OCT4B expression in PBMNCs suggests the existence of an alternative OCT4 promoter

Recently, the expression of OCT4B [NM_203289. 3], one of the two registered isoforms of the human OCT4 gene (OCT-3/4, officially POU5F1 [GeneID: 5460]), has been specifically documented in adult peripheral blood mononuclear cells (PBMNCs) (Panagopoulos et al., 2008). These data are in line with previous reports that re-evaluated OCT4 expression in PBMNCs (Liedtke et al., 2007; Kotoula et al., 2008). More recently, additional OCT4B isoforms have been identified that are regulated at the post-transcriptional (Atlasi et al., 2008) or translational levels (Wang et al., 2009). However, the demonstration of OCT4B expression in PBMNCs seems to generate the following paradox: OCT4B is expressed despite that chromatin modifications, such as histone deacetylation and methylation, at specific lysine residues render the OCT4 locus transcriptionally inactive upon cell differentiation (Golob et al., 2008; Xie et al., 2009) and despite that the promoter of the OCT4 gene is methylated in PBMCNs (Hoffmann et al., 2006; De Jong et al., 2007). The promoter region of the human OCT4 gene has been described in detail comparatively to its bovine and murine homologues (Nordhoff et al., 2001); four conserved regions (CRs) in the upstream OCT4 sequence (CR1–CR4) serve as transcription factor binding sites and are considered to regulate OCT4 expression (Nordhoff et al., 2001; De Jong et al., 2007). Importantly, the key regulatory sequences for OCT4 expression include the TATA-less minimal promoter (contained within CR1) and two enhancers, a proximal one and a distal one; routine CpG island methylation status studies mainly focus on these sequences excluding ‘‘nonregulatory upstream sequences. ’’ However, the number of CpG islands within the above regulatory sequences seems to be limited (De Jong et al., 2007). As all of the above regulatory sequences are found densely methylated in PBMNCs, and since OCT4A [NM_002701. 4] is not expressed in these cells (Panagopoulos et al., 2008), it seems that the ‘‘classical’’ promoter region upstream the 1st exon of the hOCT4 gene controls the transcription of the OCT4A isoform; OCT4B transcription may be regulated by an alternative promoter. The transcription of spliced isoforms may indeed be controlled by alternative promoters located within the gene, for example, within intronic sequences upstream of the translational start site (TSS) of the spliced variant. Such isoform-specific regulation has been reported for a variety of genes, including OCT1 (Pankratova et al., 2003), AFP (Scohy et al., 2000), CATL (Seth et al., 2003), and BRCA1 (Wardrop and Brown, 2005). Advances in bioinformatics have provided a prediction algorithm for the identification of highly conserved common regulatory motifs in established human promoters (Xie et al., 2005). Regarding the structure of the OCT4 gene, it is impressive that the 1st intron, composed out of 4048 nts, constitutes about 2/3 the whole gene length (6020 nts). Herein, in Fig. 1, we demonstrate that a region of 2000 nts upstream the TSS of OCT4B contains multiple highly conserved human promoter motives that may be ‘‘biologically meaningful’’ (Xie et al., 2005). As shown, 10 transcription factor binding sites, corresponding to seven well-known transcription factors, are identified within this region. Of note, only three motifs have a discrepant position, according to position bias, whereas four motifs tend to appear in multiple copies. According to these observations, it is tempting to speculate that the first intron of the OCT4 gene may serve as a promoter that regulates the transcription of the long OCT4B isoform, a protein of 265 amino acids (Wang et al., 2009) that remains of unknown function, or of OCT4B1 (Atlasi et al., 2008) that may serve a stemness marker (Papamichos et al., 2009). However, transcriptional activity of the aforementioned region needs to be proven first. Different patterns of OCT4 methylation in the ‘‘classic’’ promoter region have been observed in a tissue and cancer type specific manner (Cantz et al., 2007; De Jong et al., 2007). However, a