Extended Data Fig. 7: Systematic L1 perturbation influences pervasive long-range gene expression.
From: LINE-1 transcription activates long-range gene expression
a, Schematic of triple sgRNAs targeting L1Hs 5′ UTR, used in CRISPRa/i. b, RT–qPCR results showing L1Hs expression upon CRISPRa/i in NCCIT. P, unpaired two-tailed t-test. Data represent median ± SEM. n = 2 biological replicates × 3 technical replicates. RT, reverse transcription. NC, non-target control. c, Bar plots showing counts of indicated L1s (blue + yellow + gray), counts of L1s targeted by triple sgRNAs (blue + yellow) and counts of L1s with increased H3K27ac peaks upon L1 CRISPRa (blue) in NCCIT. d, Bar plots showing counts of indicated L1s with H3K27ac peaks in NCCIT (blue + yellow + gray), counts of L1s targeted by triple sgRNAs (blue + yellow) and counts of L1s with decreased H3K27ac peaks upon L1 CRISPRi (blue). e, Gene expression change upon L1 CRISPRa/i in NCCIT. Red dots, genes whose expression increased upon CRISPRa (padj < 0.1, DESeq2 analysis) and decreased or remained silenced upon CRISPRi (termed as the L1-regulated genes). f, Plotted as in e, with genes separated into deciles by distance from the nearest L1 with increased H3K27ac peaks upon L1 CRISPRa. g, Box plot showing gene expression change in different deciles, binned as in f. n = 2 biological replicates. Center lines represent the median value, box limits represent the 25th and 75th percentiles and whiskers denote minima and maxima (1.5× the interquartile range). h,i, Representative examples of the L1-initiated chimeric transcripts in NCCIT. j, Box plots showing expression changes for the genes with Mll3/4 binding sites within indicated genomic distances in mESCs. Plots show median and interquartile range (IQR), and whiskers are 1.5-fold IQR. This suggests that the enhancer activity of L1 (shown in Fig. 3d,e) is comparable to that of canonical enhancers regulated by Mll3/4. k, RNA-seq browser tracks at NRN1 and its cognate L1PA2 locus. l, RNA-seq browser tracks at SESN3, its cognate L1Hs and nearby gene locus. m, RT–qPCR results show that CRISPRa targeting the SESN3-L1, rather than one adjacent locus, increases SESN3 expression. Data represent median ± SEM. n = 2 biological replicates × 3 technical replicates. P, two-tailed t-test. ****P < 0.0001. n, IGV tracks in NCCIT after CRISPRa against L1 (data from this study) and LTR5Hs (result from ref. 29). This suggests the importance of L1 transcription, rather than the adjacent LTR5Hs, in the long-range gene activation of HMGCS1.
