Figure 6. F-Msgn1 binds directly to regulatory elements to transactivate multiple Notch pathway genes.

(a,c,e,g,i) F-Msgn1 immunoprecipitation in differentiating EBs enriches for chromatin at regulatory regions of Notch signaling genes. The y-axis denotes the peak values, and peaks are numbered according to their position of the peak midpoint relative to the transcriptional start site. Gene loci are depicted at the bottom of each panel and the arrowheads indicate orientation. Pink arrows indicate the ChIP-seq enhancer elements tested in the luciferase reporter assays depicted in the column on the right. (b,d,f,h,j) Sequences identified by F-Msgn1 ChIP-seq were tested for functional activity in luciferase reporter assays in NIH3T3 cells. Putative enhancer constructs are schematized at the top with red boxes indicating identified E-box consensus sequences. Luciferase reporter results are depicted in the graphs as fold changes. With the exception of Dll1-2313, all reporter constructs were strongly activated by Msgn1 expression. Dll1-2313 represents a weak F-Msgn1 ChIP-seq peak that falls below, and therefore empirically validates, the applied p-value threshold of 10e-5.