Fig. 6

RSINE1 elements have enhancer activity in vitro. A Fold change luminescence when compared to an empty vector control after normalization to Renilla luciferase signal of enhancer reporter plasmids with various RSINE1-derived sequences inserted downstream of the luciferase gene. Included are RSINE1 consensus as is or with “evolved” E-Box and RORE motifs at highlighted locations (all motifs were changed to the canonical motif with no other changes), an RSINE1 element upstream of the promoter of Aaed1 (see Additional file 1: Fig. S6B), and an RSINE1 upstream of the promoter of 4833420G17Rik and Tmem267 (see Additional file 1: Fig. S6A). Tmem267 and Aaed1 RSINE1s were cloned as the RSINE1 alone, with 100 bp of flanking sequence, and with the RSINE1 deleted from the flanking region as a reconstruction of the ancestral state. Experiments are representative of two biological transfection replicates with four technical replicates each. Significance was assessed using pairwise two-tailed t-tests compared to the empty vector. B A model of how RSINE1 has fine-tuned the circadian regulatory landscape of the murine lineage by distributing imperfect CR/NR binding sites with moderate regulatory activities that may modulate the activity of existing nearby circadian regulatory elements. NRs, nuclear receptors; CRs, circadian regulators; uTF, ubiquitous TF; tsTF, tissue-specific TF