Human cytomegalovirus extensively re-organizes the human genome, diminishing TEAD1 transcription factor activity.

Human cytomegalovirus (HCMV) infects up to 80% of the world's population. Here, we show that HCMV infection leads to widespread changes in human chromatin accessibility and chromatin looping, with hundreds of thousands of genomic regions affected 48 hours after infection. Integrative analyses reveal HCMV-induced perturbation of Hippo signaling through drastic reduction of TEAD1 transcription factor activity. We confirm extensive concordant loss of TEAD1 binding, active H3K27ac histone marks, and chromatin looping interactions upon infection. Our data position TEAD1 at the top of a hierarchy involving multiple altered important developmental pathways. HCMV infection reduces TEAD1 activity through four distinct mechanisms: closing of TEAD1-bound chromatin, reduction of YAP1 and phosphorylated YAP1 levels, reduction of TEAD1 transcript and protein levels, and alteration of TEAD1 exon-6 usage. Altered TEAD1-based mechanisms are highly enriched at genetic risk loci associated with eye and ear development, providing mechanistic insight into HCMV's established roles in these processes.

Macrophage memories of early-life injury drive neonatal nociceptive priming.

The developing peripheral nervous and immune systems are functionally distinct from those of adults. These systems are vulnerable to early-life injury, which influences outcomes related to nociception following subsequent injury later in life (i.e., "neonatal nociceptive priming"). The underpinnings of this phenomenon are unclear, although previous work indicates that macrophages are trained by inflammation and injury. Our findings show that macrophages are both necessary and partially sufficient to drive neonatal nociceptive priming, possibly due to a long-lasting remodeling in chromatin structure. The p75 neurotrophic factor receptor is an important effector in regulating neonatal nociceptive priming through modulation of the inflammatory profile of rodent and human macrophages. This "pain memory" is long lasting in females and can be transferred to a naive host to alter sex-specific pain-related behaviors. This study reveals a mechanism by which acute, neonatal post-surgical pain drives a peripheral immune-related predisposition to persistent pain following a subsequent injury.

Filaggrin loss-of-function variants are associated with atopic dermatitis phenotypes in a diverse, early-life prospective cohort.

Loss-of-function (LoF) variants in the filaggrin (FLG) gene are the strongest known genetic risk factor for atopic dermatitis (AD), but the impact of these variants on AD outcomes is poorly understood. We comprehensively identified genetic variants through targeted region sequencing of FLG in children participating in the Mechanisms of Progression of Atopic Dermatitis to Asthma in Children cohort. Twenty FLG LoF variants were identified, including 1 novel variant and 9 variants not previously associated with AD. FLG LoF variants were found in the cohort. Among these children, the presence of 1 or more FLG LoF variants was associated with moderate/severe AD compared with those with mild AD. Children with FLG LoF variants had a higher SCORing for Atopic Dermatitis (SCORAD) and higher likelihood of food allergy within the first 2.5 years of life. LoF variants were associated with higher transepidermal water loss (TEWL) in both lesional and nonlesional skin. Collectively, our study identifies established and potentially novel AD-associated FLG LoF variants and associates FLG LoF variants with higher TEWL in lesional and nonlesional skin.

Non-coding autoimmune risk variant defines role for ICOS in T peripheral helper cell development.

Fine-mapping and functional studies implicate rs117701653, a non-coding single nucleotide polymorphism in the CD28/CTLA4/ICOS locus, as a risk variant for rheumatoid arthritis and type 1 diabetes. Here, using DNA pulldown, mass spectrometry, genome editing and eQTL analysis, we establish that the disease-associated risk allele is functional, reducing affinity for the inhibitory chromosomal regulator SMCHD1 to enhance expression of inducible T-cell costimulator (ICOS) in memory CD4+ T cells from healthy donors. Higher ICOS expression is paralleled by an increase in circulating T peripheral helper (Tph) cells and, in rheumatoid arthritis patients, of blood and joint fluid Tph cells as well as circulating plasmablasts. Correspondingly, ICOS ligation and carriage of the rs117701653 risk allele accelerate T cell differentiation into CXCR5-PD-1high Tph cells producing IL-21 and CXCL13. Thus, mechanistic dissection of a functional non-coding variant in human autoimmunity discloses a previously undefined pathway through which ICOS regulates Tph development and abundance.

Shared and distinct interactions of type 1 and type 2 Epstein-Barr Nuclear Antigen 2 with the human genome.

BACKGROUND: There are two major genetic types of Epstein-Barr Virus (EBV): type 1 (EBV-1) and type 2 (EBV-2). EBV functions by manipulating gene expression in host B cells, using virus-encoded gene regulatory proteins including Epstein-Barr Nuclear Antigen 2 (EBNA2). While type 1 EBNA2 is known to interact with human transcription factors (hTFs) such as RBPJ, EBF1, and SPI1 (PU.1), type 2 EBNA2 shares only ~ 50% amino acid identity with type 1 and thus may have distinct binding partners, human genome binding locations, and functions. RESULTS: In this study, we examined genome-wide EBNA2 binding in EBV-1 and EBV-2 transformed human B cells to identify shared and unique EBNA2 interactions with the human genome, revealing thousands of type-specific EBNA2 ChIP-seq peaks. Computational predictions based on hTF motifs and subsequent ChIP-seq experiments revealed that both type 1 and 2 EBNA2 co-occupy the genome with SPI1 and AP-1 (BATF and JUNB) hTFs. However, type 1 EBNA2 showed preferential co-occupancy with EBF1, and type 2 EBNA2 preferred RBPJ. These differences in hTF co-occupancy revealed possible mechanisms underlying type-specific gene expression of known EBNA2 human target genes: MYC (shared), CXCR7 (type 1 specific), and CD21 (type 2 specific). Both type 1 and 2 EBNA2 binding events were enriched at systemic lupus erythematosus (SLE) and multiple sclerosis (MS) risk loci, while primary biliary cholangitis (PBC) risk loci were specifically enriched for type 2 peaks. CONCLUSIONS: This study reveals extensive type-specific EBNA2 interactions with the human genome, possible differences in EBNA2 interaction partners, and a possible new role for type 2 EBNA2 in autoimmune disorders. Our results highlight the importance of considering EBV type in the control of human gene expression and disease-related investigations.

Accessible chromatin maps of inflammatory bowel disease intestine nominate cell-type mediators of genetic disease risk.

Inflammatory Bowel Disease ( IBD ) is a chronic and often debilitating autoinflammatory condition, with an increasing incidence in children. Standard-of-care therapies lead to sustained transmural healing and clinical remission in fewer than one-third of patients. For children, TNFα inhibition remains the only FDA-approved biologic therapy, providing an even greater urgency to understanding mechanisms of response. Genome-wide association studies ( GWAS ) have identified 418 independent genetic risk loci contributing to IBD, yet the majority are noncoding and their mechanisms of action are difficult to decipher. If causal, they likely alter transcription factor ( TF ) binding and downstream gene expression in particular cell types and contexts. To bridge this knowledge gap, we built a novel resource: multiome-seq (tandem single-nuclei ( sn )RNA-seq and chromatin accessibility ( snATAC )-seq) of intestinal tissue from pediatric IBD patients, where anti-TNF response was defined by endoscopic healing. From the snATAC-seq data, we generated a first-time atlas of chromatin accessibility (putative regulatory elements) for diverse intestinal cell types in the context of IBD. For cell types/contexts mediating genetic risk, we reasoned that accessible chromatin will co-localize with genetic disease risk loci. We systematically tested for significant co-localization of our chromatin accessibility maps and risk variants for 758 GWAS traits. Globally, genetic risk variants for IBD, autoimmune and inflammatory diseases are enriched in accessible chromatin of immune populations, while other traits (e.g., colorectal cancer, metabolic) are enriched in epithelial and stromal populations. This resource opens new avenues to uncover the complex molecular and cellular mechanisms mediating genetic disease risk.

Broad de-regulated U2AF1 splicing is prognostic and augments leukemic transformation via protein arginine methyltransferase activation.

The role of splicing dysregulation in cancer is underscored by splicing factor mutations; however, its impact in the absence of such rare mutations is poorly understood. To reveal complex patient subtypes and putative regulators of pathogenic splicing in Acute Myeloid Leukemia (AML), we developed a new approach called OncoSplice. Among diverse new subtypes, OncoSplice identified a biphasic poor prognosis signature that partially phenocopies U2AF1-mutant splicing, impacting thousands of genes in over 40% of adult and pediatric AML cases. U2AF1-like splicing co-opted a healthy circadian splicing program, was stable over time and induced a leukemia stem cell (LSC) program. Pharmacological inhibition of the implicated U2AF1-like splicing regulator, PRMT5, rescued leukemia mis-splicing and inhibited leukemic cell growth. Genetic deletion of IRAK4, a common target of U2AF1-like and PRMT5 treated cells, blocked leukemia development in xenograft models and induced differentiation. These analyses reveal a new prognostic alternative-splicing mechanism in malignancy, independent of splicing-factor mutations.

Systematic identification of genotype-dependent enhancer variants in eosinophilic esophagitis.

Eosinophilic esophagitis (EoE) is a rare atopic disorder associated with esophageal dysfunction, including difficulty swallowing, food impaction, and inflammation, that develops in a small subset of people with food allergies. Genome-wide association studies (GWASs) have identified 9 independent EoE risk loci reaching genome-wide significance (p < 5 × 10-8) and 27 additional loci of suggestive significance (5 × 10-8 < p < 1 × 10-5). In the current study, we perform linkage disequilibrium (LD) expansion of these loci to nominate a set of 531 variants that are potentially causal. To systematically interrogate the gene regulatory activity of these variants, we designed a massively parallel reporter assay (MPRA) containing the alleles of each variant within their genomic sequence context cloned into a GFP reporter library. Analysis of reporter gene expression in TE-7, HaCaT, and Jurkat cells revealed cell-type-specific gene regulation. We identify 32 allelic enhancer variants, representing 6 genome-wide significant EoE loci and 7 suggestive EoE loci, that regulate reporter gene expression in a genotype-dependent manner in at least one cellular context. By annotating these variants with expression quantitative trait loci (eQTL) and chromatin looping data in related tissues and cell types, we identify putative target genes affected by genetic variation in individuals with EoE. Transcription factor enrichment analyses reveal possible roles for cell-type-specific regulators, including GATA3. Our approach reduces the large set of EoE-associated variants to a set of 32 with allelic regulatory activity, providing functional insights into the effects of genetic variation in this disease.

Splicing neoantigen discovery with SNAF reveals shared targets for cancer immunotherapy.

Immunotherapy has emerged as a crucial strategy to combat cancer by "reprogramming" a patient's own immune system. Although immunotherapy is typically reserved for patients with a high mutational burden, neoantigens produced from posttranscriptional regulation may provide an untapped reservoir of common immunogenic targets for new targeted therapies. To comprehensively define tumor-specific and likely immunogenic neoantigens from patient RNA-Seq, we developed Splicing Neo Antigen Finder (SNAF), an easy-to-use and open-source computational workflow to predict splicing-derived immunogenic MHC-bound peptides (T cell antigen) and unannotated transmembrane proteins with altered extracellular epitopes (B cell antigen). This workflow uses a highly accurate deep learning strategy for immunogenicity prediction (DeepImmuno) in conjunction with new algorithms to rank the tumor specificity of neoantigens (BayesTS) and to predict regulators of mis-splicing (RNA-SPRINT). T cell antigens from SNAF were frequently evidenced as HLA-presented peptides from mass spectrometry (MS) and predict response to immunotherapy in melanoma. Splicing neoantigen burden was attributed to coordinated splicing factor dysregulation. Shared splicing neoantigens were found in up to 90% of patients with melanoma, correlated to overall survival in multiple cancer cohorts, induced T cell reactivity, and were characterized by distinct cells of origin and amino acid preferences. In addition to T cell neoantigens, our B cell focused pipeline (SNAF-B) identified a new class of tumor-specific extracellular neoepitopes, which we termed ExNeoEpitopes. ExNeoEpitope full-length mRNA predictions were tumor specific and were validated using long-read isoform sequencing and in vitro transmembrane localization assays. Therefore, our systematic identification of splicing neoantigens revealed potential shared targets for therapy in heterogeneous cancers.

The transcription factor ZEB2 drives the formation of age-associated B cells.

Age-associated B cells (ABCs) accumulate during infection, aging, and autoimmunity, contributing to lupus pathogenesis. In this study, we screened for transcription factors driving ABC formation and found that zinc finger E-box binding homeobox 2 (ZEB2) is required for human and mouse ABC differentiation in vitro. ABCs are reduced in ZEB2 haploinsufficient individuals and in mice lacking Zeb2 in B cells. In mice with toll-like receptor 7 (TLR7)-driven lupus, ZEB2 is essential for ABC formation and autoimmune pathology. ZEB2 binds to +20-kb myocyte enhancer factor 2b (Mef2b)'s intronic enhancer, repressing MEF2B-mediated germinal center B cell differentiation and promoting ABC formation. ZEB2 also targets genes important for ABC specification and function, including Itgax. ZEB2-driven ABC differentiation requires JAK-STAT (Janus kinase-signal transducer and activator of transcription), and treatment with JAK1/3 inhibitor reduces ABC accumulation in autoimmune mice and patients. Thus, ZEB2 emerges as a driver of B cell autoimmunity.

High-throughput identification of functional regulatory SNPs in systemic lupus erythematosus.

Genome-wide association studies implicate multiple loci in risk for systemic lupus erythematosus (SLE), but few contain exonic variants, rendering systematic identification of non-coding variants essential to decoding SLE genetics. We utilized SNP-seq and bioinformatic enrichment to interrogate 2180 single-nucleotide polymorphisms (SNPs) from 87 SLE risk loci for potential binding of transcription factors and related proteins from B cells. 52 SNPs that passed initial screening were tested by electrophoretic mobility shift and luciferase reporter assays. To validate the approach, we studied rs2297550 in detail, finding that the risk allele enhanced binding to the transcription factor Ikaros (IKZF1), thereby modulating expression of IKBKE. Correspondingly, primary cells from genotyped healthy donors bearing the risk allele expressed higher levels of the interferon / NF-κB regulator IKKϵ. Together, these findings define a set of likely functional non-coding lupus risk variants and identify a new regulatory pathway involving rs2297550, Ikaros, and IKKϵ implicated by human genetics in risk for SLE.

Identification of a regulatory pathway governing TRAF1 via an arthritis-associated non-coding variant.

TRAF1/C5 was among the first loci shown to confer risk for inflammatory arthritis in the absence of an associated coding variant, but its genetic mechanism remains undefined. Using Immunochip data from 3,939 patients with juvenile idiopathic arthritis (JIA) and 14,412 control individuals, we identified 132 plausible common non-coding variants, reduced serially by single-nucleotide polymorphism sequencing (SNP-seq), electrophoretic mobility shift, and luciferase studies to the single variant rs7034653 in the third intron of TRAF1. Genetically manipulated experimental cells and primary monocytes from genotyped donors establish that the risk G allele reduces binding of Fos-related antigen 2 (FRA2), encoded by FOSL2, resulting in reduced TRAF1 expression and enhanced tumor necrosis factor (TNF) production. Conditioning on this JIA variant eliminated attributable risk for rheumatoid arthritis, implicating a mechanism shared across the arthritis spectrum. These findings reveal that rs7034653, FRA2, and TRAF1 mediate a pathway through which a non-coding functional variant drives risk of inflammatory arthritis in children and adults.

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.

Effector memory T cells induce innate inflammation by triggering DNA damage and a non-canonical STING pathway in dendritic cells.

Cognate interaction between CD4+ effector memory T (TEM) cells and dendritic cells (DCs) induces innate inflammatory cytokine production, resulting in detrimental autoimmune pathology and cytokine storms. While TEM cells use tumor necrosis factor (TNF) superfamily ligands to activate DCs, whether TEM cells prompt other DC-intrinsic changes that influence the innate inflammatory response has never been investigated. We report the surprising discovery that TEM cells trigger double-strand DNA breaks via mitochondrial reactive oxygen species (ROS) production in interacting DCs. Initiation of the DNA damage response in DCs induces activation of a cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS)-independent, non-canonical stimulator of interferon genes (STING)-TNF receptor-associated factor 6 (TRAF6)-nuclear factor κB (NF-κB) signaling axis. Consequently, STING-deficient DCs display reduced NF-κB activation and subsequent defects in transcriptional induction and functional production of interleukin-1ß (IL-1ß) and IL-6 following their interaction with TEM cells. The discovery of TEM cell-induced innate inflammation through DNA damage and a non-canonical STING-NF-κB pathway presents this pathway as a potential target to alleviate T cell-driven inflammation in autoimmunity and cytokine storms.

Lineage tracing of newly accrued nuclei in skeletal myofibers uncovers distinct transcripts and interplay between nuclear populations.

Multinucleated skeletal muscle cells have an obligatory need to acquire additional nuclei through fusion with activated skeletal muscle stem cells when responding to both developmental and adaptive growth stimuli. A fundamental question in skeletal muscle biology has been the reason underlying this need for new nuclei in syncytial cells that already harbor hundreds of nuclei. To begin to answer this long-standing question, we utilized nuclear RNA-sequencing approaches and developed a lineage tracing strategy capable of defining the transcriptional state of recently fused nuclei and distinguishing this state from that of pre-existing nuclei. Our findings reveal the presence of conserved markers of newly fused nuclei both during development and after a hypertrophic stimulus in the adult. However, newly fused nuclei also exhibit divergent gene expression that is determined by the myogenic environment to which they fuse. Moreover, accrual of new nuclei through fusion is required for nuclei already resident in adult myofibers to mount a normal transcriptional response to a load-inducing stimulus. We propose a model of mutual regulation in the control of skeletal muscle development and adaptations, where newly fused and pre-existing myonuclear populations influence each other to maintain optimal functional growth.

After the Infection: A Survey of Pathogens and Non-communicable Human Disease.

There are many well-established relationships between pathogens and human disease, but far fewer when focusing on non-communicable diseases (NCDs). We leverage data from The UK Biobank and TriNetX to perform a systematic survey across 20 pathogens and 426 diseases, focused primarily on NCDs. To this end, we assess the association between disease status and infection history proxies. We identify 206 pathogen-disease pairs that replicate in both cohorts. We replicate many established relationships, including Helicobacter pylori with several gastroenterological diseases, and connections between Epstein-Barr virus with multiple sclerosis and lupus. Overall, our approach identified evidence of association for 15 of the pathogens and 96 distinct diseases, including a currently controversial link between human cytomegalovirus (CMV) and ulcerative colitis (UC). We validate this connection through two orthogonal analyses, revealing increased CMV gene expression in UC patients and enrichment for UC genetic risk signal near human genes that have altered expression upon CMV infection. Collectively, these results form a foundation for future investigations into mechanistic roles played by pathogens in disease.

VExD: a curated resource for human gene expression alterations following viral infection.

Much of the host antiviral response is mediated through changes to host gene expression levels. Likewise, viruses induce changes to host gene expression levels in order to promote the viral life cycle and evade the host immune system. However, there is no resource that specifically collects human gene expression levels pre- and post-virus infection. Further, public gene expression repositories do not contain enough specialized metadata to easily find relevant experiments. Here, we present the Virus Expression Database (VExD), a freely available website and database, that collects human gene expression datasets in response to viral infection. VExD contains ∼8,000 uniformly processed samples obtained from 289 studies examining 51 distinct human viruses. We show that the VExD processing pipeline captures known antiviral responses in the form of interferon-stimulated genes. We further show that the datasets collected in VExD can be used to quickly identify supporting data for experiments performed in human cells or model organisms. VExD is freely available at https://vexd.cchmc.org/.

A functional mechanism for a non-coding variant near AGTR2 associated with risk for preterm birth.

BACKGROUND: Preterm birth (PTB), defined as delivery before 37 gestational weeks, imposes significant public health burdens. A recent maternal genome-wide association study of spontaneous PTB identified a noncoding locus near the angiotensin II receptor type 2 (AGTR2) gene. Genotype-Tissue Expression data revealed that alleles associated with decreased AGTR2 expression in the uterus were linked to an increased risk of PTB and shortened gestational duration. We hypothesized that a causative variant in this locus modifies AGTR2 expression by altering transcription factor (TF) binding. METHODS: To investigate this hypothesis, we performed bioinformatics analyses and functional characterizations at the implicated locus. Potential causal single nucleotide polymorphisms (SNPs) were prioritized, and allele-dependent binding of TFs was predicted. Reporter assays were employed to assess the enhancer activity of the top PTB-associated non-coding variant, rs7889204, and its impact on TF binding. RESULTS: Our analyses revealed that rs7889204, a top PTB-associated non-coding genetic variant is one of the strongest eQTLs for the AGTR2 gene in uterine tissue samples. We observed differential binding of CEBPB (CCAAT enhancer binding protein beta) and HOXA10 (homeobox A10) to the alleles of rs7889204. Reporter assays demonstrated decreased enhancer activity for the rs7889204 risk "C" allele. CONCLUSION: Collectively, these results demonstrate that decreased AGTR2 expression caused by reduced transcription factor binding increases the risk for PTB and suggest that enhancing AGTR2 activity may be a preventative measure in reducing PTB risk.

Single cell transcriptomic analysis of HPV16-infected epithelium identifies a keratinocyte subpopulation implicated in cancer.

Persistent HPV16 infection is a major cause of the global cancer burden. The viral life cycle is dependent on the differentiation program of stratified squamous epithelium, but the landscape of keratinocyte subpopulations which support distinct phases of the viral life cycle has yet to be elucidated. Here, single cell RNA sequencing of HPV16 infected compared to uninfected organoids identifies twelve distinct keratinocyte populations, with a subset mapped to reconstruct their respective 3D geography in stratified squamous epithelium. Instead of conventional terminally differentiated cells, an HPV-reprogrammed keratinocyte subpopulation (HIDDEN cells) forms the surface compartment and requires overexpression of the ELF3/ESE-1 transcription factor. HIDDEN cells are detected throughout stages of human carcinogenesis including primary human cervical intraepithelial neoplasias and HPV positive head and neck cancers, and a possible role in promoting viral carcinogenesis is supported by TCGA analyses. Single cell transcriptome information on HPV-infected versus uninfected epithelium will enable broader studies of the role of individual keratinocyte subpopulations in tumor virus infection and cancer evolution.