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. 2018 Nov 6;9:524. doi: 10.3389/fgene.2018.00524

FIGURE 1.

FIGURE 1

(A) Four possible cis-regulatory mechanisms mediated by lncRNA genes. Cis-regulation of nearby genes can be achieved directly by the lncRNA transcript, by cis-regulatory elements overlapping with the lncRNA gene such as an enhancer (Groff et al., 2016), or the lncRNA promoter itself (Engreitz et al., 2016; Paralkar et al., 2016), or by the transcription of the lncRNA (Latos et al., 2012). Different types of genetic perturbation can provide insights into the underlying mechanisms, such as: (B) deletion of the lncRNA promoter, which can affect the transcription of the regulated gene due to the lack of lncRNA transcript, transcription or absence of direct cis-regulation by the lncRNA promoter (Engreitz et al., 2016; Paralkar et al., 2016); (C) insertion of a poly-adenylation signal (PAS) downstream of the transcription start site, which prevents the transcription of the lncRNA through downstream DNA sequences (Sleutels et al., 2002; Ohhata et al., 2007; Yin et al., 2015; Anderson et al., 2016; Engreitz et al., 2016; Paralkar et al., 2016); (D) over-expression of a lncRNA from its endogenous locus can be achieved by CRISPR-activation (CRISPRa) (Gilbert et al., 2014; Konermann et al., 2015; Joung et al., 2017); it should be noted that the CRISPR-activation of the lncRNA promoter can also increase a direct cis-regulatory activity of the lncRNA promoter; (E) deletion of the full lncRNA transcript, which leaves the lncRNA promoter as a potential cis-regulatory element; note that deletion of several exons of the lncRNA will also remove any intronic enhancer (Groff et al., 2016); (F) lncRNA knock-down using short hairpin RNA (siRNA/shRNA) or antisense oligonucleotides, testing the functionality of the lncRNA transcript itself; (G) RNA Polymerase II “roadblock” by CRISPR-dCas9 downstream of the transcriptional start site of the lncRNA (Gilbert et al., 2013; Qi et al., 2013); this selectively blocks the transcription of the lncRNA, helping to discriminate a cis-regulatory function from the transcription/transcript or from genomic cis-regulatory elements.