Genomics of Immune Diseases and New Therapies.

Genomic DNA sequencing technologies have been one of the great advances of the 21st century, having decreased in cost by seven orders of magnitude and opening up new fields of investigation throughout research and clinical medicine. Genomics coupled with biochemical investigation has allowed the molecular definition of a growing number of new genetic diseases that reveal new concepts of immune regulation. Also, defining the genetic pathogenesis of these diseases has led to improved diagnosis, prognosis, genetic counseling, and, most importantly, new therapies. We highlight the investigational journey from patient phenotype to treatment using the newly defined XMEN disease, caused by the genetic loss of the MAGT1 magnesium transporter, as an example. This disease illustrates how genomics yields new fundamental immunoregulatory insights as well as how research genomics is integrated into clinical immunology. At the end, we discuss two other recently described diseases, CHAI/LATAIE (CTLA-4 deficiency) and PASLI (PI3K dysregulation), as additional examples of the journey from unknown immunological diseases to new precision medicine treatments using genomics.

How One Thing Led to Another.

I started research in high school, experimenting on immunological tolerance to transplantation antigens. This led to studies of the thymus as the site of maturation of T cells, which led to the discovery, isolation, and clinical transplantation of purified hematopoietic stem cells (HSCs). The induction of immune tolerance with HSCs has led to isolation of other tissue-specific stem cells for regenerative medicine. Our studies of circulating competing germline stem cells in colonial protochordates led us to document competing HSCs. In human acute myelogenous leukemia we showed that all preleukemic mutations occur in HSCs, and determined their order; the final mutations occur in a multipotent progenitor derived from the preleukemic HSC clone. With these, we discovered that CD47 is an upregulated gene in all human cancers and is a "don't eat me" signal; blocking it with antibodies leads to cancer cell phagocytosis. CD47 is the first known gene common to all cancers and is a target for cancer immunotherapy.

Genetic manipulation of gut microbes enables single-gene interrogation in a complex microbiome.

Hundreds of microbiota genes are associated with host biology/disease. Unraveling the causal contribution of a microbiota gene to host biology remains difficult because many are encoded by nonmodel gut commensals and not genetically targetable. A general approach to identify their gene transfer methodology and build their gene manipulation tools would enable mechanistic dissections of their impact on host physiology. We developed a pipeline that identifies the gene transfer methods for multiple nonmodel microbes spanning five phyla, and we demonstrated the utility of their genetic tools by modulating microbiome-derived short-chain fatty acids and bile acids in vitro and in the host. In a proof-of-principle study, by deleting a commensal gene for bile acid synthesis in a complex microbiome, we discovered an intriguing role of this gene in regulating colon inflammation. This technology will enable genetically engineering the nonmodel gut microbiome and facilitate mechanistic dissection of microbiota-host interactions.

Receptor binding and complex structures of human ACE2 to spike RBD from omicron and delta SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic continues worldwide with many variants arising, some of which are variants of concern (VOCs). A recent VOC, omicron (B.1.1.529), which obtains a large number of mutations in the receptor-binding domain (RBD) of the spike protein, has risen to intense scientific and public attention. Here, we studied the binding properties between the human receptor ACE2 (hACE2) and the VOC RBDs and resolved the crystal and cryoelectron microscopy structures of the omicron RBD-hACE2 complex as well as the crystal structure of the delta RBD-hACE2 complex. We found that, unlike alpha, beta, and gamma, omicron RBD binds to hACE2 at a similar affinity to that of the prototype RBD, which might be due to compensation of multiple mutations for both immune escape and transmissibility. The complex structures of omicron RBD-hACE2 and delta RBD-hACE2 reveal the structural basis of how RBD-specific mutations bind to hACE2.

Tau interactome maps synaptic and mitochondrial processes associated with neurodegeneration.

Tau (MAPT) drives neuronal dysfunction in Alzheimer disease (AD) and other tauopathies. To dissect the underlying mechanisms, we combined an engineered ascorbic acid peroxidase (APEX) approach with quantitative affinity purification mass spectrometry (AP-MS) followed by proximity ligation assay (PLA) to characterize Tau interactomes modified by neuronal activity and mutations that cause frontotemporal dementia (FTD) in human induced pluripotent stem cell (iPSC)-derived neurons. We established interactions of Tau with presynaptic vesicle proteins during activity-dependent Tau secretion and mapped the Tau-binding sites to the cytosolic domains of integral synaptic vesicle proteins. We showed that FTD mutations impair bioenergetics and markedly diminished Tau's interaction with mitochondria proteins, which were downregulated in AD brains of multiple cohorts and correlated with disease severity. These multimodal and dynamic Tau interactomes with exquisite spatial resolution shed light on Tau's role in neuronal function and disease and highlight potential therapeutic targets to block Tau-mediated pathogenesis.

GSDMB is increased in IBD and regulates epithelial restitution/repair independent of pyroptosis.

Gasdermins are a family of structurally related proteins originally described for their role in pyroptosis. Gasdermin B (GSDMB) is currently the least studied, and while its association with genetic susceptibility to chronic mucosal inflammatory disorders is well established, little is known about its functional relevance during active disease states. Herein, we report increased GSDMB in inflammatory bowel disease, with single-cell analysis identifying epithelial specificity to inflamed colonocytes/crypt top colonocytes. Surprisingly, mechanistic experiments and transcriptome profiling reveal lack of inherent GSDMB-dependent pyroptosis in activated epithelial cells and organoids but instead point to increased proliferation and migration during in vitro wound closure, which arrests in GSDMB-deficient cells that display hyper-adhesiveness and enhanced formation of vinculin-based focal adhesions dependent on PDGF-A-mediated FAK phosphorylation. Importantly, carriage of disease-associated GSDMB SNPs confers functional defects, disrupting epithelial restitution/repair, which, altogether, establishes GSDMB as a critical factor for restoration of epithelial barrier function and the resolution of inflammation.

SnapShot: Tumor evolution.

Understanding how tumors grow and evolve over time is crucial to help shed light on the underlying reasons why treatments fail and tumors metastasize. This SnapShot provides a brief introduction into the main concepts of tumor evolution. To view this SnapShot, open or download the PDF.

NMR spectroscopy captures the essential role of dynamics in regulating biomolecular function.

Biomolecules are in constant motion. To understand how they function, and why malfunctions can cause disease, it is necessary to describe their three-dimensional structures in terms of dynamic conformational ensembles. Here, we demonstrate how nuclear magnetic resonance (NMR) spectroscopy provides an essential, dynamic view of structural biology that captures biomolecular motions at atomic resolution. We focus on examples that emphasize the diversity of biomolecules and biochemical applications that are amenable to NMR, such as elucidating functional dynamics in large molecular machines, characterizing transient conformations implicated in the onset of disease, and obtaining atomic-level descriptions of intrinsically disordered regions that make weak interactions involved in liquid-liquid phase separation. Finally, we discuss the pivotal role that NMR has played in driving forward our understanding of the biomolecular dynamics-function paradigm.

Hallmarks of environmental insults.

Environmental insults impair human health around the world. Contaminated air, water, soil, food, and occupational and household settings expose humans of all ages to a plethora of chemicals and environmental stressors. We propose eight hallmarks of environmental insults that jointly underpin the damaging impact of environmental exposures during the lifespan. Specifically, they include oxidative stress and inflammation, genomic alterations and mutations, epigenetic alterations, mitochondrial dysfunction, endocrine disruption, altered intercellular communication, altered microbiome communities, and impaired nervous system function. They provide a framework to understand why complex mixtures of environmental exposures induce severe health effects even at relatively modest concentrations.

Genome design of hybrid potato.

Reinventing potato from a clonally propagated tetraploid into a seed-propagated diploid, hybrid potato, is an important innovation in agriculture. Due to deleterious mutations, it has remained a challenge to develop highly homozygous inbred lines, a prerequisite to breed hybrid potato. Here, we employed genome design to develop a generation of pure and fertile potato lines and thereby the uniform, vigorous F1s. The metrics we applied in genome design included the percentage of genome homozygosity and the number of deleterious mutations in the starting material, the number of segregation distortions in the S1 population, the haplotype information to infer the break of tight linkage between beneficial and deleterious alleles, and the genome complementarity of the parental lines. This study transforms potato breeding from a slow, non-accumulative mode into a fast-iterative one, thereby potentiating a broad spectrum of benefits to farmers and consumers.

Developmental and temporal characteristics of clonal sperm mosaicism.

Throughout development and aging, human cells accumulate mutations resulting in genomic mosaicism and genetic diversity at the cellular level. Mosaic mutations present in the gonads can affect both the individual and the offspring and subsequent generations. Here, we explore patterns and temporal stability of clonal mosaic mutations in male gonads by sequencing ejaculated sperm. Through 300× whole-genome sequencing of blood and sperm from healthy men, we find each ejaculate carries on average 33.3 ± 12.1 (mean ± SD) clonal mosaic variants, nearly all of which are detected in serial sampling, with the majority absent from sampled somal tissues. Their temporal stability and mutational signature suggest origins during embryonic development from a largely immutable stem cell niche. Clonal mosaicism likely contributes a transmissible, predicted pathogenic exonic variant for 1 in 15 men, representing a life-long threat of transmission for these individuals and a significant burden on human population health.

Top-Down Control of Sweet and Bitter Taste in the Mammalian Brain.

Hardwired circuits encoding innate responses have emerged as an essential feature of the mammalian brain. Sweet and bitter evoke opposing predetermined behaviors. Sweet drives appetitive responses and consumption of energy-rich food sources, whereas bitter prevents ingestion of toxic chemicals. Here we identified and characterized the neurons in the brainstem that transmit sweet and bitter signals from the tongue to the cortex. Next we examined how the brain modulates this hardwired circuit to control taste behaviors. We dissect the basis for bitter-evoked suppression of sweet taste and show that the taste cortex and amygdala exert strong positive and negative feedback onto incoming bitter and sweet signals in the brainstem. Finally we demonstrate that blocking the feedback markedly alters responses to ethologically relevant taste stimuli. These results illustrate how hardwired circuits can be finely regulated by top-down control and reveal the neural basis of an indispensable behavioral response for all animals.

A modular master regulator landscape controls cancer transcriptional identity.

Despite considerable efforts, the mechanisms linking genomic alterations to the transcriptional identity of cancer cells remain elusive. Integrative genomic analysis, using a network-based approach, identified 407 master regulator (MR) proteins responsible for canalizing the genetics of individual samples from 20 cohorts in The Cancer Genome Atlas (TCGA) into 112 transcriptionally distinct tumor subtypes. MR proteins could be further organized into 24 pan-cancer, master regulator block modules (MRBs), each regulating key cancer hallmarks and predictive of patient outcome in multiple cohorts. Of all somatic alterations detected in each individual sample, >50% were predicted to induce aberrant MR activity, yielding insight into mechanisms linking tumor genetics and transcriptional identity and establishing non-oncogene dependencies. Genetic and pharmacological validation assays confirmed the predicted effect of upstream mutations and MR activity on downstream cellular identity and phenotype. Thus, co-analysis of mutational and gene expression profiles identified elusive subtypes and provided testable hypothesis for mechanisms mediating the effect of genetic alterations.

Enhanced prime editing systems by manipulating cellular determinants of editing outcomes.

While prime editing enables precise sequence changes in DNA, cellular determinants of prime editing remain poorly understood. Using pooled CRISPRi screens, we discovered that DNA mismatch repair (MMR) impedes prime editing and promotes undesired indel byproducts. We developed PE4 and PE5 prime editing systems in which transient expression of an engineered MMR-inhibiting protein enhances the efficiency of substitution, small insertion, and small deletion prime edits by an average 7.7-fold and 2.0-fold compared to PE2 and PE3 systems, respectively, while improving edit/indel ratios by 3.4-fold in MMR-proficient cell types. Strategic installation of silent mutations near the intended edit can enhance prime editing outcomes by evading MMR. Prime editor protein optimization resulted in a PEmax architecture that enhances editing efficacy by 2.8-fold on average in HeLa cells. These findings enrich our understanding of prime editing and establish prime editing systems that show substantial improvement across 191 edits in seven mammalian cell types.

Chromatin and gene-regulatory dynamics of the developing human cerebral cortex at single-cell resolution.

Genetic perturbations of cortical development can lead to neurodevelopmental disease, including autism spectrum disorder (ASD). To identify genomic regions crucial to corticogenesis, we mapped the activity of gene-regulatory elements generating a single-cell atlas of gene expression and chromatin accessibility both independently and jointly. This revealed waves of gene regulation by key transcription factors (TFs) across a nearly continuous differentiation trajectory, distinguished the expression programs of glial lineages, and identified lineage-determining TFs that exhibited strong correlation between linked gene-regulatory elements and expression levels. These highly connected genes adopted an active chromatin state in early differentiating cells, consistent with lineage commitment. Base-pair-resolution neural network models identified strong cell-type-specific enrichment of noncoding mutations predicted to be disruptive in a cohort of ASD individuals and identified frequently disrupted TF binding sites. This approach illustrates how cell-type-specific mapping can provide insights into the programs governing human development and disease.

Ancient and modern genomes unravel the evolutionary history of the rhinoceros family.

Only five species of the once-diverse Rhinocerotidae remain, making the reconstruction of their evolutionary history a challenge to biologists since Darwin. We sequenced genomes from five rhinoceros species (three extinct and two living), which we compared to existing data from the remaining three living species and a range of outgroups. We identify an early divergence between extant African and Eurasian lineages, resolving a key debate regarding the phylogeny of extant rhinoceroses. This early Miocene (∼16 million years ago [mya]) split post-dates the land bridge formation between the Afro-Arabian and Eurasian landmasses. Our analyses also show that while rhinoceros genomes in general exhibit low levels of genome-wide diversity, heterozygosity is lowest and inbreeding is highest in the modern species. These results suggest that while low genetic diversity is a long-term feature of the family, it has been particularly exacerbated recently, likely reflecting recent anthropogenic-driven population declines.

Cell-cycle-dependent EBNA1-DNA crosslinking promotes replication termination at oriP and viral episome maintenance.

Epstein-Barr virus (EBV) is an oncogenic human herpesvirus that persists as a multicopy episome in proliferating host cells. Episome maintenance is strictly dependent on EBNA1, a sequence-specific DNA-binding protein with no known enzymatic activities. Here, we show that EBNA1 forms a cell cycle-dependent DNA crosslink with the EBV origin of plasmid replication oriP. EBNA1 tyrosine 518 (Y518) is essential for crosslinking to oriP and functionally required for episome maintenance and generation of EBV-transformed lymphoblastoid cell lines (LCLs). Mechanistically, Y518 is required for replication fork termination at oriP in vivo and for formation of SDS-resistant complexes in vitro. EBNA1-DNA crosslinking corresponds to single-strand endonuclease activity specific to DNA structures enriched at replication-termination sites, such as 4-way junctions. These findings reveal that EBNA1 forms tyrosine-dependent DNA-protein crosslinks and single-strand cleavage at oriP required for replication termination and viral episome maintenance.

A natural single-guide RNA repurposes Cas9 to autoregulate CRISPR-Cas expression.

CRISPR-Cas systems provide prokaryotes with acquired immunity against viruses and plasmids, but how these systems are regulated to prevent autoimmunity is poorly understood. Here, we show that in the S. pyogenes CRISPR-Cas system, a long-form transactivating CRISPR RNA (tracr-L) folds into a natural single guide that directs Cas9 to transcriptionally repress its own promoter (Pcas). Further, we demonstrate that Pcas serves as a critical regulatory node. De-repression causes a dramatic 3,000-fold increase in immunization rates against viruses; however, heightened immunity comes at the cost of increased autoimmune toxicity. Using bioinformatic analyses, we provide evidence that tracrRNA-mediated autoregulation is widespread in type II-A CRISPR-Cas systems. Collectively, we unveil a new paradigm for the intrinsic regulation of CRISPR-Cas systems by natural single guides, which may facilitate the frequent horizontal transfer of these systems into new hosts that have not yet evolved their own regulatory strategies.

TDP-43 condensation properties specify its RNA-binding and regulatory repertoire.

Mutations causing amyotrophic lateral sclerosis (ALS) often affect the condensation properties of RNA-binding proteins (RBPs). However, the role of RBP condensation in the specificity and function of protein-RNA complexes remains unclear. We created a series of TDP-43 C-terminal domain (CTD) variants that exhibited a gradient of low to high condensation propensity, as observed in vitro and by nuclear mobility and foci formation. Notably, a capacity for condensation was required for efficient TDP-43 assembly on subsets of RNA-binding regions, which contain unusually long clusters of motifs of characteristic types and density. These "binding-region condensates" are promoted by homomeric CTD-driven interactions and required for efficient regulation of a subset of bound transcripts, including autoregulation of TDP-43 mRNA. We establish that RBP condensation can occur in a binding-region-specific manner to selectively modulate transcriptome-wide RNA regulation, which has implications for remodeling RNA networks in the context of signaling, disease, and evolution.

Cyclic CMP and cyclic UMP mediate bacterial immunity against phages.

The cyclic pyrimidines 3',5'-cyclic cytidine monophosphate (cCMP) and 3',5'-cyclic uridine monophosphate (cUMP) have been reported in multiple organisms and cell types. As opposed to the cyclic nucleotides 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP), which are second messenger molecules with well-established regulatory roles across all domains of life, the biological role of cyclic pyrimidines has remained unclear. Here we report that cCMP and cUMP are second messengers functioning in bacterial immunity against viruses. We discovered a family of bacterial pyrimidine cyclase enzymes that specifically synthesize cCMP and cUMP following phage infection and demonstrate that these molecules activate immune effectors that execute an antiviral response. A crystal structure of a uridylate cyclase enzyme from this family explains the molecular mechanism of selectivity for pyrimidines as cyclization substrates. Defense systems encoding pyrimidine cyclases, denoted here Pycsar (pyrimidine cyclase system for antiphage resistance), are widespread in prokaryotes. Our results assign clear biological function to cCMP and cUMP as immunity signaling molecules in bacteria.