Figure 4.

Effects of ATXN2 PDs on ATXN2-luc expression and identification of an ATXN2 5′-UTR region (+87 to +109) accounting for the majority of ATXN2 expression. (A and B) Effect of increasing ATXN2 PD on ATXN2-luc expression. (A) The long ATXN2 upstream sequence contained in plasmid pGL2-L5A3 reduced ATXN2-luc expression similarly in HEK293 and SH-SY5Y cells compared with the shorter upstream sequence in pGL2-5A3. (B) Further decreases in the ATXN2 promoter length in pGL2-5A3 correlated with increases in ATXN2-luc expression. A plateau where no successive increased expression was observed between −816 and −351 in HEK293 cells and −555 and −55 in SH-SY5Y cells. PD12 spans the smaller +87 to +109 5′-UTR region and resulted in expression reduction in both cell lines. (C) Effect of ATXN2 IPDs in pGL2-5A3 on ATXN2-luc expression. Only deletions IPD16 and IPD23 including the +87 to +109 5′-UTR region caused reduced expression in both HEK293 and SH-SY5Y cells compared with the pGL2-5A3 plasmid that contains no interstitial deletion. For each of (A)–(C), a graphic representation is provided (left) where the upstream/promoter sequence present is represented by a black bar and for (C) where the sequence that has been interstitially deleted is represented by a white box. Relative expression in HEK293 and SH-SY5Y cells for each deletion (right), controlled with SV40-Renilla luciferase. Nucleotides are numbered such that +1 corresponds to the transcriptional start site or first base in the 5′-UTR. Values are mean ± SD from three independent transfections each read in triplicate.