Transcriptome-wide Mapping Reveals Widespread Dynamic-Regulated Pseudouridylation of ncRNA and mRNA

Pseudouridine is the most abundant RNA modification, yet except for a few well-studied cases, little is known about the modified positions and their function(s). Here, we develop Ψ-seq for transcriptome-wide quantitative mapping of pseudouridine. We validate Ψ-seq with spike-ins and de novo identification of previously reported positions and discover hundreds of unique sites in human and yeast mRNAs and snoRNAs. Perturbing pseudouridine synthases (PUS) uncovers which pseudouridine synthase modifies each site and their target sequence features. mRNA pseudouridinylation depends on both site-specific and snoRNA-guided pseudouridine synthases. Upon heat shock in yeast, Pus7p-mediated pseudouridylation is induced at >200 sites, and PUS7 deletion decreases the levels of otherwise pseudouridylated mRNA, suggesting a role in enhancing transcript stability. rRNA pseudouridine stoichiometries are conserved but reduced in cells from dyskeratosis congenita patients, where the PUS DKC1 is mutated. Our work identifies an enhanced, transcriptome-wide scope for pseudouridine and methods to dissect its underlying mechanisms and function

Transcript-indexed ATAC-seq for precision immune profiling.

T cells create vast amounts of diversity in the genes that encode their T cell receptors (TCRs), which enables individual clones to recognize specific peptide-major histocompatibility complex (MHC) ligands. Here we combined sequencing of the TCR-encoding genes with assay for transposase-accessible chromatin with sequencing (ATAC-seq) analysis at the single-cell level to provide information on the TCR specificity and epigenomic state of individual T cells. By using this approach, termed transcript-indexed ATAC-seq (T-ATAC-seq), we identified epigenomic signatures in immortalized leukemic T cells, primary human T cells from healthy volunteers and primary leukemic T cells from patient samples. In peripheral blood CD4+ T cells from healthy individuals, we identified cis and trans regulators of naive and memory T cell states and found substantial heterogeneity in surface-marker-defined T cell populations. In patients with a leukemic form of cutaneous T cell lymphoma, T-ATAC-seq enabled identification of leukemic and nonleukemic regulatory pathways in T cells from the same individual by allowing separation of the signals that arose from the malignant clone from the background T cell noise. Thus, T-ATAC-seq is a new tool that enables analysis of epigenomic landscapes in clonal T cells and should be valuable for studies of T cell malignancy, immunity and immunotherapy

GWAS for systemic sclerosis identifies multiple risk loci and highlights fibrotic and vasculopathy pathways

Systemic sclerosis (SSc) is an autoimmune disease that shows one of the highest mortality rates among rheumatic diseases. We perform a large genome-wide association study (GWAS), and meta-analysis with previous GWASs, in 26,679 individuals and identify 27 independent genome-wide associated signals, including 13 new risk loci. The novel associations nearly double the number of genome-wide hits reported for SSc thus far. We define 95% credible sets of less than 5 likely causal variants in 12 loci. Additionally, we identify specific SSc subtype-associated signals. Functional analysis of high-priority variants shows the potential function of SSc signals, with the identification of 43 robust target genes through HiChIP. Our results point towards molecular pathways potentially involved in vasculopathy and fibrosis, two main hallmarks in SSc, and highlight the spectrum of critical cell types for the disease. This work supports a better understanding of the genetic basis of SSc and provides directions for future functional experiments

Lineage-specific dynamic and pre-established enhancer–promoter contacts cooperate in terminal differentiation

Chromosome conformation is an important feature of metazoan gene regulation; however, enhancer–promoter contact remodeling during cellular differentiation remains poorly understood. To address this, genome-wide promoter capture Hi-C (CHi-C) was performed during epidermal differentiation. Two classes of enhancer–promoter contacts associated with differentiation-induced genes were identified. The first class ('gained') increased in contact strength during differentiation in concert with enhancer acquisition of the H3K27ac activation mark. The second class ('stable') were pre-established in undifferentiated cells, with enhancers constitutively marked by H3K27ac. The stable class was associated with the canonical conformation regulator cohesin, whereas the gained class was not, implying distinct mechanisms of contact formation and regulation. Analysis of stable enhancers identified a new, essential role for a constitutively expressed, lineage-restricted ETS-family transcription factor, EHF, in epidermal differentiation. Furthermore, neither class of contacts was observed in pluripotent cells, suggesting that lineage-specific chromatin structure is established in tissue progenitor cells and is further remodeled in terminal differentiation

Transcriptome-Wide Mapping of 5-methylcytidine RNA Modifications in Bacteria, Archaea, and Yeast Reveals m⁵C within Archaeal mRNAs

<div><p>The presence of 5-methylcytidine (m⁵C) in tRNA and rRNA molecules of a wide variety of organisms was first observed more than 40 years ago. However, detection of this modification was limited to specific, abundant, RNA species, due to the usage of low-throughput methods. To obtain a high resolution, systematic, and comprehensive transcriptome-wide overview of m⁵C across the three domains of life, we used bisulfite treatment on total RNA from both gram positive (<i>B. subtilis</i>) and gram negative (<i>E. coli</i>) bacteria, an archaeon (<i>S. solfataricus</i>) and a eukaryote (<i>S. cerevisiae</i>), followed by massively parallel sequencing. We were able to recover most previously documented m⁵C sites on rRNA in the four organisms, and identified several novel sites in yeast and archaeal rRNAs. Our analyses also allowed quantification of methylated m⁵C positions in 64 tRNAs in yeast and archaea, revealing stoichiometric differences between the methylation patterns of these organisms. Molecules of tRNAs in which m⁵C was absent were also discovered. Intriguingly, we detected m⁵C sites within archaeal mRNAs, and identified a consensus motif of AU<b><u>C</u></b>GANGU that directs methylation in <i>S. solfataricus</i>. Our results, which were validated using m⁵C-specific RNA immunoprecipitation, provide the first evidence for mRNA modifications in archaea, suggesting that this mode of post-transcriptional regulation extends beyond the eukaryotic domain.</p></div

Transcriptome-wide interrogation of RNA secondary structure in living cells with icSHAPE

icSHAPE (in vivo click selective 2-hydroxyl acylation and profiling experiment) captures RNA secondary structure at a transcriptome-wide level by measuring nucleotide flexibility at base resolution. Living cells are treated with the icSHAPE chemical NAI-N3 followed by selective chemical enrichment of NAI-N3-modified RNA, which provides an improved signal-to-noise ratio compared with similar methods leveraging deep sequencing. Purified RNA is then reverse-transcribed to produce cDNA, with SHAPE-modified bases leading to truncated cDNA. After deep sequencing of cDNA, computational analysis yields flexibility scores for every base across the starting RNA population. The entire experimental procedure can be completed in ∼5 d, and the sequencing and bioinformatics data analysis take an additional 4-5 d with no extensive computational skills required. Comparing in vivo and in vitro icSHAPE measurements can reveal in vivo RNA-binding protein imprints or facilitate the dissection of RNA post-transcriptional modifications. icSHAPE reactivities can additionally be used to constrain and improve RNA secondary structure prediction models

Modified m⁵C bases in mRNAs.

<p>Modified m⁵C bases in mRNAs.</p

Flow of the bisulfite-treatment/RNA-seq experiment.

<p>(A) Shown is a schematic representation of RNA molecules; colored boxes represent single bases. Red, blue, green and black represent C, U/T, A, and G residues, respectively. m⁵C modified cytosine bases are marked by a black circle. Total RNA is bisulfite-treated, leading to deamination of ‘C’ residues into ‘U’, except for methylated residues. Bisulfite-treated RNA is reverse transcribed into cDNA, which is then sequenced via an Illumina HiSeq system. This yields short sequence reads, representing random fragments of the sequenced RNAs. Resulting reads are mapped to the genome using an algorithm that allows mapping of ‘T’ residues in the sequenced cDNAs onto genomic ‘C's. Residues in which ‘C's are found to be consistently non-modified are declared as m⁵C (red arrow). (B) Flowchart of data analysis and artifact filtering (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003602#s4" target="_blank">Methods</a>).</p