Alternative promoters regulate transcription of the gene that encodes stem cell surface protein AC133

Abstract

AC133 is a member of a novel family of cell surface proteins with 5 transmembrane domains. The function of AC133 is unknown. Although AC133 mRNA is detected in different tissues, its expression in the hematopoietic system is restricted to CD34 ؉ stem cells. AC133 is also expressed on stem cells of other tissues, including endothelial progenitor cells. However, despite the potential importance of AC133 to the field of stem cell biology, nothing is known about the transcriptional regulation of AC133 expression. In this report we showed that the human AC133 gene has at least 9 distinctive 5-untranslated region (UTR) exons, resulting in the formation of at least 7 alternatively spliced 5-UTR isoforms of AC133 mRNA, which are expressed in a tissue-dependent manner. We found that transcription of these AC133 isoforms is controlled by 5 alternative promoters, and we demonstrated their activity on AC133-expressing cell lines using a luciferase reporter system. We also showed that in vitro methylation of 2 of these AC133 promoters completely suppresses their activity, suggesting that methylation plays a role in their regulation. Identification of tissue-specific AC133 promoters may provide a novel method to isolate tissuespecific stem and progenitor cells. (Blood.

Epigenetic regulation of CD133 and tumorigenicity of CD133+ ovarian cancer cells

“…We examined the methylation status of 15 CpG cytosines using bisulfite sequencing to assess the possibility of a direct epigenetic influence on CD133 expression. These CpG sites are located from À366 to À191 bp 5 0 of P2 promoter-specific exon 1B and CD133 þ cells in ovarian cancer T Baba et al partially overlap (13 of 15 CpGs) with the region tested for influence of methylation on transcription in vitro (Shmelkov et al, 2004). As shown in Figures 4a and b, 34 cell lines exhibited complete or partial methylation of cytosines in this region.…”

“…CD133 was previously characterized as having five alternative promoters (P1-P5) that are active in a tissue-dependent manner (Shmelkov et al, 2004). The P1, P2 and P3 promoters are located within a 1540-bp CpG island (17.4% CpG, observed to expected CpG ratio ¼ 0.82), whereas promoters P4 and P5 are not encompassed by CpG-rich sequences.…”

“…Using an in vitro reporter system, Shmelkov and colleagues determined that P1 and P2, whose exon 1A-specific and exon 1B-specific exons, respectively, are located 244 bp apart, are inactive when methylated, suggesting that they may be epigenetically regulated. Because CD133 promoter 2 is the only active promoter in ovarian tissues (Shmelkov et al, 2004), and CD133 expression in our ovarian cancer cell lines was highly variable, we investigated the possibility that this variability is due to promoter methylation. Of the 40 ovarian cancer cell lines treated for 72 h with the DNA methyltransferase inhibitor, decitabine, 27 exhibited an increase in CD133 mRNA levels (P ¼ 0.01, two-tailed paired t-test; Supplementary Figure S7).…”

Promoter hypomethylation regulates CD133 expression in human gliomas

“…CD133 was found localized in microvilli and protruding plasma membrane, thus interacting with membrane cholesterol [20]. The CD133 gene is also known to possess five promoters yielding different spliced transcripts and five alternative first exons that are expressed in a tissue-specific manner [21]. However, the mechanisms of CD133 expression and its regulation remain largely unclear.…”

“…The translational initiation site is positioned in exon 2, and exon 1 produces alternatively spliced 5′-untranslated regions (5′-UTRs), designated as exons 1A, 1B, 1C, 1D1, 1D2, 1D3, 1E1, 1E2, 1E3, and 1E4. Transcription at these sites is controlled by five alternative promoters known as P1, P2, P3, P4, and P5 (Figure 2A upper scheme) [21]. To determine the first exon transcribed in glioma cells, we utilized RT-PCR to investigate the expression pattern of CD133 exon 1s in 22 glioma specimens ( Figure 1A panel-iv-viii).…”