Expression profiling by high throughput sequencing Genome binding/occupancy profiling by high throughput sequencing Other
Summary
Mammalian genomes are pervasively transcribed to produce thousands of spliced long noncoding RNAs (lncRNAs), whose functions remain poorly understood. Because recent evidence has implicated several specific lncRNA loci in the local regulation of gene expression, we sought to determine whether such local regulation is a property of many lncRNA loci. We used genetic manipulations to dissect 12 genomic loci that produce lncRNAs and found that 5 of these loci influence the expression of a neighboring gene in cis. Surprisingly, however, none of these effects required the specific lncRNA transcripts themselves and instead involved general processes associated with their production, including enhancer-like activity of gene promoters, the process of transcription, and the splicing of the transcript. Interestingly, such effects are not limited to lncRNA loci: we found similar effects on local gene expression at 4 of 6 protein-coding loci. These results demonstrate that ‘crosstalk’ among neighboring genes is a prevalent phenomenon that can involve multiple mechanisms and cis regulatory signals, including a novel role for RNA splice sites. These mechanisms may coordinate the expression of neighboring genes and explain the function and evolution of some genomic loci that produce lncRNAs.
Overall design
We developed a genetic approach based on classical cis-trans tests to distinguish between (i) primary effects on the expression of nearby genes resulting from direct local functions of the lncRNA locus and (ii) secondary effects on nearby genes resulting from indirect downstream consequences of the lncRNA acting elsewhere in the cell. We generated clonal cell lines carrying heterozygous genetic modifications at lncRNA loci and compared the expression of nearby genes within 1 megabase on the cis and trans alleles (i.e., on the modified and unmodified homologous chromosomes) in the same cells. Changes in neighboring gene expression that involve only the cis allele likely result from local functions, while changes that involve both the cis and trans alleles likely result as downstream effects of non-local functions. We performed genetic modifications in 129/Castaneus F1 hybrid mouse embryonic stem cells (mESCs) that contain ~1 polymorphic site every 140 basepairs (bp), enabling us to distinguish the two alleles using RNA sequencing, and we checked for consistency between knockouts on each genetic background to control for potential haplotype-specific effects. We initially characterized allele-specific gene expression using RNA sequencing combined with hybrid selection, and we further characterized a subset of clones in selected loci using global run-on sequencing (GRO-seq), chromatin immunoprecipitation (ChIP-Seq), and ATAC-Seq.
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