Laf4/Aff3, a Gene Involved in Intellectual Disability, Is Required for Cellular Migration in the Mouse Cerebral Cortex
Figure 5
Identification of Laf4 as a potential transcriptional regulator of Mdga2.
(A) qRT-PCR analysis of four genes originally shown to be up-regulated from microarray transcriptome profiling of FACS-sorted primary cortical cells over-expressing Laf4 (Laf4-HA-IRES-GFP) compared to an empty control (HA-IRES-GFP) vector. Significant up-regulation of Gatad1, Mdga2, Ndufs and Scna3 was confirmed in Laf4 over-expressing cells (n = 3 biological replicates *p<0.05, **p<0.01, 2-way ANOVA Bonferroni’s multiple comparison test). (B) ChIP from cortical cells transfected and FACS-sorted as in (A) was performed using an anti-HA antibody to HA-tagged Laf4-IRES-GFP or empty vector (HA-IRES-GFP). qPCR showed specific binding of Laf4 to Mdga2 at the beginning of the coding region (primer pair 3 and 4 shown in (C)) and at the end of the first exon/beginning of the first intron (primer pair 4). Laf4 did not appear to bind to Mdga1 for the primers tested. (n = 3 biological replicates; *p<0.05, ***p<0.001). 1-way ANOVA Bonferroni’s multiple comparison test used to compare each primer sets to primer 5 (Mdga2) or 3 (Mdga1) used as control primer set. (D) qRT-PCR analysis of Mdga2 levels in cortical cells transfected with shRNA-Laf4 vector (*p<0.01, 1-way ANOVA Bonferroni’s multiple comparison test).