Figure 4

Minimal regions within the CSE1 responsible for transcriptional repression of Peg3 and Usp29. (A) Evolutionary conservation of the CSE1 is demonstrated through aligning the sequences derived from six mammals: human, rhesus, chimpanzee, mouse, cow, and dog. The bases in red indicate differences from the human CSE1. The hyphens (-) indicate insertions/deletions. (B) A series of 6-bp-segments were mutated on the mouse CSE1 starting from 5' to the 3'-ends. The intact sequence of the mouse CSE1 is shown on top and the following sequences represent the mutated versions of the CSE1 with the changed bases marked in blue. (C&D) The mutated CSE1 was analyzed for the promoter activity after subcloning into the IRES-β-Geo promoterless vector system. The Peg3-DMR promoter containing a series of mutated CSE1 sequences was analyzed in two different directions: Peg3 (C) and Usp29 (D) directions. The construct layout on the top region of each graph shows the transcriptional direction of the mouse Peg3-DMR containing CSE1 (open square) and YY1 binding sites (open oval). The thick vertical lines indicate the positions of the 1st exons of Peg3 and Usp29. Each construct was analyzed more than three times. The averaged value for each construct was first compared with that of the reference construct, in this case Construct 1+C+YF, and the subsequent fold difference was presented with S.D. in the graph. These assays were conducted using two cell lines, Neuro2A and HeLa. The result set shown in the graph was derived from Neuro2A cells but the result set from HeLa cells also showed very similar patterns [see Additional file 1].