Figure 3

CSE1 functions as a transcriptional repressor for Peg3-DMR. The top panel shows the genomic layout of the mouse Peg3-DMR containing the two evolutionarily conserved sequence elements, CSE1 (open square) and YY1 binding sites (open oval). The thick vertical lines indicate the positions of the 1^st and 2^nd exons of Peg3. The transcriptional involvement of CSE1 in mouse Peg3-DMR was analyzed using the IRES-β-Geo promoterless vector system. (A) The first series of assays were conducted with the ten individual constructs that differ by the presence/absence of CSE1 (with or without C in their names), the intact versus mutated YY1 binding sites (-Y or -mY), and the orientation of YY1 binding sites (-F or -R). (B) The second series of assays investigating the position- and orientation-dependency of CSE1 on the Peg3 promoter activity were conducted by three additional constructs containing the reverse orientation of CSE1 (-CR & -C3'R) and the 3'-end position of CSE1 (-C3'F & -C3'R). Each construct was analyzed more than three times as shown with the average value with S.D. The averaged value for each construct was compared with that of the reference constructs, and subsequently presented in the graph as fold differences. Two sets of assays were conducted with two different reference constructs: Construct 1+C (A) and Construct 1+CF+YF (B). In order to compare the different values derived from the two different series, the value for the control construct of the second set (1+CF+YF) was adjusted to 1.7 as derived from the first set (1+C+YF). These assays were conducted using three cell lines, Neuro2A, NIH3T3, and HeLa, and they all showed a similar pattern as the above set of assays using the Neuro2A cell line [see Additional file 1].