In Vivo Characterization of Linc-p21 Reveals Functional cis -Regulatory DNA Elements

Abstract

Summary The Linc-p21 locus, encoding a long non-coding RNA, plays an important role in p53 signalling, cell cycle regulation, and tumour 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.

Linear models enable powerful differential activity analysis in massively parallel reporter assays

“…Characterization studies examine thousands of different putative regulatory elements that have a wide variety of sequence features and try to correlate these sequence features with measured activity levels (Grossman et al, 2017;Guo et al, 2017;Safra et al, 2017;Levo et al, 2017;Maricque, Dougherty, and Cohen, 2017;Groff et al, 2016;Ernst et al, 2016;White, Kwasnieski, et al, 2016;Ferreira et al, 2016;Fiore and Cohen, 2016;Farley et al, 2015;Kamps-Hughes et al, 2015;Dickel et al, 2014;Kwasnieski, Fiore, et al, 2014;Mogno, Kwasnieski, and Cohen, 2013;Gisselbrecht et al, 2013;White, Myers, et al, 2013;Smith et al, 2013). Typical statistical analyses use regression to study the impact of multiple features simultaneously.…”

Molecular functions and biological roles of long non‐coding RNAs in human physiology and disease

“…This notion is further supported by detailed studies of selected lncRNA loci that most likely confer their gene regulatory functions via the process of transcription (i.e. independent of the resulting transcript), via DNA enhancer elements within the lncRNA locus, or via promoter competition between the promoter of the lncRNA and the neighboring protein‐coding gene . Taking these findings into consideration, it becomes apparent that the molecular mechanism of each lncRNA locus needs to be carefully analyzed on a case‐by‐case basis before an actual functionality can be ascribed to the encoded RNA transcript.…”

“…Dependent on the abundance of lncRNAs and associated spatial constraints, this regulatory mode of action can affect the expression of either neighboring genes in cis or distant genes in trans (Figure ) . Lately, the general validity of this concept has been challenged by several studies reporting that, in many cases, it is not the mature lncRNA transcript itself but rather the sole act of transcription or DNA elements within the lncRNA locus that is likely to confer the gene regulatory function . Because active regulatory DNA elements, such as promoters and enhancers, are known to be commonly transcribed, many nuclear lncRNAs may represent enhancer RNAs (eRNAs) that do not function in an RNA‐ and sequence‐dependent manner .…”

On the Origin of lncRNAs: Missing Link Found

“…Despite these seemingly strong discriminating criteria, many recent reports have brought into question the distinction between eRNAs and lncRNAs. In some instances, the bulk of the biological activity of lncRNA loci was found to be due to an enhancer DNA element near the lncRNA transcription start site [2, 3]. In other cases, lncRNA loci were shown to actually work locally in an RNA-independent fashion to facilitate chromatin accessibility or regulate enhancer action by the mere act of transcription [4, 5].…”

“…In other cases, lncRNA loci were shown to actually work locally in an RNA-independent fashion to facilitate chromatin accessibility or regulate enhancer action by the mere act of transcription [4, 5]. Examples of such ‘lncRNA-to-eRNA converts’ are linc-p21, now considered an eRNA derived from a p53 enhancer element in the human CDKN1A (p21) locus [2], and Lockd, an RNA derived from the human CDKN1B (p27) locus [3]. Based on these observations, I recently hypothesized an evolutionary path from eRNAs to lncRNAs [6], whereby spurious splicing and polyadenylation of ancestral eRNAs, due to the accidental presence of splicing and 3′ processing sequences in the vicinity of the respective enhancers, would lead to the stabilization of the eRNAs.…”