Genome-wide CRISPR Screens Reveal Host Factors Critical for SARS-CoV-2 Infection.

Identification of host genes essential for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may reveal novel therapeutic targets and inform our understanding of coronavirus disease 2019 (COVID-19) pathogenesis. Here we performed genome-wide CRISPR screens in Vero-E6 cells with SARS-CoV-2, Middle East respiratory syndrome CoV (MERS-CoV), bat CoV HKU5 expressing the SARS-CoV-1 spike, and vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike. We identified known SARS-CoV-2 host factors, including the receptor ACE2 and protease Cathepsin L. We additionally discovered pro-viral genes and pathways, including HMGB1 and the SWI/SNF chromatin remodeling complex, that are SARS lineage and pan-coronavirus specific, respectively. We show that HMGB1 regulates ACE2 expression and is critical for entry of SARS-CoV-2, SARS-CoV-1, and NL63. We also show that small-molecule antagonists of identified gene products inhibited SARS-CoV-2 infection in monkey and human cells, demonstrating the conserved role of these genetic hits across species. This identifies potential therapeutic targets for SARS-CoV-2 and reveals SARS lineage-specific and pan-CoV host factors that regulate susceptibility to highly pathogenic CoVs.

Antisense lncRNA Transcription Mediates DNA Demethylation to Drive Stochastic Protocadherin α Promoter Choice.

Stochastic activation of clustered Protocadherin (Pcdh) α, ß, and γ genes generates a cell-surface identity code in individual neurons that functions in neural circuit assembly. Here, we show that Pcdhα gene choice involves the activation of an antisense promoter located in the first exon of each Pcdhα alternate gene. Transcription of an antisense long noncoding RNA (lncRNA) from this antisense promoter extends through the sense promoter, leading to DNA demethylation of the CTCF binding sites proximal to each promoter. Demethylation-dependent CTCF binding to both promoters facilitates cohesin-mediated DNA looping with a distal enhancer (HS5-1), locking in the transcriptional state of the chosen Pcdhα gene. Uncoupling DNA demethylation from antisense transcription by Tet3 overexpression in mouse olfactory neurons promotes CTCF binding to all Pcdhα promoters, resulting in proximity-biased DNA looping of the HS5-1 enhancer. Thus, antisense transcription-mediated promoter demethylation functions as a mechanism for distance-independent enhancer/promoter DNA looping to ensure stochastic Pcdhα promoter choice.

Transcription elongation defects link oncogenic SF3B1 mutations to targetable alterations in chromatin landscape.

Transcription and splicing of pre-messenger RNA are closely coordinated, but how this functional coupling is disrupted in human diseases remains unexplored. Using isogenic cell lines, patient samples, and a mutant mouse model, we investigated how cancer-associated mutations in SF3B1 alter transcription. We found that these mutations reduce the elongation rate of RNA polymerase II (RNAPII) along gene bodies and its density at promoters. The elongation defect results from disrupted pre-spliceosome assembly due to impaired protein-protein interactions of mutant SF3B1. The decreased promoter-proximal RNAPII density reduces both chromatin accessibility and H3K4me3 marks at promoters. Through an unbiased screen, we identified epigenetic factors in the Sin3/HDAC/H3K4me pathway, which, when modulated, reverse both transcription and chromatin changes. Our findings reveal how splicing factor mutant states behave functionally as epigenetic disorders through impaired transcription-related changes to the chromatin landscape. We also present a rationale for targeting the Sin3/HDAC complex as a therapeutic strategy.

Catalytic and non-catalytic mechanisms of histone H4 lysine 20 methyltransferase SUV420H1.

SUV420H1 di- and tri-methylates histone H4 lysine 20 (H4K20me2/H4K20me3) and plays crucial roles in DNA replication, repair, and heterochromatin formation. It is dysregulated in several cancers. Many of these processes were linked to its catalytic activity. However, deletion and inhibition of SUV420H1 have shown distinct phenotypes, suggesting that the enzyme likely has uncharacterized non-catalytic activities. Our cryoelectron microscopy (cryo-EM), biochemical, biophysical, and cellular analyses reveal how SUV420H1 recognizes its nucleosome substrates, and how histone variant H2A.Z stimulates its catalytic activity. SUV420H1 binding to nucleosomes causes a dramatic detachment of nucleosomal DNA from the histone octamer, which is a non-catalytic activity. We hypothesize that this regulates the accessibility of large macromolecular complexes to chromatin. We show that SUV420H1 can promote chromatin condensation, another non-catalytic activity that we speculate is needed for its heterochromatin functions. Together, our studies uncover and characterize the catalytic and non-catalytic mechanisms of SUV420H1, a key histone methyltransferase that plays an essential role in genomic stability.

Precision analysis of mutant U2AF1 activity reveals deployment of stress granules in myeloid malignancies.

Splicing factor mutations are common among cancers, recently emerging as drivers of myeloid malignancies. U2AF1 carries hotspot mutations in its RNA-binding motifs; however, how they affect splicing and promote cancer remain unclear. The U2AF1/U2AF2 heterodimer is critical for 3' splice site (3'SS) definition. To specifically unmask changes in U2AF1 function in vivo, we developed a crosslinking and immunoprecipitation procedure that detects contacts between U2AF1 and the 3'SS AG at single-nucleotide resolution. Our data reveal that the U2AF1 S34F and Q157R mutants establish new 3'SS contacts at -3 and +1 nucleotides, respectively. These effects compromise U2AF2-RNA interactions, resulting predominantly in intron retention and exon exclusion. Integrating RNA binding, splicing, and turnover data, we predicted that U2AF1 mutations directly affect stress granule components, which was corroborated by single-cell RNA-seq. Remarkably, U2AF1-mutant cell lines and patient-derived MDS/AML blasts displayed a heightened stress granule response, pointing to a novel role for biomolecular condensates in adaptive oncogenic strategies.

A chromatin-dependent role of the fragile X mental retardation protein FMRP in the DNA damage response.

Fragile X syndrome, a common form of inherited intellectual disability, is caused by loss of the fragile X mental retardation protein FMRP. FMRP is present predominantly in the cytoplasm, where it regulates translation of proteins that are important for synaptic function. We identify FMRP as a chromatin-binding protein that functions in the DNA damage response (DDR). Specifically, we show that FMRP binds chromatin through its tandem Tudor (Agenet) domain in vitro and associates with chromatin in vivo. We also demonstrate that FMRP participates in the DDR in a chromatin-binding-dependent manner. The DDR machinery is known to play important roles in developmental processes such as gametogenesis. We show that FMRP occupies meiotic chromosomes and regulates the dynamics of the DDR machinery during mouse spermatogenesis. These findings suggest that nuclear FMRP regulates genomic stability at the chromatin interface and may impact gametogenesis and some developmental aspects of fragile X syndrome.

Acetyl-methyllysine marks chromatin at active transcription start sites.

Lysine residues in histones and other proteins can be modified by post-translational modifications that encode regulatory information1. Lysine acetylation and methylation are especially important for regulating chromatin and gene expression2-4. Pathways involving these post-translational modifications are targets for clinically approved therapeutics to treat human diseases. Lysine methylation and acetylation are generally assumed to be mutually exclusive at the same residue. Here we report cellular lysine residues that are both methylated and acetylated on the same side chain to form Nε-acetyl-Nε-methyllysine (Kacme). We show that Kacme is found on histone H4 (H4Kacme) across a range of species and across mammalian tissues. Kacme is associated with marks of active chromatin, increased transcriptional initiation and is regulated in response to biological signals. H4Kacme can be installed by enzymatic acetylation of monomethyllysine peptides and is resistant to deacetylation by some HDACs in vitro. Kacme can be bound by chromatin proteins that recognize modified lysine residues, as we demonstrate with the crystal structure of acetyllysine-binding protein BRD2 bound to a histone H4Kacme peptide. These results establish Kacme as a cellular post-translational modification with the potential to encode information distinct from methylation and acetylation alone and demonstrate that Kacme has all the hallmarks of a post-translational modification with fundamental importance to chromatin biology.

STL-seq reveals pause-release and termination kinetics for promoter-proximal paused RNA polymerase II transcripts.

Despite the critical regulatory function of promoter-proximal pausing, the influence of pausing kinetics on transcriptional control remains an active area of investigation. Here, we present Start-TimeLapse-seq (STL-seq), a method that captures the genome-wide kinetics of short, capped RNA turnover and reveals principles of regulation at the pause site. By measuring the rates of release into elongation and premature termination through the inhibition of pause release, we determine that pause-release rates are highly variable, and most promoter-proximal paused RNA polymerase II molecules prematurely terminate (∼80%). The preferred regulatory mechanism upon a hormonal stimulus (20-hydroxyecdysone) is to influence pause-release rather than termination rates. Transcriptional shutdown occurs concurrently with the induction of promoter-proximal termination under hyperosmotic stress, but paused transcripts from TATA box-containing promoters remain stable, demonstrating an important role for cis-acting DNA elements in pausing. STL-seq dissects the kinetics of pause release and termination, providing an opportunity to identify mechanisms of transcriptional regulation.

Hyperosmotic stress alters the RNA polymerase II interactome and induces readthrough transcription despite widespread transcriptional repression.

Stress-induced readthrough transcription results in the synthesis of downstream-of-gene (DoG)-containing transcripts. The mechanisms underlying DoG formation during cellular stress remain unknown. Nascent transcription profiles during DoG induction in human cell lines using TT-TimeLapse sequencing revealed widespread transcriptional repression upon hyperosmotic stress. Yet, DoGs are produced regardless of the transcriptional level of their upstream genes. ChIP sequencing confirmed that stress-induced redistribution of RNA polymerase (Pol) II correlates with the transcriptional output of genes. Stress-induced alterations in the Pol II interactome are observed by mass spectrometry. While certain cleavage and polyadenylation factors remain Pol II associated, Integrator complex subunits dissociate from Pol II under stress leading to a genome-wide loss of Integrator on DNA. Depleting the catalytic subunit of Integrator using siRNAs induces hundreds of readthrough transcripts, whose parental genes partially overlap those of stress-induced DoGs. Our results provide insights into the mechanisms underlying DoG production and how Integrator activity influences DoG transcription.

p53 Activates the Long Noncoding RNA Pvt1b to Inhibit Myc and Suppress Tumorigenesis.

The tumor suppressor p53 transcriptionally activates target genes to suppress cellular proliferation during stress. p53 has also been implicated in the repression of the proto-oncogene Myc, but the mechanism has remained unclear. Here, we identify Pvt1b, a p53-dependent isoform of the long noncoding RNA (lncRNA) Pvt1, expressed 50 kb downstream of Myc, which becomes induced by DNA damage or oncogenic signaling and accumulates near its site of transcription. We show that production of the Pvt1b RNA is necessary and sufficient to suppress Myc transcription in cis without altering the chromatin organization of the locus. Inhibition of Pvt1b increases Myc levels and transcriptional activity and promotes cellular proliferation. Furthermore, Pvt1b loss accelerates tumor growth, but not tumor progression, in an autochthonous mouse model of lung cancer. These findings demonstrate that Pvt1b acts at the intersection of the p53 and Myc transcriptional networks to reinforce the anti-proliferative activities of p53.

Phosphorylation of the nuclear poly(A) binding protein (PABPN1) during mitosis protects mRNA from hyperadenylation and maintains transcriptome dynamics.

Polyadenylation controls mRNA biogenesis, nucleo-cytoplasmic export, translation and decay. These processes are interdependent and coordinately regulated by poly(A)-binding proteins (PABPs), yet how PABPs are themselves regulated is not fully understood. Here, we report the discovery that human nuclear PABPN1 is phosphorylated by mitotic kinases at four specific sites during mitosis, a time when nucleoplasm and cytoplasm mix. To understand the functional consequences of phosphorylation, we generated a panel of stable cell lines inducibly over-expressing PABPN1 with point mutations at these sites. Phospho-inhibitory mutations decreased cell proliferation, highlighting the importance of PABPN1 phosphorylation in cycling cells. Dynamic regulation of poly(A) tail length and RNA stability have emerged as important modes of gene regulation. We therefore employed long-read sequencing to determine how PABPN1 phospho-site mutants affected poly(A) tails lengths and TimeLapse-seq to monitor mRNA synthesis and decay. Widespread poly(A) tail lengthening was observed for phospho-inhibitory PABPN1 mutants. In contrast, expression of phospho-mimetic PABPN1 resulted in shorter poly(A) tails with increased non-A nucleotides, in addition to increased transcription and reduced stability of a distinct cohort of mRNAs. Taken together, PABPN1 phosphorylation remodels poly(A) tails and increases mRNA turnover, supporting the model that enhanced transcriptome dynamics reset gene expression programs across the cell cycle.

bakR: uncovering differential RNA synthesis and degradation kinetics transcriptome-wide with Bayesian hierarchical modeling.

Differential expression analysis of RNA sequencing (RNA-seq) data can identify changes in cellular RNA levels, but provides limited information about the kinetic mechanisms underlying such changes. Nucleotide recoding RNA-seq methods (NR-seq; e.g., TimeLapse-seq, SLAM-seq, etc.) address this shortcoming and are widely used approaches to identify changes in RNA synthesis and degradation kinetics. While advanced statistical models implemented in user-friendly software (e.g., DESeq2) have ensured the statistical rigor of differential expression analyses, no such tools that facilitate differential kinetic analysis with NR-seq exist. Here, we report the development of Bayesian analysis of the kinetics of RNA (bakR; https:// github.com/simonlabcode/bakR), an R package to address this need. bakR relies on Bayesian hierarchical modeling of NR-seq data to increase statistical power by sharing information across transcripts. Analyses of simulated data confirmed that bakR implementations of the hierarchical model outperform attempts to analyze differential kinetics with existing models. bakR also uncovers biological signals in real NR-seq data sets and provides improved analyses of existing data sets. This work establishes bakR as an important tool for identifying differential RNA synthesis and degradation kinetics.

Polycomb proteins remain bound to chromatin and DNA during DNA replication in vitro.

The transcriptional status of a gene can be maintained through multiple rounds of cell division during development. This epigenetic effect is believed to reflect heritable changes in chromatin folding and histone modifications or variants at target genes, but little is known about how these chromatin features are inherited through cell division. A particular challenge for maintaining transcription states is DNA replication, which disrupts or dilutes chromatin-associated proteins and histone modifications. PRC1-class Polycomb group protein complexes are essential for development and are thought to heritably silence transcription by altering chromatin folding and histone modifications. It is not known whether these complexes and their effects are maintained during DNA replication or subsequently re-established. We find that when PRC1-class Polycomb complex-bound chromatin or DNA is replicated in vitro, Polycomb complexes remain bound to replicated templates. Retention of Polycomb proteins through DNA replication may contribute to maintenance of transcriptional silencing through cell division.

Jumonji modulates polycomb activity and self-renewal versus differentiation of stem cells.

Trimethylation on histone H3 lysine 27 (H3K27me3) by Polycomb repressive complex 2 (PRC2) regulates the balance between self-renewal and differentiation of embryonic stem cells (ESCs). The mechanisms controlling the activity and recruitment of PRC2 are largely unknown. Here we demonstrate that the founding member of the Jumonji family, JMJ (JUMONJI or JARID2), is associated with PRC2, colocalizes with PRC2 and H3K27me3 on chromatin, and modulates PRC2 function. In vitro JMJ inhibits PRC2 methyltransferase activity, consistent with increased H3K27me3 marks at PRC2 targets in Jmj(-/-) ESCs. Paradoxically, JMJ is required for efficient binding of PRC2, indicating that the interplay of PRC2 and JMJ fine-tunes deposition of the H3K27me3 mark. During differentiation, activation of genes marked by H3K27me3 and lineage commitments are delayed in Jmj(-/-) ESCs. Our results demonstrate that dynamic regulation of Polycomb complex activity orchestrated by JMJ balances self-renewal and differentiation, highlighting the involvement of chromatin dynamics in cell-fate transitions.

Internally controlled RNA sequencing comparisons using nucleoside recoding chemistry.

Quantitative comparisons of RNA levels from different samples can lead to new biological understanding if they are able to distinguish biological variation from variable sample preparation. These challenges are pronounced in comparisons that require complex biochemical manipulations (e.g. isolating polysomes to study translation). Here, we present Transcript Regulation Identified by Labeling with Nucleoside Analogues in Cell Culture (TILAC), an internally controlled approach for quantitative comparisons of RNA content. TILAC uses two metabolic labels, 4-thiouridine (s4U) and 6-thioguanosine (s6G), to differentially label RNAs in cells, allowing experimental and control samples to be pooled prior to downstream biochemical manipulations. TILAC leverages nucleoside recoding chemistry to generate characteristic sequencing signatures for each label and uses statistical modeling to compare the abundance of RNA transcripts between samples. We verified the performance of TILAC in transcriptome-scale experiments involving RNA polymerase II inhibition and heat shock. We then applied TILAC to quantify changes in mRNA association with actively translating ribosomes during sodium arsenite stress and discovered a set of transcripts that are translationally upregulated, including MCM2 and DDX5. TILAC is broadly applicable to uncover differences between samples leading to improved biological insights.

m6A mRNA methylation controls T cell homeostasis by targeting the IL-7/STAT5/SOCS pathways.

N6-methyladenosine (m6A) is the most common and abundant messenger RNA modification, modulated by 'writers', 'erasers' and 'readers' of this mark. In vitro data have shown that m6A influences all fundamental aspects of mRNA metabolism, mainly mRNA stability, to determine stem cell fates. However, its in vivo physiological function in mammals and adult mammalian cells is still unknown. Here we show that the deletion of m6A 'writer' protein METTL3 in mouse T cells disrupts T cell homeostasis and differentiation. In a lymphopaenic mouse adoptive transfer model, naive Mettl3-deficient T cells failed to undergo homeostatic expansion and remained in the naive state for up to 12 weeks, thereby preventing colitis. Consistent with these observations, the mRNAs of SOCS family genes encoding the STAT signalling inhibitory proteins SOCS1, SOCS3 and CISH were marked by m6A, exhibited slower mRNA decay and showed increased mRNAs and levels of protein expression in Mettl3-deficient naive T cells. This increased SOCS family activity consequently inhibited IL-7-mediated STAT5 activation and T cell homeostatic proliferation and differentiation. We also found that m6A has important roles for inducible degradation of Socs mRNAs in response to IL-7 signalling in order to reprogram naive T cells for proliferation and differentiation. Our study elucidates for the first time, to our knowledge, the in vivo biological role of m6A modification in T-cell-mediated pathogenesis and reveals a novel mechanism of T cell homeostasis and signal-dependent induction of mRNA degradation.

Tracking Distinct RNA Populations Using Efficient and Reversible Covalent Chemistry.

We describe a chemical method to label and purify 4-thiouridine (s(4)U)-containing RNA. We demonstrate that methanethiosulfonate (MTS) reagents form disulfide bonds with s(4)U more efficiently than the commonly used HPDP-biotin, leading to higher yields and less biased enrichment. This increase in efficiency allowed us to use s(4)U labeling to study global microRNA (miRNA) turnover in proliferating cultured human cells without perturbing global miRNA levels or the miRNA processing machinery. This improved chemistry will enhance methods that depend on tracking different populations of RNA, such as 4-thiouridine tagging to study tissue-specific transcription and dynamic transcriptome analysis (DTA) to study RNA turnover.

The long noncoding RNAs NEAT1 and MALAT1 bind active chromatin sites.

Mechanistic roles for many lncRNAs are poorly understood, in part because their direct interactions with genomic loci and proteins are difficult to assess. Using a method to purify endogenous RNAs and their associated factors, we mapped the genomic binding sites for two highly expressed human lncRNAs, NEAT1 and MALAT1. We show that NEAT1 and MALAT1 localize to hundreds of genomic sites in human cells, primarily over active genes. NEAT1 and MALAT1 exhibit colocalization to many of these loci, but display distinct gene body binding patterns at these sites, suggesting independent but complementary functions for these RNAs. We also identified numerous proteins enriched by both lncRNAs, supporting complementary binding and function, in addition to unique associated proteins. Transcriptional inhibition or stimulation alters localization of NEAT1 on active chromatin sites, implying that underlying DNA sequence does not target NEAT1 to chromatin, and that localization responds to cues involved in the transcription process.

TimeLapse-seq: adding a temporal dimension to RNA sequencing through nucleoside recoding.

RNA sequencing (RNA-seq) offers a snapshot of cellular RNA populations, but not temporal information about the sequenced RNA. Here we report TimeLapse-seq, which uses oxidative-nucleophilic-aromatic substitution to convert 4-thiouridine into cytidine analogs, yielding apparent U-to-C mutations that mark new transcripts upon sequencing. TimeLapse-seq is a single-molecule approach that is adaptable to many applications and reveals RNA dynamics and induced differential expression concealed in traditional RNA-seq.

Carbodiimide reagents for the chemical probing of RNA structure in cells.

Deciphering the conformations of RNAs in their cellular environment allows identification of RNA elements with potentially functional roles within biological contexts. Insight into the conformation of RNA in cells has been achieved using chemical probes that were developed to react specifically with flexible RNA nucleotides, or the Watson-Crick face of single-stranded nucleotides. The most widely used probes are either selective SHAPE (2'-hydroxyl acylation and primer extension) reagents that probe nucleotide flexibility, or dimethyl sulfate (DMS), which probes the base-pairing at adenine and cytosine but is unable to interrogate guanine or uracil. The constitutively charged carbodiimide N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMC) is widely used for probing G and U nucleotides, but has not been established for probing RNA in cells. Here, we report the use of a smaller and conditionally charged reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), as a chemical probe of RNA conformation, and the first reagent validated for structure probing of unpaired G and U nucleotides in intact cells. We showed that EDC demonstrates similar reactivity to CMC when probing transcripts in vitro. We found that EDC specifically reacted with accessible nucleotides in the 7SK noncoding RNA in intact cells. We probed structured regions within the Xist lncRNA with EDC and integrated these data with DMS probing data. Together, EDC and DMS allowed us to refine predicted structure models for the 3' extension of repeat C within Xist. These results highlight how complementing DMS probing experiments with EDC allows the analysis of Watson-Crick base-pairing at all four nucleotides of RNAs in their cellular context.