Alexander A. Shishkin

Genetic and Epigenetic Fine-Mapping of Causal Autoimmune Disease Variants

Genome-wide association studies have identified loci underlying human diseases, but the causal nucleotide changes and mechanisms remain largely unknown. Here we developed a fine-mapping algorithm to identify candidate causal variants for 21 autoimmune diseases from genotyping data. We integrated these predictions with transcription and cis-regulatory element annotations, derived by mapping RNA and chromatin in primary immune cells, including resting and stimulated CD4+ T-cell subsets, regulatory T cells, CD8+ T cells, B cells, and monocytes. We find that ∼90% of causal variants are non-coding, with ∼60% mapping to immune-cell enhancers, many of which gain histone acetylation and transcribe enhancer-associated RNA upon immune stimulation. Causal variants tend to occur near binding sites for master regulators of immune differentiation and stimulus-dependent gene activation, but only 10–20% directly alter recognizable transcription factor binding motifs. Rather, most non-coding risk variants, including those that alter gene expression, affect non-canonical sequence determinants not well-explained by current gene regulatory models.

The Xist lncRNA exploits three-dimensional genome architecture to spread across the X-chromosome*

Introduction Mammalian genomes encode thousands of large noncoding RNAs (lncRNAs), many of which regulate gene expression, interact with chromatin regulatory complexes, and are thought to play a role in localizing these complexes to target loci across the genome. A paradigm for this class of lncRNAs is Xist, which orchestrates mammalian X-chromosome inactivation (XCI) by coating and silencing one X chromosome in females. Despite the central role of RNA-chromatin interactions in this process, the mechanisms by which Xist localizes to DNA and spreads across the X chromosome remain unknown. Upon activation, Xist spreads from its genomic locus to sites in close three-dimensional proximity. Xist modifies chromatin architecture at these sites, thereby repositioning these regions into the Xist compartment (red cloud) and pulling new regions (green, yellow) of the chromosome into closer proximity. These structural changes allow Xist to access new sites and spread across the entire chromosome. Methods We developed a biochemical method called RNA antisense purification (RAP) to map the localization of a lncRNA across the genome. RAP uses long biotinylated antisense RNA probes to hybridize to and capture a target lncRNA and associated genomic DNA, enabling high-resolution mapping of lncRNA binding sites through high-throughput DNA sequencing. We applied RAP to study the localization of Xist during the initiation and maintenance of XCI. Results We show that during the maintenance of XCI, Xist binds broadly across the X chromosome, lacking defined localization sites. Xist preferentially localizes to broad gene-dense regions and excludes genes that escape XCI. At the initiation of XCI in mouse embryonic stem cells, Xist initially transfers to distal regions across the X chromosome that are not defined by specific sequences. Instead, Xist RNA identifies these regions using a proximity-guided search mechanism, exploiting the three-dimensional conformation of the X chromosome to spread to distal regions in close spatial proximity to the Xist genomic locus. Initially, Xist is excluded from actively transcribed genes and accumulates on the periphery of regions containing many active genes. Xist requires its silencing domain to spread across these regions and access the entire chromosome. Discussion Our data suggest a model for how Xist can integrate its two functions—localization to DNA and silencing of gene expression—to coat the entire X chromosome. In this model, Xist exploits three-dimensional conformation to identify and localize to initial target sites and leads to repositioning of these regions into the growing Xist compartment. These structural changes effectively pull new regions of the chromosome closer to the Xist genomic locus, allowing Xist RNA to spread to these newly accessible sites by proximity transfer. This localization strategy capitalizes on the abilities of a lncRNA to act while tethered to its transcription locus and to interact with chromatin regulatory proteins to modify chromatin structure. Beyond Xist, other lncRNAs may use a similar strategy to locate regulatory targets in three-dimensional proximity and to alter chromatin structure to establish local nuclear compartments containing co-regulated targets. Understanding Xist-ance Large noncoding RNAs (lncRNAs) are increasingly appreciated to play important roles in the cell. A number of lncRNAs act to target chromatin regulatory complexes to their sites of action. Engreitz et al. (p. 10.1126/science.1237973, published online 4 July; see the Perspective by Dimond and Fraser) found that the mouse Xist lncRNA, which initiates X-chromosome inactivation, was transferred from its site of transcription to distant sites on the X chromosome purely through their close three-dimensional proximity to the Xist gene. Xist initially localized to the periphery of active genes on the X chromosome but gradually spread across them using its A-repeat domain, until the Xist RNA bound broadly across the inactive X chromosome in differentiated female cells. A large noncoding RNA uses folds within the chromosome to drive the spread of a chromatin repressive complex. [Also see Perspective by Dimond and Fraser] 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.

Transcriptional and epigenetic dynamics during specification of human embryonic stem cells.

Differentiation of human embryonic stem cells (hESCs) provides a unique opportunity to study the regulatory mechanisms that facilitate cellular transitions in a human context. To that end, we performed comprehensive transcriptional and epigenetic profiling of populations derived through directed differentiation of hESCs representing each of the three embryonic germ layers. Integration of whole-genome bisulfite sequencing, chromatin immunoprecipitation sequencing, and RNA sequencing reveals unique events associated with specification toward each lineage. Lineage-specific dynamic alterations in DNA methylation and H3K4me1 are evident at putative distal regulatory elements that are frequently bound by pluripotency factors in the undifferentiated hESCs. In addition, we identified germ-layer-specific H3K27me3 enrichment at sites exhibiting high DNA methylation in the undifferentiated state. A better understanding of these initial specification events will facilitate identification of deficiencies in current approaches, leading to more faithful differentiation strategies as well as providing insights into the rewiring of human regulatory programs during cellular transitions.

High-Resolution Mapping Reveals a Conserved, Widespread, Dynamic mRNA Methylation Program in Yeast Meiosis

N(6)-methyladenosine (m(6)A) is the most ubiquitous mRNA base modification, but little is known about its precise location, temporal dynamics, and regulation. Here, we generated genomic maps of m(6)A sites in meiotic yeast transcripts at nearly single-nucleotide resolution, identifying 1,308 putatively methylated sites within 1,183 transcripts. We validated eight out of eight methylation sites in different genes with direct genetic analysis, demonstrated that methylated sites are significantly conserved in a related species, and built a model that predicts methylated sites directly from sequence. Sites vary in their methylation profiles along a dense meiotic time course and are regulated both locally, via predictable methylatability of each site, and globally, through the core meiotic circuitry. The methyltransferase complex components localize to the yeast nucleolus, and this localization is essential for mRNA methylation. Our data illuminate a conserved, dynamically regulated methylation program in yeast meiosis and provide an important resource for studying the function of this epitranscriptomic modification.

Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP)

As RNA-binding proteins (RBPs) play essential roles in cellular physiology by interacting with target RNA molecules, binding site identification by UV crosslinking and immunoprecipitation (CLIP) of ribonucleoprotein complexes is critical to understanding RBP function. However, current CLIP protocols are technically demanding and yield low-complexity libraries with high experimental failure rates. We have developed an enhanced CLIP (eCLIP) protocol that decreases requisite amplification by ∼1,000-fold, decreasing discarded PCR duplicate reads by ∼60% while maintaining single-nucleotide binding resolution. By simplifying the generation of paired IgG and size-matched input controls, eCLIP improves specificity in the discovery of authentic binding sites. We generated 102 eCLIP experiments for 73 diverse RBPs in HepG2 and K562 cells (available at https://www.encodeproject.org), demonstrating that eCLIP enables large-scale and robust profiling, with amplification and sample requirements similar to those of ChIP-seq. eCLIP enables integrative analysis of diverse RBPs to reveal factor-specific profiles, common artifacts for CLIP and RNA-centric perspectives on RBP activity.

Large-Scale Expansions of Friedreich's Ataxia GAA Repeats in Yeast

Large-scale expansions of DNA repeats are implicated in numerous hereditary disorders in humans. We describe a yeast experimental system to analyze large-scale expansions of triplet GAA repeats responsible for the human disease Friedreichs ataxia. When GAA repeats were placed into an intron of the chimeric URA3 gene, their expansions caused gene inactivation, which was detected on the selective media. We found that the rates of expansions of GAA repeats increased exponentially with their lengths. These rates were only mildly dependent on the repeats orientation within the replicon, whereas the repeat-mediated replication fork stalling was exquisitely orientation dependent. Expansion rates were significantly elevated upon inactivation of the replication fork stabilizers, Tof1 and Csm3, but decreased in the knockouts of postreplication DNA repair proteins, Rad6 and Rad5, and the DNA helicase Sgs1. We propose a model for large-scale repeat expansions based on template switching during replication fork progression through repetitive DNA.

Replisome stalling and stabilization at CGG repeats, which are responsible for chromosomal fragility

Expanded CGG repeats cause chromosomal fragility and hereditary neurological disorders in humans. Replication forks stall at CGG repeats in a length-dependent manner in primate cells and in yeast. Saccharomyces cerevisiae proteins Tof1 and Mrc1 facilitate replication fork progression through CGG repeats. Remarkably, the fork-stabilizing role of Mrc1 does not involve its checkpoint function. Thus, chromosomal fragility might occur when forks stalled at expanded CGG repeats escape the S-phase checkpoint.

Simultaneous generation of many RNA-seq libraries in a single reaction

Although RNA-seq is a powerful tool, the considerable time and cost associated with library construction has limited its utilization for various applications. RNAtag-Seq, an approach to generate multiple RNA-seq libraries in a single reaction, lowers time and cost per sample, and it produces data on prokaryotic and eukaryotic samples that are comparable to those generated by traditional strand-specific RNA-seq approaches.

Evolutionary analysis across mammals reveals distinct classes of long non-coding RNAs

BackgroundRecent advances in transcriptome sequencing have enabled the discovery of thousands of long non-coding RNAs (lncRNAs) across many species. Though several lncRNAs have been shown to play important roles in diverse biological processes, the functions and mechanisms of most lncRNAs remain unknown. Two significant obstacles lie between transcriptome sequencing and functional characterization of lncRNAs: identifying truly non-coding genes from de novo reconstructed transcriptomes, and prioritizing the hundreds of resulting putative lncRNAs for downstream experimental interrogation.ResultsWe present slncky, a lncRNA discovery tool that produces a high-quality set of lncRNAs from RNA-sequencing data and further uses evolutionary constraint to prioritize lncRNAs that are likely to be functionally important. Our automated filtering pipeline is comparable to manual curation efforts and more sensitive than previously published computational approaches. Furthermore, we developed a sensitive alignment pipeline for aligning lncRNA loci and propose new evolutionary metrics relevant for analyzing sequence and transcript evolution. Our analysis reveals that evolutionary selection acts in several distinct patterns, and uncovers two notable classes of intergenic lncRNAs: one showing strong purifying selection on RNA sequence and another where constraint is restricted to the regulation but not the sequence of the transcript.ConclusionOur results highlight that lncRNAs are not a homogenous class of molecules but rather a mixture of multiple functional classes with distinct biological mechanism and/or roles. Our novel comparative methods for lncRNAs reveals 233 constrained lncRNAs out of tens of thousands of currently annotated transcripts, which we make available through the slncky Evolution Browser.

Role of DNA Polymerases in Repeat-Mediated Genome Instability

Expansions of simple DNA repeats cause numerous hereditary diseases in humans. We analyzed the role of DNA polymerases in the instability of Friedreichs ataxia (GAA)(n) repeats in a yeast experimental system. The elementary step of expansion corresponded to ~160 bp in the wild-type strain, matching the size of Okazaki fragments in yeast. This step increased when DNA polymerase α was mutated, suggesting a link between the scale of expansions and Okazaki fragment size. Expandable repeats strongly elevated the rate of mutations at substantial distances around them, a phenomenon we call repeat-induced mutagenesis (RIM). Notably, defects in the replicative DNA polymerases δ and ε strongly increased rates for both repeat expansions and RIM. The increases in repeat-mediated instability observed in DNA polymerase δ mutants depended on translesion DNA polymerases. We conclude that repeat expansions and RIM are two sides of the same replicative mechanism.