TBR1 Directly Represses Fezf2 to Control the Laminar Origin and Development of the Corticospinal Tract. Mus musculus

Our independent analyses using mRNA-Seq, quantitative RT-PCR, and in situ hybridization confirmed a significant up-regulation of Fezf2 in Tbr1-/- neocortex. However, analysis by immunostaining and immunoblotting revealed that SOX5 protein levels were relatively unaltered in late embryonic and neonatal Tbr1-/- cortex. This led us to the hypothesis that TBR1 regulates Fezf2 transcription via direct binding to regulatory sequences near Fezf2. To identify genome-wide TBR1 binding sites in an unbiased and hypothesis-independent manner, we analyzed TBR1-immu-noprecipitated chromatin using deep sequencing (ChIP-Seq). We tested several available anti-TBR1 antibodies and found that none was suitable for immunoprecipitating chromatin of sufficient quality for ChIP-Seq. Thus, we generated a V5-TBR1fusion construct and expressed it in N2A cells. V5-TBR1 was immunoprecipitated using an anti-V5 antibody. DNA-Seq was performed on the Illumina GAIIx platform.