Porcine OCT4 upstream region-based reporter system could work similar to human reporter analysis during porcine reprogramming
The reprogramming process and epigenetic reversion were checked in mouse using an Oct4 reporter system [41–43]. Transgenic mice were generated that had a mouse Oct4 upstream region-based dual reporter system to check the control of enhancers in embryonic and germ cell development. The dual reporter system worked and distinguished mouse naïve and primed stem cells [19, 44]. The mouse Oct4 reporter system worked very well by cell-type and developmental stage because the activation of mouse Oct4 enhancers were distinguished clearly by pluripotent states in mouse pluripotent cells. However, researchers attempted to establish naïve-state human pluripotent stem cells using a hOCT4 upstream region-based reporter system. Some results indicated that human OCT4 enhancers did not express and clearly silence genes, similar to mouse Oct4 enhancers, in naïve or primed state pluripotent stem cells [20, 45]. This experiment identified that porcine OCT4 enhancers acted differently in naïve and primed pluripotency states in mouse pluripotent stem cells and functioned in porcine pluripotent stem cells. Our results found that porcine-induced pluripotent stem cells in FGF- or LIF-dependent conditions were similar to the primed pluripotent state. When porcine OCT4 enhancers acted in porcine pluripotent stem cells, the distal and proximal enhancers worked similar to a human OCT4-derived reporter system rather than a mouse reporter system. Porcine OCT4 expression was similar to human OCT4 expression rather than mouse Oct4 expression [46]. Our results indicated that porcine OCT4 enhancers and other reporter systems also worked similar to human, not mouse systems.
The characteristics of mouse Oct4, human OCT4 and porcine OCT4 exhibited many differences, such as low homology of sequences, but various features of porcine OCT4 and its enhancers were more similar to human OCT4 than the mouse Oct4. Nucleotide sequences from the porcine OCT4 upstream region were more similar to the human OCT4 upstream region rather than counterpart of mouse Oct4. In all conserved regions and a 3.2-kb upstream region, total nucleotide, including distal and proximal enhancers, homology between human and pig is higher than between mouse and pig [29]. Enhancers are key contributors to gene expression patterns via various mechanisms [14]. Research of Oct4 in mouse indicated that distal enhancers activated Oct4 expression in mouse embryonic stem cell, morula, ICM and primordial germ cells, and proximal enhancers inhibited the expression of Oct4 in the epiblast [11]. However, the OCT4 gene was expressed in the ICM and trophectoderm, and it was epiblast-specific in human [47] and pig [48]. These results indicated that the expression of distal and proximal enhancers in mouse Oct4 was clearly distinct during embryo development, but not in human and pig.
Our previous research found that putative transcription factor binding sites exist in porcine OCT4 upstream regions. One of the 4 conserved regions is conserved region 4 (CR4), which is farthest from the core promoter and has distal enhancer site 2A and a binding domain for Oct4/Sox2 [29]. OCT4 and SOX2 are required in embryo development and pluripotent stem cells, and these genes were controlled in embryo and stem cells. These genes work together closely, and they have a negative feedback loop balance [49]. During embryo development, CDX2 in TE inhibits OCT4 and SOX2 expression by affecting the OCT4 upstream CR4 region [50]. Oct4 and Cdx2 were specifically expressed in ICM and TE, respectively, in mouse. Oct4 was excluded from TE, and Cdx2 was excluded from ICM. However, OCT4 and CDX2 are coexpressed for a relatively long time in TE cells in human and pig [48, 51]. These results indicated that the expression and mechanism of transcription binding factors in the OCT4 distal enhancer region may be different between species. This research supports our results that porcine enhancers and our porcine-based reporter system were more similar to human rather than mouse.
Applications of porcine OCT4 upstream region-based reporter system could help investigation of porcine-specific pluripotency
Pluripotency, naïve and primed markers vary from species to species. For example, naïve markers in mouse and human are not applicable in pig [34], and various human-specific pluripotent markers were reported [35]. Our results also indicated that naïve and primed markers in mouse and human were not applicable in porcine embryo and pluripotent stem cells. Despite the importance of porcine-specific markers for naïve and primed conditions, this research remains an ongoing endeavor. Under these circumstances, a porcine reporter system would help identify new porcine-specific pluripotent state markers. Our OCT4 reporter system would help distinguish between naïve and primed pluripotent states and identify porcine-specific markers in porcine embryo and pluripotent stem cells. In the LIF-dependent condition, the activity of a distal enhancer was slightly higher than in the FGF-dependent condition. However, the expression level of distal and proximal enhancers in both conditions differed in naïve-state mouse embryonic stem cells, and it was rather similar to the primed-state mouse embryonic carcinoma (Fig. 3B). This difference means that the porcine-induced pluripotent stem cells in both conditions were closer to a primed pluripotent state than naïve. Our reporter system was applied in porcine pluripotent cells, a distinction between the naïve and primed states may be performed in the current situation where the porcine-specific naïve and primed pluripotent markers were not identified. The porcine OCT4 upstream region has various transcription factor binding sites, and these transcription factors may relate to species-specific pluripotency [29]. Our reporter system will also help find the naïve and primed markers after separating the cells in naïve or primed states.
During the reprogramming process, pluripotent marker genes with unique expression patterns were found. DAX1 affected OCT4 via LRH-1 mediated activation. The LRH-1 (NR5A2) binding site in the OCT4 promoter region was confirmed using chromatin immunoprecipitation. DAX1 is related to OCT4 expression [52], and its expression was similar to the expression pattern of the enhancers of porcine OCT4 enhancers. Therefore, the activities of OCT4 enhancers affect its promoter region, and our results indicated that DAX1 was a representative OCT4 enhancer in the FGF condition. Two genes (SALL4 and CRIPTO) thought to be related to FGF and LIF signaling were also found, and these genes may be candidates for the marker genes identified in porcine-induced pluripotent stem cells that were defined as primed. SALL4 affects OCT4 expression via binding its distal enhancer DE2A in CR4, which is one of the 4 OCT4 upstream conserved regions [38]. Considering that the basic fibroblast growth factor signaling pathway maintains the stemness of primed pluripotent stem cells [53], and SALL4 was highly expressed in FGF-dependent reprogramming conditions in pig, the distal enhancer of porcine OCT4 upstream region played an important role. Although the expression was lower than the proximal enhancer in primed state porcine pluripotent stem cells, unlike the mouse Oct4 distal enhancer, which is specifically activated in naïve state pluripotent stem cells. Further verification of the OCT4 enhancers is required to analyze the unique mechanism of porcine enhancers and establish naïve state pluripotent stem cells in porcine-specific enhancer studies and research on reporter systems.