Multiple causal variants underlie genetic associations in humans.
Science (New York, N.Y.)
2022; 375 (6586): 1247-1254
Associations between genetic variation and traits are often in noncoding regions with strong linkage disequilibrium (LD), where a single causal variant is assumed to underlie the association. We applied a massively parallel reporter assay (MPRA) to functionally evaluate genetic variants in high, local LD for independent cis-expression quantitative trait loci (eQTL). We found that 17.7% of eQTLs exhibit more than one major allelic effect in tight LD. The detected regulatory variants were highly and specifically enriched for activating chromatin structures and allelic transcription factor binding. Integration of MPRA profiles with eQTL/complex trait colocalizations across 114 human traits and diseases identified causal variant sets demonstrating how genetic association signals can manifest through multiple, tightly linked causal variants.
View details for DOI 10.1126/science.abj5117
View details for PubMedID 35298243
Transgenic Drosophila lines for LexA-dependent gene and growth regulation.
G3 (Bethesda, Md.)
Conditional expression of short hairpin RNAs (shRNAs) with binary genetic systems is an indispensable tool for studying gene function. Addressing mechanisms underlying cell-cell communication in vivo benefits from simultaneous use of two independent gene expression systems. To complement the abundance of existing Gal4/UAS-based resources in Drosophila, we and others have developed LexA/LexAop-based genetic tools. Here, we describe experimental and pedagogical advances that promote the efficient conversion of Drosophila Gal4 lines to LexA lines, and the generation of LexAop-shRNA lines to suppress gene function. We developed a CRISPR/Cas9-based knock-in system to replace Gal4 coding sequences with LexA, and a LexAop-based shRNA expression vector to achieve shRNA-mediated gene silencing. We demonstrate the use of these approaches to achieve targeted genetic loss-of-function in multiple tissues. We also detail our development of secondary school curricula that enable students to create transgenic flies, thereby magnifying the production of well-characterized LexA/LexAop lines for the scientific community. The genetic tools and teaching methods presented here provide LexA/LexAop resources that complement existing resources to study intercellular communication coordinating metazoan physiology and development.
View details for DOI 10.1093/g3journal/jkac018
View details for PubMedID 35100369
Dual functions of Aire CARD multimerization in the transcriptional regulation of T cell tolerance.
2020; 11 (1): 1625
Aggregate-like biomolecular assemblies are emerging as new conformational states with functionality. Aire, a transcription factor essential for central T cell tolerance, forms large aggregate-like assemblies visualized as nuclear foci. Here we demonstrate that Aire utilizes its caspase activation recruitment domain (CARD) to form filamentous homo-multimers in vitro, and this assembly mediates foci formation and transcriptional activity. However, CARD-mediated multimerization also makes Aire susceptible to interaction with promyelocytic leukemia protein (PML) bodies, sites of many nuclear processes including protein quality control of nuclear aggregates. Several loss-of-function Aire mutants, including those causing autoimmune polyendocrine syndrome type-1, form foci with increased PML body association. Directing Aire to PML bodies impairs the transcriptional activity of Aire, while dispersing PML bodies with a viral antagonist restores this activity. Our study thus reveals a new regulatory role of PML bodies in Aire function, and highlights the interplay between nuclear aggregate-like assemblies and PML-mediated protein quality control.
View details for DOI 10.1038/s41467-020-15448-w
View details for PubMedID 32242017
View details for PubMedCentralID PMC7118133
Transcriptomic signatures across human tissues identify functional rare genetic variation.
Science (New York, N.Y.)
2020; 369 (6509)
Rare genetic variants are abundant across the human genome, and identifying their function and phenotypic impact is a major challenge. Measuring aberrant gene expression has aided in identifying functional, large-effect rare variants (RVs). Here, we expanded detection of genetically driven transcriptome abnormalities by analyzing gene expression, allele-specific expression, and alternative splicing from multitissue RNA-sequencing data, and demonstrate that each signal informs unique classes of RVs. We developed Watershed, a probabilistic model that integrates multiple genomic and transcriptomic signals to predict variant function, validated these predictions in additional cohorts and through experimental assays, and used them to assess RVs in the UK Biobank, the Million Veterans Program, and the Jackson Heart Study. Our results link thousands of RVs to diverse molecular effects and provide evidence to associate RVs affecting the transcriptome with human traits.
View details for DOI 10.1126/science.aaz5900
View details for PubMedID 32913073
An origin of the immunogenicity of in vitro transcribed RNA.
Nucleic acids research
2018; 46 (10): 5239-5249
The emergence of RNA-based therapeutics demands robust and economical methods to produce RNA with few byproducts from aberrant activity. While in vitro transcription using the bacteriophage T7 RNA polymerase is one such popular method, its transcripts are known to display an immune-stimulatory activity that is often undesirable and uncontrollable. We here showed that the immune-stimulatory activity of T7 transcript is contributed by its aberrant activity to initiate transcription from a promoter-less DNA end. This activity results in the production of an antisense RNA that is fully complementary to the intended sense RNA product, and consequently a long double-stranded RNA (dsRNA) that can robustly stimulate a cytosolic pattern recognition receptor, MDA5. This promoter-independent transcriptional activity of the T7 RNA polymerase was observed for a wide range of DNA sequences and lengths, but can be suppressed by altering the transcription reaction with modified nucleotides or by reducing the Mg2+ concentration. The current work thus not only offers a previously unappreciated mechanism by which T7 transcripts stimulate the innate immune system, but also shows that the immune-stimulatory activity can be readily regulated.
View details for DOI 10.1093/nar/gky177
View details for PubMedID 29534222
View details for PubMedCentralID PMC6007322
Breaching Self-Tolerance to Alu Duplex RNA Underlies MDA5-Mediated Inflammation.
2018; 172 (4): 797-810.e13
Aberrant activation of innate immune receptors can cause a spectrum of immune disorders, such as Aicardi-Goutières syndrome (AGS). One such receptor is MDA5, a viral dsRNA sensor that induces antiviral immune response. Using a newly developed RNase-protection/RNA-seq approach, we demonstrate here that constitutive activation of MDA5 in AGS results from the loss of tolerance to cellular dsRNAs formed by Alu retroelements. While wild-type MDA5 cannot efficiently recognize Alu-dsRNAs because of its limited filament formation on imperfect duplexes, AGS variants of MDA5 display reduced sensitivity to duplex structural irregularities, assembling signaling-competent filaments on Alu-dsRNAs. Moreover, we identified an unexpected role of an RNA-rich cellular environment in suppressing aberrant MDA5 oligomerization, highlighting context dependence of self versus non-self discrimination. Overall, our work demonstrates that the increased efficiency of MDA5 in recognizing dsRNA comes at a cost of self-recognition and implicates a unique role of Alu-dsRNAs as virus-like elements that shape the primate immune system.
View details for DOI 10.1016/j.cell.2017.12.016
View details for PubMedID 29395326
View details for PubMedCentralID PMC5807104
A translational consideration of intercellular adhesion molecule-1 biology in the perioperative setting.
Translational perioperative and pain medicine
2016; 1 (2): 17-23
Intercellular adhesion molecule-1 (ICAM-1) is a critical adhesion molecule involved in leukocyte recruitment. Since its discovery in 1986, a large number of studies have been performed to elucidate its role in vitro and in vivo. Here, we review its role in leukocyte recruitment and consider future steps to take that will enhance our understanding of ICAM-1 biology and its translational application in the perioperative setting.
View details for PubMedID 27182533
View details for PubMedCentralID PMC4863998