Figure 7. Msgn1 binds directly to clock enhancers in ESCs and in vivo and synergizes with Notch to activate Lfng.

(a) F-Msgn1 ChIP-seq peaks map to demonstrated clock enhancers in blocks A and B in the 5′ regulatory region of Lfng in differentiating EBs. Blocks A, B (and C) represent regions of sequence homology conserved between mouse and human^31,34. (b) ChIP-qPCR analysis performed on PSMs or head tissue dissected from wildtype embryos demonstrate that endogenous Msgn1 binds strongly to Blocks A and B of the Lfng enhancer in the PSM, relative to the head, and not to control regions in intron 1. Y axis represents fold change. Abbreviations: C, control ascites fluid; αM, anti-Msgn1. (c) Lfng-luciferase reporter constructs containing up to 2.3kb of the Lfng enhancer/promoter are depicted on the left. Circles depict Rbpjk binding sites while triangles represent E-boxes. The luciferase activity for each construct, cotransfected with control (purple), Msgn1 (green), and/or activated Notch (NICD, blue) expression constructs in 3T3 cells is expressed as a fold change on the right. Msgn1 plus NICD (red) synergistically activate the full length (FL) 2.3kb Lfng construct 7.4 fold, and the block A construct 49.7 fold. The A block is the only element capable of negotiating synergistic interactions between Msgn1 and NICD. (d) The cyclic transcriptional repressor Hes7 (orange) completely blocks the Msgn1/NICD synergism on the FL Lfng enhancer construct and requires the B block for maximal inhibitory activity. The combined activities of Hes7 with NICD (yellow), Hes7 and Msgn1 (pink), and Hes7 with Msgn1+NICD (gray) are illustrated.