Figure 3. α1ACT is a transcription factor that regulates neural gene expression through an AT-rich element.

(A) ChIP and quantitative real-time PCR verification of DNA sequences identified by ChIP-cloning. PC12 cells were transfected with empty vector or FLAG-tagged α1ACT_WT. (B) Relative enrichment was calculated as the ratio between the net intensity of each bound sample normalized to its input sample, and the vehicle control sample normalized to vehicle control input sample (n ≥ 3). (C) Enhancer activity of BTG1 gene fragments. Positions of the fragments are indicated. (D) Promoter activity of GRN gene fragments. Positions of the fragments are indicated. (E) Consensus sequence analyzed by CLC main workbench Version 6.5 among the α1ACT ChIP-targeted sequences. Consensus sequence was predicted and labeled in red. (F) EMSA demonstrates the formation of displaceable nucleoprotein complex with the α1ACT and BTG1 WT (517-630nt) element. Lane 1 is biotin-labeled BTG1 WT probe. Lanes 2 and 5 show the three major complexes formed between BTG1 probe and α1ACT_WT nuclear protein. Lane 8 shows the fourth complex formed between BTG1 probe and α1ACT_SCA6 nuclear protein. (G) EMSA shows that the AT-rich probe (531-553nt) forms three complexes in the absence of competitor. These complexes were displaced by excess unlabeled AT-rich sequence and partially abolished by AT-rich Mut1 and Mut3. The super-shifted bands were only seen in α1ACT_WT–FLAG nuclear extracts treated with FLAG-M2 antibodies, but not in lane of pCDNA3 nuclear extracts. (H) EMSA shows that the TTATAA region is critical for the formation of nucleoprotein complexes with AT-rich element. (I) α1ACT_WT significantly increases BTG1 enhancer activity through intact TTATAA region. Plasmid pRL-TK is used as transfection efficiency control. Data are mean ± SEM, n ≥ 3 (each involving triplicate assays, *p<0.05 vs. control construct) (see also Figure S3).