정원줄기세포 리프로그래밍 관련 E2f4 기능 연구

Abstract

Spermatogonial stem cells (SSCs) are the germ cells located along the basement membrane of semiferous tubules in testes, which can differentiate into spermatozoa. Recently, SSCs have been reported to reprogram into multipotent SSCs (mSSCs), which hold ES cell-like characteristics, under defined culture conditions. However, the mechanisms by which SSCs reprogram to mSSCs are not fully elucidated. To identify a mechanism of how SSCs acquire pluripotency,Iestablished intermediate SSCs (iSSCs), which are the transitional cells from SSCs to mSSCs, and examined the gene expression patterns using the data of gene expression profilings of SSCs, iSSCs, and mSSCs. From gene profiling data analysis, I built the network of differently expressed genes and transcription factors (TFs) that influence SSC reprogramming, and found twelve TFs and their target genes that might regulate SSC reprogramming. Various cellular pathways may control the SSC reprogramming. Among them, three processes, such as cell cycle, aerobic glycolysis, and epigenetic regulation, that may play a key role in SSC reprogramming. Especially, E2f4 that involves in the three pathways may control SSC reprogramming. To test this idea, E2f4 KO ESCs were generated by the replacement of endogenous E2f4 gene to HR donor containing a puromycin cassette using CRSPR/Cas9 recombination technique. E2f4 KO cells displayed the diminished cell proliferation with increasing S phase and decreasing G2/M phase compared to wild type R1 cells. Additionally, E2f4 KO cells increased the protein and mRNA levels of Oct4 compared to the control. As reported previous, E2f4 KO cells differentiated hematopoietic lineage at considerable rate. To identify the molecular roles of E2f4 on the above phenotypes, I investigated the functional study how E2f4 maintains pluripotency in ESCs. E2f4 associated with Oct4 but not Sox2 and Nanog in vitro and in vivo. EMSA data demonstrated that E2f4 formed a heterodimer with Oct4 in vitro and in vivo. E2f4 is known to be a transcriptional repressor. As anticipated, E2f4 suppressed Oct4 transcriptional activity through a heterodimers. Therefore, the expression level of Oct4 was higher in E2f4 KO cells compared to wild type cells. Likewise, the functional enrichment analysis using gene expression profilings in E2f4 KO and Oct4 KD cells demonstrated that the expression patterns of Oct4 target genes were anti-correlated between E2f4 KO and Oct4 KD cells. However, at the translational level, E2f4 stabilized Oct4 proteins through inhibiting Oct4 ubiquitination by direct E2f4 - Oct4 interaction. In addition, Plk1 associated with and phosphorylated E2f4. The interaction of E2f4 with Plk1 may control ES cell cycle through tubulin association to recruit segregating chromosomes. In summary, my data demonstrate that E2f4 plays a key role in regulating Oct4 transcriptional activity and ES-like cell cycle with Plk1 during SSC reprogramming. Collectively, E2f4 could be a pluripotent factor to activate SSC reprogramming.