic environment（weak H3K4me3 and H3K9ac signal,stronger H3K27me3 signal and weak Pol II enrichment）.In the three groups of binding sites,we discovered the same changes in key histone modifications upon MRG15knockdown.H3K4me3 and H3K9ac signals were downregulated and Pol II enrichment was increased.This change is consistent with changes in histone modifications across the genome.In other words,changes in histone modifications did not correlate with changes in MRG15 target gene expression levels.This finding suggests that MRG15 in hESCs does not affect gene expression through histone modifications alteration,but rather that the target gene’s epigenetic environment affects MRG15 binding and thus MRG15 transcriptional regulation.In conclusion,we thoroughly investigated the mechanism of MRG15 during hESC differentiation and identified the key downstream genes regulated by MRG15.Our findings add a new dimension to the regulatory mechanism in hESCs differentiation.