Neighborhood regulation by lncRNA promoters, transcription, and splicing

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 splicing. These mechanisms may explain the function and evolution of some genomic loci that produce lncRNAs

Deep coverage whole genome sequences and plasma lipoprotein(a) in individuals of European and African ancestries.

Lipoprotein(a), Lp(a), is a modified low-density lipoprotein particle that contains apolipoprotein(a), encoded by LPA, and is a highly heritable, causal risk factor for cardiovascular diseases that varies in concentrations across ancestries. Here, we use deep-coverage whole genome sequencing in 8392 individuals of European and African ancestry to discover and interpret both single-nucleotide variants and copy number (CN) variation associated with Lp(a). We observe that genetic determinants between Europeans and Africans have several unique determinants. The common variant rs12740374 associated with Lp(a) cholesterol is an eQTL for SORT1 and independent of LDL cholesterol. Observed associations of aggregates of rare non-coding variants are largely explained by LPA structural variation, namely the LPA kringle IV 2 (KIV2)-CN. Finally, we find that LPA risk genotypes confer greater relative risk for incident atherosclerotic cardiovascular diseases compared to directly measured Lp(a), and are significantly associated with measures of subclinical atherosclerosis in African Americans

Publisher Correction: Deep coverage whole genome sequences and plasma lipoprotein(a) in individuals of European and African ancestries.

The original version of this article contained an error in the name of the author Ramachandran S. Vasan, which was incorrectly given as Vasan S. Ramachandran. This has now been corrected in both the PDF and HTML versions of the article

Publisher Correction: Deep coverage whole genome sequences and plasma lipoprotein(a) in individuals of European and African ancestries.

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Topological organization of multichromosomal regions by the long intergenic noncoding RNA Firre

RNA, including long noncoding RNA (lncRNA), is known to be an abundant and important structural component of the nuclear matrix. However, the molecular identities, functional roles and localization dynamics of lncRNAs that influence nuclear architecture remain poorly understood. Here, we describe one lncRNA, Firre, that interacts with the nuclear-matrix factor hnRNPU through a 156-bp repeating sequence and localizes across an ~5-Mb domain on the X chromosome. We further observed Firre localization across five distinct trans-chromosomal loci, which reside in spatial proximity to the Firre genomic locus on the X chromosome. Both genetic deletion of the Firre locus and knockdown of hnRNPU resulted in loss of colocalization of these trans-chromosomal interacting loci. Thus, our data suggest a model in which lncRNAs such as Firre can interface with and modulate nuclear architecture across chromosomes

Genome regulation by long noncoding Ribonucleic acids

Thesis: Ph. D. in Bioinformatics and Integrative Genomics, Harvard-MIT Program in Health Sciences and Technology, 2016.Cataloged from PDF version of thesis.Includes bibliographical references.Our genomes encode the molecular information that gives rise to life, yet we are just beginning to unravel how this information is organized, interpreted, and regulated. While the human genome contains -20,000 protein-coding genes, mammalian genomes also produce thousands of long non-coding RNAs (lncRNAs), some of which are now known to play essential roles in diverse biological processes including cellular differentiation and human disease. Recent studies show that many lncRNAs localize to the nucleus and interact with chromatin regulatory complexes, suggesting that some lncRNAs may represent a crucial missing component in our understanding of genome regulation. To test whether lncRNAs localize to and regulate specific sites in the genome, we developed genome-wide approaches to map lncRNA interactions with chromatin. Through studies of three conserved lncRNAs, we demonstrate that lncRNAs can exploit the three-dimensional architecture of the genome to identify their regulatory targets and, in turn, actively manipulate genome architecture to form subcompartments containing co-regulated genes. Thus, lncRNAs have unique capabilities as dynamic regulators that can locally amplify epigenetic signals. We next explored whether this model might apply to other long noncoding RNAs, many of which are not conserved across species and thus whose functions remain unclear. Through genetic dissection of their local regulatory functions, we show that many of these genomic loci participate in the local regulation of gene expression, but that these functions do not involve the IncRNA transcripts themselves. Instead, multiple mechanisms associated with RNA production including their promoters, the process of transcription, and RNA splicing - act in local networks of regulatory connections between spatially proximal genes, both protein-coding and noncoding. These findings reveal novel mechanistic explanations for the functions and evolution of noncoding transcription in mammalian genomes. Together these studies suggest a model in which mammalian gene regulation is organized into local neighborhoods defined by the spatial architecture of the genome. Within these neighborhoods, lncRNAs and DNA regulatory elements may function cooperatively to coordinate local gene expression. Dissecting this fundamental model for genome regulation may enable manipulation of the processes that interpret our genome sequence and galvanize efforts to develop new treatments for human disease.by Jesse M. Engreitz.Ph. D. in Bioinformatics and Integrative Genomic

CRISPR-SURF: discovering regulatory elements by deconvolution of CRISPR tiling screen data

Tiling screens that use CRISPR-Cas technologies provide a powerful approach for the mapping of regulatory elements to phenotypes of interest1–6. Here we present CRISPR screening uncharacterized region function (CRISPR-SURF), a deconvolution framework that can be used to identify functional regulatory regions in the genome from data generated by CRISPR-Cas nuclease, CRISPR interference (CRISPRi), or CRISPR activation (CRISPRa) tiling screens. CRISPR-SURF can be run as a stand-alone command line utility (https://github.com/pinellolab/CRISPR-SURF) or as a web application (http://crisprsurf.pinellolab.org/)National Human Genome Research Institute (U.S.) (Career Development Award R00 HG008399)National Human Genome Research Institute (U.S.). Centers of Excellence in Genomic Science (Grant RM1HG009490)National Institutes of Health (U.S.) (Grant R35 GM118158)National Institutes of Health (U.S.) (Grant RM1 HG009490)National Human Genome Research Institute (U.S.) (Grant 1K99HG009917-01

The Xist lncRNA Exploits Three-Dimensional Genome Architecture to Spread Across the X Chromosome

Many large noncoding RNAs (lncRNAs) regulate chromatin, but the mechanisms by which they localize to genomic targets remain unexplored. We investigated the localization mechanisms of the Xist lncRNA during X-chromosome inactivation (XCI), a paradigm of lncRNA-mediated chromatin regulation. During the maintenance of XCI, Xist binds broadly across the X chromosome. During initiation of XCI, Xist initially transfers to distal regions across the X chromosome that are not defined by specific sequences. Instead, Xist identifies these regions by exploiting the three-dimensional conformation of the X chromosome. Xist requires its silencing domain to spread across actively transcribed regions and thereby access the entire chromosome. These findings suggest a model in which Xist coats the X chromosome by searching in three dimensions, modifying chromosome structure, and spreading to newly accessible locations.Hertz FoundationAmerican Society for Engineering Education. National Defense Science and Engineering Graduate FellowshipBroad Institute of MIT and Harvar

Systematic dissection of genomic features determining transcription factor binding and enhancer function

Enhancers regulate gene expression through the binding of sequence-specific transcription factors (TFs) to cognate motifs. Various features influence TF binding and enhancer function—including the chromatin state of the genomic locus, the affinities of the binding site, the activity of the bound TFs, and interactions among TFs. However, the precise nature and relative contributions of these features remain unclear. Here, we used massively parallel reporter assays (MPRAs) involving 32,115 natural and synthetic enhancers, together with high-throughput in vivo binding assays, to systematically dissect the contribution of each of these features to the binding and activity of genomic regulatory elements that contain motifs for PPARγ, a TF that serves as a key regulator of adipogenesis. We show that distinct sets of features govern PPARγ binding vs. enhancer activity. PPARγ binding is largely governed by the affinity of the specific motif site and higher-order features of the larger genomic locus, such as chromatin accessibility. In contrast, the enhancer activity of PPARγ binding sites depends on varying contributions from dozens of TFs in the immediate vicinity, including interactions between combinations of these TFs. Different pairs of motifs follow different interaction rules, including subadditive, additive, and superadditive interactions among specific classes of TFs, with both spatially constrained and flexible grammars. Our results provide a paradigm for the systematic characterization of the genomic features underlying regulatory elements, applicable to the design of synthetic regulatory elements or the interpretation of human genetic variation.National Human Genome Research Institute (U.S.) (Grant 2U54HG003067-10)National Institute of General Medical Sciences (U.S.) (Grant T32GM007753