A PRMT5-ZNF326 axis mediates innate immune activation upon replication stress.

DNA replication stress (RS) is a widespread phenomenon in carcinogenesis, causing genomic instability and extensive chromatin alterations. DNA damage leads to activation of innate immune signaling, but little is known about transcriptional regulators mediating such signaling upon RS. Using a chemical screen, we identified protein arginine methyltransferase 5 (PRMT5) as a key mediator of RS-dependent induction of interferon-stimulated genes (ISGs). This response is also associated with reactivation of endogenous retroviruses (ERVs). Using quantitative mass spectrometry, we identify proteins with PRMT5-dependent symmetric dimethylarginine (SDMA) modification induced upon RS. Among these, we show that PRMT5 targets and modulates the activity of ZNF326, a zinc finger protein essential for ISG response. Our data demonstrate a role for PRMT5-mediated SDMA in the context of RS-induced transcriptional induction, affecting physiological homeostasis and cancer therapy.

Circadian control of tumor immunosuppression affects efficacy of immune checkpoint blockade.

The circadian clock is a critical regulator of immunity, and this circadian control of immune modulation has an essential function in host defense and tumor immunosurveillance. Here we use a single-cell RNA sequencing approach and a genetic model of colorectal cancer to identify clock-dependent changes to the immune landscape that control the abundance of immunosuppressive cells and consequent suppression of cytotoxic CD8+ T cells. Of these immunosuppressive cell types, PD-L1-expressing myeloid-derived suppressor cells (MDSCs) peak in abundance in a rhythmic manner. Disruption of the epithelial cell clock regulates the secretion of cytokines that promote heightened inflammation, recruitment of neutrophils and the subsequent development of MDSCs. We also show that time-of-day anti-PD-L1 delivery is most effective when synchronized with the abundance of immunosuppressive MDSCs. Collectively, these data indicate that circadian gating of tumor immunosuppression informs the timing and efficacy of immune checkpoint inhibitors.

Leveraging inter-individual transcriptional correlation structure to infer discrete signaling mechanisms across metabolic tissues.

Inter-organ communication is a vital process to maintain physiologic homeostasis, and its dysregulation contributes to many human diseases. Given that circulating bioactive factors are stable in serum, occur naturally, and are easily assayed from blood, they present obvious focal molecules for therapeutic intervention and biomarker development. Recently, studies have shown that secreted proteins mediating inter-tissue signaling could be identified by 'brute force' surveys of all genes within RNA-sequencing measures across tissues within a population. Expanding on this intuition, we reasoned that parallel strategies could be used to understand how individual genes mediate signaling across metabolic tissues through correlative analyses of gene variation between individuals. Thus, comparison of quantitative levels of gene expression relationships between organs in a population could aid in understanding cross-organ signaling. Here, we surveyed gene-gene correlation structure across 18 metabolic tissues in 310 human individuals and 7 tissues in 103 diverse strains of mice fed a normal chow or high-fat/high-sucrose (HFHS) diet. Variation of genes such as FGF21, ADIPOQ, GCG, and IL6 showed enrichments which recapitulate experimental observations. Further, similar analyses were applied to explore both within-tissue signaling mechanisms (liver PCSK9) and genes encoding enzymes producing metabolites (adipose PNPLA2), where inter-individual correlation structure aligned with known roles for these critical metabolic pathways. Examination of sex hormone receptor correlations in mice highlighted the difference of tissue-specific variation in relationships with metabolic traits. We refer to this resource as gene-derived correlations across tissues (GD-CAT) where all tools and data are built into a web portal enabling users to perform these analyses without a single line of code (gdcat.org). This resource enables querying of any gene in any tissue to find correlated patterns of genes, cell types, pathways, and network architectures across metabolic organs.

The anticancer compound JTE-607 reveals hidden sequence specificity of the mRNA 3' processing machinery.

JTE-607 is an anticancer and anti-inflammatory compound and its active form, compound 2, directly binds to and inhibits CPSF73, the endonuclease for the cleavage step in pre-messenger RNA (pre-mRNA) 3' processing. Surprisingly, compound 2-mediated inhibition of pre-mRNA cleavage is sequence specific and the drug sensitivity is predominantly determined by sequences flanking the cleavage site (CS). Using massively parallel in vitro assays, we identified key sequence features that determine drug sensitivity. We trained a machine learning model that can predict poly(A) site (PAS) relative sensitivity to compound 2 and provide the molecular basis for understanding the impact of JTE-607 on PAS selection and transcription termination genome wide. We propose that CPSF73 and associated factors bind to the CS region in a sequence-dependent manner and the interaction affinity determines compound 2 sensitivity. These results have not only elucidated the mechanism of action of JTE-607, but also unveiled an evolutionarily conserved sequence specificity of the mRNA 3' processing machinery.

Nuclear RNA catabolism controls endogenous retroviruses, gene expression asymmetry, and dedifferentiation.

Endogenous retroviruses (ERVs) are remnants of ancient parasitic infections and comprise sizable portions of most genomes. Although epigenetic mechanisms silence most ERVs by generating a repressive environment that prevents their expression (heterochromatin), little is known about mechanisms silencing ERVs residing in open regions of the genome (euchromatin). This is particularly important during embryonic development, where induction and repression of distinct classes of ERVs occur in short temporal windows. Here, we demonstrate that transcription-associated RNA degradation by the nuclear RNA exosome and Integrator is a regulatory mechanism that controls the productive transcription of most genes and many ERVs involved in preimplantation development. Disrupting nuclear RNA catabolism promotes dedifferentiation to a totipotent-like state characterized by defects in RNAPII elongation and decreased expression of long genes (gene-length asymmetry). Our results indicate that RNA catabolism is a core regulatory module of gene networks that safeguards RNAPII activity, ERV expression, cell identity, and developmental potency.

Isoform-resolved transcriptome of the human preimplantation embryo.

Human preimplantation development involves extensive remodeling of RNA expression and splicing. However, its transcriptome has been compiled using short-read sequencing data, which fails to capture most full-length mRNAs. Here, we generate an isoform-resolved transcriptome of early human development by performing long- and short-read RNA sequencing on 73 embryos spanning the zygote to blastocyst stages. We identify 110,212 unannotated isoforms transcribed from known genes, including highly conserved protein-coding loci and key developmental regulators. We further identify 17,964 isoforms from 5,239 unannotated genes, which are largely non-coding, primate-specific, and highly associated with transposable elements. These isoforms are widely supported by the integration of published multi-omics datasets, including single-cell 8CLC and blastoid studies. Alternative splicing and gene co-expression network analyses further reveal that embryonic genome activation is associated with splicing disruption and transient upregulation of gene modules. Together, these findings show that the human embryo transcriptome is far more complex than currently known, and will act as a valuable resource to empower future studies exploring development.

WNTinib is a multi-kinase inhibitor with specificity against ß-catenin mutant hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide. ß-Catenin (CTNNB1)-mutated HCC represents 30% of cases of the disease with no precision therapeutics available. Using chemical libraries derived from clinical multi-kinase inhibitor (KI) scaffolds, we screened HCC organoids to identify WNTinib, a KI with exquisite selectivity in CTNNB1-mutated human and murine models, including patient samples. Multiomic and target engagement analyses, combined with rescue experiments and in vitro and in vivo efficacy studies, revealed that WNTinib is superior to clinical KIs and inhibits KIT/mitogen-activated protein kinase (MAPK) signaling at multiple nodes. Moreover, we demonstrate that reduced engagement on BRAF and p38α kinases by WNTinib relative to several multi-KIs is necessary to avoid compensatory feedback signaling-providing a durable and selective transcriptional repression of mutant ß-catenin/Wnt targets through nuclear translocation of the EZH2 transcriptional repressor. Our studies uncover a previously unknown mechanism to harness the KIT/MAPK/EZH2 pathway to potently and selectively antagonize CTNNB1-mutant HCC with an unprecedented wide therapeutic index.

Generation of host-directed and virus-specific antivirals using targeted protein degradation promoted by small molecules and viral RNA mimics.

Targeted protein degradation (TPD), as exemplified by proteolysis-targeting chimera (PROTAC), is an emerging drug discovery platform. PROTAC molecules, which typically contain a target protein ligand linked to an E3 ligase ligand, recruit a target protein to the E3 ligase to induce its ubiquitination and degradation. Here, we applied PROTAC approaches to develop broad-spectrum antivirals targeting key host factors for many viruses and virus-specific antivirals targeting unique viral proteins. For host-directed antivirals, we identified a small-molecule degrader, FM-74-103, that elicits selective degradation of human GSPT1, a translation termination factor. FM-74-103-mediated GSPT1 degradation inhibits both RNA and DNA viruses. Among virus-specific antivirals, we developed viral RNA oligonucleotide-based bifunctional molecules (Destroyers). As a proof of principle, RNA mimics of viral promoter sequences were used as heterobifunctional molecules to recruit and target influenza viral polymerase for degradation. This work highlights the broad utility of TPD to rationally design and develop next-generation antivirals.

The anti-cancer compound JTE-607 reveals hidden sequence specificity of the mRNA 3' processing machinery.

JTE-607 is a small molecule compound with anti-inflammation and anti-cancer activities. Upon entering the cell, it is hydrolyzed to Compound 2, which directly binds to and inhibits CPSF73, the endonuclease for the cleavage step in pre-mRNA 3' processing. Although CPSF73 is universally required for mRNA 3' end formation, we have unexpectedly found that Compound 2- mediated inhibition of pre-mRNA 3' processing is sequence-specific and that the sequences flanking the cleavage site (CS) are a major determinant for drug sensitivity. By using massively parallel in vitro assays, we have measured the Compound 2 sensitivities of over 260,000 sequence variants and identified key sequence features that determine drug sensitivity. A machine learning model trained on these data can predict the impact of JTE-607 on poly(A) site (PAS) selection and transcription termination genome-wide. We propose a biochemical model in which CPSF73 and other mRNA 3' processing factors bind to RNA of the CS region in a sequence-specific manner and the affinity of such interaction determines the Compound 2 sensitivity of a PAS. As the Compound 2-resistant CS sequences, characterized by U/A-rich motifs, are prevalent in PASs from yeast to human, the CS region sequence may have more fundamental functions beyond determining drug resistance. Together, our study not only characterized the mechanism of action of a compound with clinical implications, but also revealed a previously unknown and evolutionarily conserved sequence-specificity of the mRNA 3' processing machinery.

Clonally expanded CD8 T cells characterize amyotrophic lateral sclerosis-4.

Amyotrophic lateral sclerosis (ALS) is a heterogenous neurodegenerative disorder that affects motor neurons and voluntary muscle control1. ALS heterogeneity includes the age of manifestation, the rate of progression and the anatomical sites of symptom onset. Disease-causing mutations in specific genes have been identified and define different subtypes of ALS1. Although several ALS-associated genes have been shown to affect immune functions2, whether specific immune features account for ALS heterogeneity is poorly understood. Amyotrophic lateral sclerosis-4 (ALS4) is characterized by juvenile onset and slow progression3. Patients with ALS4 show motor difficulties by the time that they are in their thirties, and most of them require devices to assist with walking by their fifties. ALS4 is caused by mutations in the senataxin gene (SETX). Here, using Setx knock-in mice that carry the ALS4-causative L389S mutation, we describe an immunological signature that consists of clonally expanded, terminally differentiated effector memory (TEMRA) CD8 T cells in the central nervous system and the blood of knock-in mice. Increased frequencies of antigen-specific CD8 T cells in knock-in mice mirror the progression of motor neuron disease and correlate with anti-glioma immunity. Furthermore, bone marrow transplantation experiments indicate that the immune system has a key role in ALS4 neurodegeneration. In patients with ALS4, clonally expanded TEMRA CD8 T cells circulate in the peripheral blood. Our results provide evidence of an antigen-specific CD8 T cell response in ALS4, which could be used to unravel disease mechanisms and as a potential biomarker of disease state.

Emergency drug use in a pandemic: Harsh lessons from COVID-19.

The scientific and clinical communities have both experienced several harsh lessons on clinical care management and drug development during the COVID-19 pandemic. Here, we discuss several key lessons learned and describe a framework within which our two communities can work together and invest in to improve future pandemic responses.

A small nucleosome from a weird virus with a fat genome.

Valencia-Sánchez et al. (2021) and Liu et al. (2021) provide structural and biological insights about the existence and importance of a nucleosome-like particle in a family of giant viruses.

HNRNPM controls circRNA biogenesis and splicing fidelity to sustain cancer cell fitness.

High spliceosome activity is a dependency for cancer cells, making them more vulnerable to perturbation of the splicing machinery compared to normal cells. To identify splicing factors important for prostate cancer (PCa) fitness, we performed pooled shRNA screens in vitro and in vivo. Our screens identified heterogeneous nuclear ribonucleoprotein M (HNRNPM) as a regulator of PCa cell growth. RNA- and eCLIP-sequencing identified HNRNPM binding to transcripts of key homeostatic genes. HNRNPM binding to its targets prevents aberrant exon inclusion and backsplicing events. In both linear and circular mis-spliced transcripts, HNRNPM preferentially binds to GU-rich elements in long flanking proximal introns. Mimicry of HNRNPM-dependent linear-splicing events using splice-switching-antisense-oligonucleotides was sufficient to inhibit PCa cell growth. This suggests that PCa dependence on HNRNPM is likely a result of mis-splicing of key homeostatic coding and non-coding genes. Our results have further been confirmed in other solid tumors. Taken together, our data reveal a role for HNRNPM in supporting cancer cell fitness. Inhibition of HNRNPM activity is therefore a potential therapeutic strategy in suppressing growth of PCa and other solid tumors.

Unconventional viral gene expression mechanisms as therapeutic targets.

Unlike the human genome that comprises mostly noncoding and regulatory sequences, viruses have evolved under the constraints of maintaining a small genome size while expanding the efficiency of their coding and regulatory sequences. As a result, viruses use strategies of transcription and translation in which one or more of the steps in the conventional gene-protein production line are altered. These alternative strategies of viral gene expression (also known as gene recoding) can be uniquely brought about by dedicated viral enzymes or by co-opting host factors (known as host dependencies). Targeting these unique enzymatic activities and host factors exposes vulnerabilities of a virus and provides a paradigm for the design of novel antiviral therapies. In this Review, we describe the types and mechanisms of unconventional gene and protein expression in viruses, and provide a perspective on how future basic mechanistic work could inform translational efforts that are aimed at viral eradication.

DNMT3A haploinsufficiency causes dichotomous DNA methylation defects at enhancers in mature human immune cells.

DNMT3A encodes an enzyme that carries out de novo DNA methylation, which is essential for the acquisition of cellular identity and specialized functions during cellular differentiation. DNMT3A is the most frequently mutated gene in age-related clonal hematopoiesis. As such, mature immune cells harboring DNMT3A mutations can be readily detected in elderly persons. Most DNMT3A mutations associated with clonal hematopoiesis are heterozygous and predicted to cause loss of function, indicating that haploinsufficiency is the predominant pathogenic mechanism. Yet, the impact of DNMT3A haploinsufficiency on the function of mature immune cells is poorly understood. Here, we demonstrate that DNMT3A haploinsufficiency impairs the gain of DNA methylation at decommissioned enhancers, while simultaneously and unexpectedly impairing DNA demethylation of newly activated enhancers in mature human myeloid cells. The DNA methylation defects alter the activity of affected enhancers, leading to abnormal gene expression and impaired immune response. These findings provide insights into the mechanism of immune dysfunction associated with clonal hematopoiesis and acquired DNMT3A mutations.

Human Virus Transcriptional Regulators.

Viral genomes encode transcriptional regulators that alter the expression of viral and host genes. Despite an emerging role in human diseases, a thorough annotation of human viral transcriptional regulators (vTRs) is currently lacking, limiting our understanding of their molecular features and functions. Here, we provide a comprehensive catalog of 419 vTRs belonging to 20 different virus families. Using this catalog, we characterize shared and unique cellular genes, proteins, and pathways targeted by particular vTRs and discuss the role of vTRs in human disease pathogenesis. Our study provides a unique and valuable resource for the fields of virology, genomics, and human disease genetics.

A Quantitative Genetic Interaction Map of HIV Infection.

We have developed a platform for quantitative genetic interaction mapping using viral infectivity as a functional readout and constructed a viral host-dependency epistasis map (vE-MAP) of 356 human genes linked to HIV function, comprising >63,000 pairwise genetic perturbations. The vE-MAP provides an expansive view of the genetic dependencies underlying HIV infection and can be used to identify drug targets and study viral mutations. We found that the RNA deadenylase complex, CNOT, is a central player in the vE-MAP and show that knockout of CNOT1, 10, and 11 suppressed HIV infection in primary T cells by upregulating innate immunity pathways. This phenotype was rescued by deletion of IRF7, a transcription factor regulating interferon-stimulated genes, revealing a previously unrecognized host signaling pathway involved in HIV infection. The vE-MAP represents a generic platform that can be used to study the global effects of how different pathogens hijack and rewire the host during infection.

Comparative Flavivirus-Host Protein Interaction Mapping Reveals Mechanisms of Dengue and Zika Virus Pathogenesis.

Mosquito-borne flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), are a growing public health concern. Systems-level analysis of how flaviviruses hijack cellular processes through virus-host protein-protein interactions (PPIs) provides information about their replication and pathogenic mechanisms. We used affinity purification-mass spectrometry (AP-MS) to compare flavivirus-host interactions for two viruses (DENV and ZIKV) in two hosts (human and mosquito). Conserved virus-host PPIs revealed that the flavivirus NS5 protein suppresses interferon stimulated genes by inhibiting recruitment of the transcription complex PAF1C and that chemical modulation of SEC61 inhibits DENV and ZIKV replication in human and mosquito cells. Finally, we identified a ZIKV-specific interaction between NS4A and ANKLE2, a gene linked to hereditary microcephaly, and showed that ZIKV NS4A causes microcephaly in Drosophila in an ANKLE2-dependent manner. Thus, comparative flavivirus-host PPI mapping provides biological insights and, when coupled with in vivo models, can be used to unravel pathogenic mechanisms.