Fig. 1.

Identification of a novel alternative splice variant in the human DNMT3B locus whose expression correlates with pluripotency. (A) Schematic showing the splicing structure of the human DNMT3B gene with 23 exons. Translation begins in exon 2 (open boxes are untranslated exons). The alternatively spliced regions examined in the paper are denoted below this with bold arrows. A blowup of the exon 2–7 region of DNMT3B shows the location of the PWWP domain, the primers used in semi-quantitative RT-PCR (bold arrows below exons) and quantitative RT-PCR (bold arrows above exons). ‘*’ putative sumoylation site. (B) Representative ethidium bromide stained agarose gel photo showing expression of the newly identified splice variants DNMT3BΔ5 and DNMT3BΔ(4+5). Ectopic expression of DNMT3B3Δ5 in HCT116 cells yields a stable protein product of the expected molecular weight. DNMT3B1, DNMT3B3, and DNMT3B3Δ5 were expressed in HCT116 cells following transient transfection and detected with the Express antibody epitope tag (right). ‘*’ non-specific amplification product confirmed by DNA sequencing. (C) DNMT3BΔ5 expression is regulated during differentiation. Expression of DNMT3B isoforms during differentiation of NCCIT EC cells over twenty days with retinoic acid, relative to GAPDH expression. (D) Quantitative RT-PCR analysis of DNMT3B1-6 and DNMT3BΔ5 expression, relative to GAPDH, in human pluripotent cell lines (left panel), two neural stem cell lines (SCP’s) and one glioma tumor initiating cell line (H1228, middle panel), and the relative expression for each of the cell lines (right panel). ‘hES’ human ES cells.