To identify proteins that physically associate with PHF6, we used an approach of immunoprecipitation followed by mass spectrometry (IP/MS). We used a rigorous proteomics method that compares a specific IP/MS data set against a large set of unrelated parallel

IP/MS data sets, thus distinguishing high-confidence candidate interacting proteins (HCIPs) from background proteins (Behrends et al., 2010; Litterman et al., 2011; Sowa et al., 2009). Remarkably, AZD6738 concentration all four core components of the PAF1 transcription elongation complex, PAF1, LEO1, CDC73, and CTR9, were found as robust HCIPs of PHF6 (Figure 3A). We validated the interaction of HA-PHF6 and the endogenous PAF1 complex in coimmunoprecipitation analyses in cells (Figure 3B). Importantly, we also found that endogenous PHF6 associated with all four components of the endogenous PAF1 complex in mouse cerebral cortex at E17, coinciding temporally with migration of neurons to the superficial layers (Figure 3C). These data suggest that the PAF1 complex might represent a physiological interacting partner of PHF6. The PAF1 transcription elongation complex

was first identified in yeast as an RNA polymerase II-associated complex and plays a critical role this website in efficient transcriptional elongation along chromatin (Kim et al., 2010; Rondón et al., 2004; Shi et al., 1996). All four components of the complex were highly expressed during early development in primary cortical neurons and the cerebral cortex in vivo (Figure 3D). The role of the PAF1 complex in the brain has remained unexplored. We asked whether the PHF6-PAF1 interaction is functionally relevant in neuronal migration. We reasoned that if PHF6 acts via the PAF1 complex to regulate neuronal migration, loss of PAF1 would be predicted to disrupt neuronal migration. Consistent with this prediction, PAF1 knockdown by two distinct shRNAs substantially impaired neuronal migration in the cerebral cortex

in vivo, phenocopying the effect of PHF6 knockdown (Figures 3E, 3F, 3G, and 3H). Notably, knockdown of PAF1 led to downregulation of the other components of the PAF1 complex (Figure 3F) (Chen et al., 2009), suggesting that the intact PAF1 complex is required for proper neuronal migration. Collectively, these data suggest that PHF6 physically associates with the PAF1 Sodium butyrate complex and thereby drives neuronal migration. The finding that PHF6 interacts with the PAF1 transcription elongation complex and thereby promotes cortical neuronal migration led us to determine whether PHF6 exerts its function via regulation of gene expression. Because the PAF1 complex promotes transcription, we reasoned that PHF6 might stimulate the expression of genes that trigger neuronal migration. We performed microarray analyses of control and PHF6 knockdown primary cortical neurons. A large number of genes were downregulated in cortical neurons upon PHF6 knockdown (Table S1).