Abigail F. Groff, Diana B. Sanchez-Gomez, Marcela M.L. Soruco, Chiara Gerhardinger, A. Rasim Barutcu et al.
The Linc-p21 locus, encoding a long non-coding RNA, plays an important role in p53 signaling, cell-cycle regulation, and tumor suppression. However, despite extensive study, confusion exists regarding its mechanism of action: is activity driven by the transcript acting in trans, in cis, or by an underlying functional enhancer? Here, using a knockout mouse model and a massively parallel enhancer assay, we delineate the functional elements at this locus. We observe that, even in tissues with no detectable Linc-p21 transcript, deletion of the locus significantly affects local gene expression, including of the cell-cycle regulator Cdkn1a. To characterize this RNA-independent regulatory effect, we systematically interrogated the underlying DNA sequence for enhancer activity at nucleotide resolution and confirmed the existence of multiple enhancer elements. Together, these data suggest that, in vivo, the cis-regulatory effects mediated by Linc-p21, in the presence or absence of transcription, are due to DNA enhancer elements.
Abigail F. Groff, Diana B. Sanchez-Gomez, Marcela M.L. Soruco, Chiara Gerhardinger, A. Rasim Barutcu, Eric Li, Lara Elcavage, Olivia Plana, Lluvia V. Sanchez, James C. Lee, Martin Sauvageau, John L. Rinn. In Vivo Characterization of Linc-p21 Reveals Functional cis-Regulatory DNA Elements. 2016-08-23. https://doi.org/10.1016/j.celrep.2016.07.050
An episomal GFP reporter MPRA tested 2,225 synthesized oligos tiling the mouse Linc-p21 locus and 500 bp of its promoter. The library contained 90-bp genomic inserts starting every 50 bp, with five unique barcodes per tile, and was assayed in C2C12 myoblasts using three RNA-library and three vector-library replicates.
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Download all 3 files (ZIP)GSE73472_family.soft.gzGSE73472_MPRA_processed_data_lincp21.tab.gzLincp21_MPRA_analysis.Rmd