In mice Oct encodes at least variants designated

In mice, Oct4 encodes at least 2 variants, designated Oct4A and putative Oct4B. Oct4A was detected in somatic stem cells and has been recognized as a gatekeeper for maintaining pluripotency in embryonic stem cells and pre-implantation embryos. However, because of the existence of pseudogene and misinterpretation of background signals, the intact open read frame (ORF) and detailed functions of Oct4B variant remain unclear (Farashahi Yazd et al., 2011; Mizuno and Kosaka, 2008). In this study, we identified and characterized a novel mouse Oct4 transcript variant from ES cells, designated as mouse Oct4B, whose products share high similarity with human OCT4B. We demonstrated that the mouse Oct4B can be translated into three distinct isoforms through endogenous IRES element and alternative initiators of the coding sequence (CDS). In addition, we have found transcript Oct4B was not able to reprogram somatic cells to induced pluripotent stem cells, suggesting it was not involved in maintaining the pluripotency of stem cells.
Results
Discussion Mouse embryonic stem (mES) cells are an immensely powerful tool for studying the early differentiation of mammalian cell lineages and regenerative medicine. Oct4A is the key regulator in maintaining ES cells pluripotency and self-renewal. However, because of pseudogene expression and alternative splicing, mouse Oct4 variants are still a controversial and unsolved issue (Farashahi Yazd, et al., 2011; Mizuno and Kosaka, 2008). In this study, we clearly demonstrated the existence of Oct4B transcript in ES cells and found that Oct4A and Oct4B have different function in reprogramming of somatic cell to iPSC and self-renewal, therefore, it is necessary to distinguish Oct4A and Oct4B variants in future research. First of all, we identified the coding sequence of Oct4B transcript, which contained 741 base pairs and encoded 247 amino acids (supplementary Tab.2 and 3). The mouse Oct4A gene is known to encode 353 amino acids and encompass 5 exons, whereas mouse Oct4B is composed of 4 exons: exon 1 contains exon 2 and 66nt of intron 1 of Oct4A, exon 2, 3 and 4 are exactly the same with exon 3, 4 and 5 of Oct4A CDS (Fig. 1D). Due to the similar nucleic S63845 sequence between Oct4A and Oct4B, therefore, primers should be designed in distinctive regions when using PCR analysis: to detect the expression level of Oct4A, primers should be set in the exon 1 of Oct4A, whereas the 66bp at 5` end of Oct4B should be included when detecting Oct4B transcript. Similar to human OCT4B, the mouse Oct4B transcript generates three isoforms: Oct4B-247aa (full-length), Oct4B-190aa, and Oct4B-164aa. The protein sequences of these isoforms are highly identical between mouse and human. Oct4B-247aa is the longest product, it\'s synthesized started from the first ATG initiation site. Compared with the mouse Oct4A, Oct4B-247aa consists of an identical POU domain and C-termini and with different NTD (Fig. 4A). Although Oct4B-190aa shares the same mRNA with Oct4B-247aa, the translation was initiated by another start codon CUG (172–174 position) under the mediation of IRES (1-171nt) element. Oct4B-164aa is the shortest isoform, and the translation was initiated by ATG (250–252 position) codon. Compared with Oct4A, products Oct4B-190aa and 164aa contain intact C-termini and partial POU domain without NTD, so it seems they are not functional copies of the Oct4 protein. Although these three isoforms are generated from a single Oct4B mRNA, the endogenous protein isoforms may not be simultaneously expressed in the same cell type, the expression of each isoform may depend on its function and the cell state (Mizuno and Kosaka, 2008). And due to the absence of distinctive primer sequence, Western Blot but not PCR should be the optimal method to distinguish these isoforms. Combined with Sox2, Klf4 and c-Myc, Oct4B was not able to reprogram somatic cell to iPSC as that of Oct4A, although it contains an identical POU domain and CTD to Oct4A, and this may attributed to the difference of NTD. Human OCT4A protein contains three domains: the N terminal domain (NTD, 1-133aa), encompassing a transcriptional activation region that is active in various cultured cell types; the POU DNA-binding domain (134aa–289aa) that binds to DNA in a sequence-specific fashion, which is essential for its proper function in ES cells (Niwa et al., 2002); and the C terminal domain (CTD, 290aa-362aa) which controls the transactivation function of OCT4 in a cell type-specific way (Brehm et al., 1997). Yet the activity of these two transactivation domains is not identical in ES cells, the NTD domain has a specific function to transactivate a suite of target genes independently of interaction with partners such as Sox2 or cellular E1A-like factors (Niwa, et al., 2002). OCT4A binds its target sequences by the recognition of POU domain and octamer motif (5`-ATGCAATG-3`), and then transactivates the downstream genes with the assistance of the NTD or CTD domain, thus maintaining pluripotency or initiating cell differentiation (Cauffman et al., 2006; Niwa, et al., 2002; Takeda et al., 1992). Compared with OCT4A, human OCT4B shares the conserved POU and CTD domains, but lacks the transactivation domain of the NTD. Moreover, amino acids 1–20 and 21–40 of the NTD have an inhibitory effect on the binding of OCT4B POU domain and target sequence (Lee, et al., 2006). However, overexpression of OCT4B does not interfere with the transactivation reaction activated by OCT4A, and so OCT4B has no effect on pluripotency (Lee, et al., 2006). The mouse Oct4B NTD is composed of 24 amino acids and has no similarity with that of Oct4A. However, it shares a high identity with the human OCT4B: the peptides 1-11aa and 12-23aa of mouse Oct4B are exactly the same as 1-11aa and 29-40aa of human OCT4B NTD which inhibits the transactivation of the POU domain (Fig. 4B). Based on this similarity, it is likely that the 1-23aa of the mouse Oct4B interrupts the binding of the POU domain with target sequence and thus prevents the activation of downstream signal pathways.