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. 2016 Nov 17;539(7629):452-455.
doi: 10.1038/nature20149. Epub 2016 Oct 26.

Local regulation of gene expression by lncRNA promoters, transcription and splicing

Affiliations

Local regulation of gene expression by lncRNA promoters, transcription and splicing

Jesse M Engreitz et al. Nature. .

Abstract

Mammalian genomes are pervasively transcribed to produce thousands of long non-coding RNAs (lncRNAs). A few of these lncRNAs have been shown to recruit regulatory complexes through RNA-protein interactions to influence the expression of nearby genes, and it has been suggested that many other lncRNAs can also act as local regulators. Such local functions could explain the observation that lncRNA expression is often correlated with the expression of nearby genes. However, these correlations have been challenging to dissect and could alternatively result from processes that are not mediated by the lncRNA transcripts themselves VSports手机版. For example, some gene promoters have been proposed to have dual functions as enhancers, and the process of transcription itself may contribute to gene regulation by recruiting activating factors or remodelling nucleosomes. Here we use genetic manipulation in mouse cell lines to dissect 12 genomic loci that produce lncRNAs and find that 5 of these loci influence the expression of a neighbouring gene in cis. Notably, none of these effects requires the specific lncRNA transcripts themselves and instead involves general processes associated with their production, including enhancer-like activity of gene promoters, the process of transcription, and the splicing of the transcript. Furthermore, such effects are not limited to lncRNA loci: we find that four out of six protein-coding loci also influence the expression of a neighbour. These results demonstrate that cross-talk among neighbouring genes is a prevalent phenomenon that can involve multiple mechanisms and cis-regulatory signals, including a role for RNA splice sites. These mechanisms may explain the function and evolution of some genomic loci that produce lncRNAs and broadly contribute to the regulation of both coding and non-coding genes. .

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Conflict of interest statement

Conflict of interest statement: The Broad Institute holds patents and has filed patent applications on technologies related to other aspects of CRISPR.

Figures

Extended Data Fig. 1.
Extended Data Fig. 1.. Expression and subcellular localization of knocked-out lncRNAs and mRNAs.
(a) Expression of lncRNAs and mRNAs in F1 129/Castaneus female mESCs, reported in fragments per kilobase per million (FPKM) in whole-cell p(A)+ RNA-seq. Cumulative fraction is plotted for all mRNAs expressed in mESCs. Large dots represent transcripts whose promoters we deleted in this study. LncRNAs and mRNAs span a >20-fold range of abundance levels. (b) Relative subcellular localization of lncRNAs and mRNAs. We sequenced p(A)+ RNA from chromatin, soluble nuclear, and cytoplasmic fractions (see Methods) and plotted the relative abundance of mature transcripts in each fraction. We selected lncRNAs that showed localization biased toward the nuclear fractions relative to most mRNAs. For comparison, we plotted 1,000 randomly selected mRNAs (light gray).
Extended Data Fig. 2.
Extended Data Fig. 2.. Generation of knockout clones and measurement of allele-specific RNA expression.
(a) Overview of knockout and measurement protocol. (b) Distribution of allelic expression ratios (number of informative reads mapping to 129S1 allele divided by the number mapping to either the 129S1 or the Castaneus allele) across active genes in mESCs. (c) Scatterplot of allelic expression ratios for genes with RPKM ≥ 2 that have more than 100 allele-informative reads across all libraries. Allelic expression ratios are consistent in RNA sequencing data before and after hybrid selection (HS). (d) Allelic expression ratios as measured by two independent methods for Blustr and (e) Sfmbt2 expression in 15 clonal cell lines containing genetic modifications in the Blustr locus. (f) Example locus showing hybrid selection strategy and RNA-seq coverage for cell lines with the indicated genotype for deletion of the Bendr promoter. Y-axis scales represent normalized read counts and are the same for all hybrid selection tracks. The absolute level of expression for any given gene varies among clonal cell lines; throughout this work, we instead consider the relative level of expression between the two alleles in heterozygous knockout cells. For similar plots of each gene studied, see http://pubs.broadinstitute.org/neighboring-genes/.
Extended Data Fig. 3.
Extended Data Fig. 3.. Read-through transcription at Meg3 and Snhg3 loci.
(a) Snhg3 promoter knockout reduces the levels of Rcc1 mRNA by 23%. However, sequencing of chromatin-associated RNA shows that transcription continues past the annotated 3’ end of Snhg3 into the downstream Rcc1 gene (see Methods). This read-through transcription creates a fusion transcript containing exons of both Snhg3 and Rcc1, as well as intergenic RNA. We note that this fusion transcript is also annotated in the syntenic human locus as an alternative isoform of RCC1. Bars: relative p(A)+ RNA expression on modified versus unmodified alleles. Error bars: 95% CI for the mean (n ≥ 2 alleles, see Table S1). (b) Meg3 promoter knockout eliminates the expression not only of Meg3 but also of two additional lncRNAs encoded downstream in a tandem orientation (Rian and Mirg). Although these three lncRNAs are annotated as separate genes, they appear to be derived from a single transcript driven by the Meg3 promoter. This is consistent with the presence of continuous chromatin-associated RNA throughout the locus and a lack of CAGE reads at the 5’ ends of Rian and Mirg3.
Extended Data Fig. 4.
Extended Data Fig. 4.. Promoter knockouts for 5 intergenic lncRNAs affect the expression of a neighboring gene.
Significance (z-score) of allele-specific expression ratios at all genes within 1 Mb of each of 5 lncRNA loci. Each dot represents a different heterozygous promoter knockout clone for a given gene. Dots are shown only for genes that are sufficiently highly expressed to assess allele-specific expression (see Methods). The y-axis is capped at –10 to +10 standard deviations from the mean. Black: knocked-out lncRNA. Blue: Gene with significant allele-specific change in gene expression (FDR < 10%). Independent clones are not expected to yield the same significance value (z-score), in part because read depth differs between samples.
Extended Data Fig. 5.
Extended Data Fig. 5.. Promoter knockouts for 4 mRNAs affect the expression of a neighboring gene.
Significance (z-score) of allele-specific expression ratios at all genes within 1 Mb of each of 4 mRNA loci. Each dot represents a different heterozygous promoter knockout clone for a given gene. Dots are shown only for genes that are sufficiently highly expressed to assess allele-specific expression (see Methods). The y-axis is capped at –10 to +10 standard deviations from the mean. Black: knocked-out lncRNA. Blue: Gene with significant allele-specific change in gene expression (FDR < 10%). Independent clones are not expected to yield the same significance value (z-score), in part because read depth differs between samples.
Extended Data Fig. 6.
Extended Data Fig. 6.. Dissecting mechanisms for how gene loci regulate a neighbor.
(a) Three categories of possible mechanisms by which a gene locus might regulate the expression of a neighbor. (b) We used two strategies to insert pAS downstream of gene promoters. In the first strategy, we inserted a 49-bp synthetic pAS (“spA”) using a single-stranded DNA oligo with 75-bp homology arms (see Methods). (c) In the second pAS insertion strategy, we cloned a donor plasmid containing a selection cassette and three different pAS sequences (see Methods). Homology arms of 300–800 bp were used to integrate the cassette. After isolating clones with successful insertions, we used a second round of transfections to remove the selection cassette, leaving behind three tandem pASs. EFS = elongation factor 1 promoter. Puro = puromycin resistance gene (pac). HSV-tk = herpes simplex virus thymidine kinase.
Extended Data Fig. 7.
Extended Data Fig. 7.. Promoters of lncRNAs and mRNAs have enhancer-like functions.
(a) Allele-specific GRO-seq signal for clones with the indicated modifications at the Bendr locus. Only reads specifically mapping to one of the two alleles are shown. Y-axis scale represents normalized read count and is the same for all tracks. (b) Allele-specific p(A)+ RNA expression for genetic modifications at the linc1405, Snhg17, Gpr19, and Slc30a9 loci. Bars: Average RNA expression on modified compared to unmodified (wild-type) alleles. Error bars: 95% CI for the mean (n ≥ 2 alleles, see Table S1). Gray arrows indicates distance from the targeted locus promoter to the affected neighboring gene. We note that, based on their location, the Snhg17 and Gpr19 pAS insertions likely allow more substantial splicing and transcription; for these loci, it is clear that the majority of the transcript is dispensable but it is possible that transcription close to the promoter may be involved in the cis regulatory function. (c) Presence (gray) or absence (white) of various chromatin marks and transcription factors in mESCs in a 1.5-kb window centered on the TSS of each targeted gene. (d) Distance from each knocked-out gene to its neighboring target gene (x-axis) versus the magnitude of the effect on the expression of the neighboring gene (% compared to wild-type, y-axis). Blue genes represent those discussed in main text; gray genes are discussed in Note S5. (e) Proximity-based contacts between the linc1405 and Eomes loci (the pair of loci separated by the greatest linear distance). The y-axis shows enrichment in a sequencing-based proximity assay in which we used antisense oligos to capture linc1405 DNA and any interacting, crosslinked proximal DNA (see Methods). TAD annotations are derived from Hi-C experiments in mESCs (see Methods). Blue arrow: focal contact between the linc1405 and Eomes loci.
Extended Data Fig. 8.
Extended Data Fig. 8.. Characterization of genetic modifications in the Blustr locus.
(a) Allele-specific GRO-seq signal for clones with the indicated modifications at the Blustr locus. Only reads specifically mapping to one of the two alleles are shown. Y-axis scale represents normalized read count and is the same for all tracks, and is magnified 5 times at the indicated location to better visualize the reads in the Sfmbt2 locus. (b) Quantification of allele-specific GRO-seq signal in the Sfmbt2 locus on alleles modified as indicated. TSS: region including the two alternative TSSs of Sfmbt2 and 2 kb downstream. Gene body: region containing the remainder of the Sfmbt2 gene locus. Pause index: ratio of TSS to gene body. Dashed gray lines indicate the 95% CI for the mean of 8 wild-type clones. (c) Schematic of the 5’ end of the Blustr locus and genotypes of two knockout clones. The 5’ splice site is located 78 bp downstream of the Blustr transcription start site (in this panel, Blustr is transcribed from left to right). One of the alleles from the two clones contains insertion of the oligo mediated by homologous recombination; the remaining three alleles contain insertions or deletions resulting from non-homologous end joining repair of sgRNA-mediated double-strand breaks, some of which also disrupt the 5’ splice site. Barplots show allele-specific RNA expression for knockout clones and control clones (+/+). (d) Schematic of the observed splice structures of Blustr RNA transcripts in p(A)+ RNA sequencing of the exon deletion clones. Each deletion removes a region including ~50–200 bp on either side of the exon, thereby removing both the exon and its splice sites. The Exon 4 deletion removes the endogenous pAS, leading to new isoforms of the lncRNA transcript that splice into two cryptic splice acceptors downstream. (e) GRO-Seq, H3K4me3 ChIP-Seq, and chromatin accessibility (ATAC-Seq FPKM) at the Blustr and Sfmbt2 promoters in cell lines with the indicated genotypes. Deletion of the first 5’ splice site leads to a significant reduction in H3K4me3, RNA polymerase occupancy, and chromatin accessibility at the Blustr promoter, as well as H3K4me3 and RNA polymerase occupancy (but not accessibility) at the Sfmbt2 promoter. (f) H3K27me3 ChIP-seq at the Blustr and Sfmbt2 loci in cell lines with the indicated genotypes. Deletion of the Blustr promoter or 5’ splice site leads to spreading of the repression-associated H3K27me3 modification across a ~30 kb region.
Extended Data Fig. 9.
Extended Data Fig. 9.. Mechanisms for crosstalk between neighboring lncRNAs and mRNAs.
Proposed mechanisms based on pAS insertion experiments and other genetic manipulations (see text). †For proposed mechanisms, see Note S5.
Extended Data Fig. 10.
Extended Data Fig. 10.. Classification of lncRNAs based on conservation and promoter location.
(a) Classification of 307 lncRNAs expressed in mESCs. “Conserved” transcripts are those that show significant evidence of capped analysis of gene expression (CAGE) data and/or p(A)+ RNA in syntenic loci (see Methods). Divergent: initiating within 500 bp of an mRNA TSS, on the opposite strand. ERV: endogenous retroviral repetitive element (see Note S9). Boxplot shows sequence-level conservation of the promoters of subsets of lncRNAs expressed in mESCs. Random intergenic regions are matched to lncRNA promoters by GC content. Positive SiPhy score indicates evolutionary constraint on functional sequences. Orange category corresponds to mouse-specific lncRNAs that appear to have evolved from ancestral regulatory elements (REs) and correspond to sequences that show evidence for DNase I hypersensitivity in human embryonic stem cells. Significance is calculated compared to random intergenic regions using a Mann-Whitney U-test. ***: P < 0.001. Whiskers represent data within 1.5× the interquartile range of the box. (b) Chromatin and RNA data for 11 mouse-specific lncRNAs that appear to have evolved from ancestral regulatory elements. In mouse, these elements show evidence for CAGE, H3K4me3, and DNase I hypersensitivity, consistent with their roles as promoters. The syntenic sequences in human do not show evidence for CAGE but nonetheless are DNase I hypersensitive and are frequently marked by H3K4me1 and/or CTCF. (c) Model for evolution of lncRNAs from pre-existing enhancers, which often initiate weak bidirectional transcription to produce eRNA. Spliced transcripts may neutrally appear through the appearance of splice signals and loss of polyadenylation signals. In some cases, transcription, splicing, or other RNA processing mechanisms may feed back and contribute to the cis regulatory function of the promoter, producing a lncRNA as a byproduct.
Fig. 1.
Fig. 1.. Many lncRNA and mRNA loci influence the expression of neighboring genes.
(a) Knocking out a promoter (black) could affect a neighboring gene (blue) directly (local) or indirectly (downstream). (b) Knockout of the linc1536 promoter. Left: genotypes. Right: allele-specific RNA expression for 129 and Castaneus (Cast) alleles normalized to 81 control clones (+/+). Error bars: 95% confidence interval (CI) for the mean (n ≥ 2 clones, see Table S1). (c) Gene neighborhoods oriented so each knocked-out gene (black) is transcribed in the positive direction. Blue neighboring genes show allele-specific changes in expression. ^see Note S3. (d) Average RNA expression on promoter knockout compared to wild-type alleles (n ≥ 2 alleles, see Table S1). *: FDR < 10%. ***: FDR < 0.1%.
Fig. 2.
Fig. 2.. Enhancer-like function of the Bendr promoter.
(a) Transcriptionally engaged RNA polymerase (GRO-Seq) and H3K4me3 occupancy (ChIP-Seq). (b) p(A)+ RNA expression upon deleting the Bendr promoter or inserting a pAS on modified versus unmodified alleles. Error bars: 95% CI for the mean (n ≥ 2 alleles, see Table S1). (c) Allele-specific GRO-seq signal for clones carrying the indicated modifications. Both clones are modified on the 129 allele, and only reads specifically mapping that allele are shown. Y-axis: normalized read count. Bar plot quantifies signal at Bend4, including 7 additional wild-type controls not shown on left.
Fig. 3.
Fig. 3.. Transcription and splicing of Blustr activates Sfmbt2 expression.
(a) p(A)+ RNA-seq, GRO-seq, and H3K4me3 ChIP-Seq in the Blustr locus. Sfmbt2 has two alternative TSSs. (b) p(A)+ RNA expression on knocked-out alleles compared to controls (arrows). Error bars: 95% CI for the mean (n ≥ 2 alleles, except for pAS +15 kb where n = 1, see Table S1). Sfmbt2 pAS comparisons: two-sided t-test P < 0.05 (*) or < 0.01 (**). (c) Allele-specific GRO-seq signal for clones carrying indicated modifications. Only reads mapping to the modified allele are shown (Cast for pAS +2 kb; 129 for others). (d) Model for how transcription in the Blustr locus activates Sfmbt2.
Fig. 4.
Fig. 4.. Evolutionary conservation of mESC lncRNAs and their promoters.
(a) Classification of a subset of lncRNAs expressed in mESCs (see Note S9, Methods). (b) 11 have promoters whose syntenic sequence corresponds to putative DNA regulatory elements (REs) marked by DNase I hypersensitivity (HS) in human ESCs. (c) Example: linc1494. (d) Enhancers and lncRNA promoters are significantly enriched for corresponding to human REs (pie chart, ***: P < 10−10, Chi-squared test versus GC-matched random regions) and show elevated sequence conservation compared to GC-matched regions (bar plot, **: P < 0.01, ***: P < 0.001, Mann-Whitney test versus ii+iii).

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