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EWSR1 is involved in chimeric proteins which play crucial roles in the development of a variety of bone and soft tissue tumors. Many of the chimeric genes involving EWSR1 have been extensively studied, whereas less is known about the wild-type (wt) gene and its regulation. As the expression of the chimeric gene is driven by the EWSR1 promoter, it is of importance to study the mechanisms regulating wt EWSR1 expression. We estimated the transcriptional activity of the EWSR1 promoter through deletion fragments driving reporter gene expression. This assay identified the 100-bp region immediately downstream of the EWSR1 transcriptional start site (+1) and the downstream region from +100 to +300 as important regions for transcriptional regulation. We also found that EWSR1 and RHBDD3, a gene located directly upstream of EWSR1 that is likely to share regulatory elements with EWSR1, were co-expressed in the tissue panels, Ewing tumor biopsies and cell lines. Thus, our results show that the EWSR1 promoter functions in a bidirectional manner, thereby regulating also RHBDD3, and identifies specific regions that strongly influence promoter activity.

POU5F1, which encodes a transcriptional factor, has two alternatively spliced transcripts, 1 and 2, as well as six pseudogenes. Transcript 1 is considered to be a key regulator of cellular pluripotency and self-renewal. The POU5F1 pseudogene, POU5F1P1 on 8q24, encodes a protein with 95% homology with the isoform 1 of POU5F1. It is located 15 kbp downstream of the SNP rs6983267, which is strongly associated with an increased risk of prostate and colon cancer, and within the amplified region in a variety of human malignancies. The previous finding of expressed sequence tags suggests that POU5F1P1 can be expressed. We showed that a putative POU5F1P1 protein is localized in the nucleus, acts as a transcriptional activator and regulates the expression in a similar way to the POU5F1 isoform 1. However, POU5F1P1 was a weaker activator than isoform 1 of POU5F1, possibly due to the amino acid substitutions.