Enhancer activity sensitive to the orientation of the gene it regulates in the chordategenome

Abstract

Enhancers are flexible in terms of their location and orientation relative to the genes they regulate. However, little is known about whether the flexibility can be applied in every combination of enhancers and genes. Enhancer detection with transposable elements is a powerful method to identify enhancers in the genome and to create marker lines expressing fluorescent proteins in a tissue-specific manner. In the chordate Ciona intestinalis, this method has been established with a Tc1/mariner superfamily transposon Minos. Previously, we created the enhancer detection line E[MiTSAdTPOG]15 (E15) that specifically expresses green fluorescent protein (GFP) in the central nervous system (CNS) after metamorphosis. In this study, we identified the causal insertion site of the transgenic line. There are two genes flanking the causal insertion of the E15 line, and the genomic region around the insertion site contains the enhancers responsible for the expression in the endostyle and gut in addition to the CNS. We found that the endostyle and gut enhancers show sensitivity to the orientation of the GFP gene for their enhancer activity. Namely, the enhancers cannot enhance the expression of GFP which is inserted at the same orientation as the E15 line, while the enhancers can enhance GFP expression inserted at the opposite orientation. The CNS enhancer can enhance GFP expression in both orientations. The DNA element adjacent to the endostyle enhancer is responsible for the orientation sensitivity of the enhancer. The different sensitivity of the enhancers to the orientation of the transgene is a cause of CNS-specific GFP expression in the E15 line.

The landscape of RNA polymerase II transcription initiation in C. elegans reveals promoter and enhancer architectures

“…However, oriented enhancer function has been documented (Swamynathan and Piatigorsky 2002;Hozumi et al 2013). Furthermore, although transcription across C. elegans enhancer regions is oriented, we do not know whether the enhancer function of these binding sites is oriented as well.…”

Shared evolutionary origin of vertebrate neural crest and cranial placodes

“…To generate, Pax3/7>Foxc , the enhancer region of Pax3/7 was inserted into the XhoI and NotI sites of pSPFoxc. The minimal enhancer of Six1/2 was amplified with the following primers (5’-tgcctgcagcgaaaacaatggtttatccg −3’ and 5’-gatcggatcctacatgtacgcgcactttaa-3’) and cloned into the PstI/BamHI restriction site of a reporter construct containing the pSPFoxAa basal promoter and Kaede 33 .…”

Enhancer adoption caused by genomic insertion elicits interdigital Shh expression and syndactyly in mouse

“…In addition to syndactyly, the inversion caused other abnormalities including increased muscle volume in the autopod and cleft palate, the latter of which is perhaps the cause of perinatal lethality. Few studies to date have documented orientation-dependent enhancer activity ( 40 , 41 ). Recently, it was reported that orientation-dependent CTCF binding sites determine topological DNA looping for promoter–enhancer interaction and thereby exert proper gene regulation ( 42 ).…”