Biology subjects

Kelly, J. A.

Publications and source records attributed to Kelly, J. A..

7 recordsLinked to original sources

bioRxiv2023

Variants in the DDX6-CXCR5 autoimmune disease risk locus influence the regulatory network in immune cells and salivary gland

Fine mapping and bioinformatic analysis of the DDX6-CXCR5 genetic risk association in Sjogrens Disease (SjD) and Systemic Lupus Erythematosus (SLE) identified five common SNPs with functional evidence in immune cell types: rs4938573, rs57494551, rs4938572, rs4936443, rs7117261. Functional interrogation of nuclear protein binding affinity, enhancer/promoter regulatory activity, and chromatin-chromatin interactions in immune, salivary gland epithelial, and kidney epithelial cells revealed cell type-specific allelic effects for all five SNPs that expanded regulation beyond effects on DDX6 and CXCR5 expression. Mapping the local chromatin regulatory network revealed several additional genes of interest, including lnc-PHLDB1-1. Collectively, functional characterization implicated the risk alleles of these SNPs as modulators of promoter and/or enhancer activities that regulate cell type-specific expression of DDX6, CXCR5, and lnc-PHLDB1-1, among others. Further, these findings emphasize the importance of exploring the functional significance of SNPs in the context of complex chromatin architecture in disease-relevant cell types and tissues.

genetics↗

bioRxiv2023

Comparison of the three-dimensional chromatin structures of adolescent and adult peripheral blood B cells: implications for the study of pediatric autoimmune diseases

Background/PurposeKnowledge of the 3D genome is essential to elucidate genetic mechanisms driving autoimmune diseases. The 3D genome is distinct for each cell type, and it is uncertain whether cell lines faithfully recapitulate the 3D architecture of primary human cells or whether developmental aspects of the pediatric immune system require use of pediatric samples. We undertook a systematic analysis of B cells and B cell lines to compare 3D genomic features encompassing risk loci for juvenile idiopathic arthritis (JIA), systemic lupus (SLE), and type 1 diabetes (T1D). MethodsWe isolated B cells from healthy individuals, ages 9-17. HiChIP was performed using CTCF antibody, and CTCF peaks were identified. CTCF loops within the pediatric were compared to three datasets: 1) self-called CTCF consensus peaks called within the pediatric samples, 2) ENCODEs publicly available GM12878 CTCF ChIP-seq peaks, and 3) ENCODEs primary B cell CTCF ChIP-seq peaks from two adult females. Differential looping was assessed within the pediatric samples and each of the three peak datasets. ResultsThe number of consensus peaks called in the pediatric samples was similar to that identified in ENCODEs GM12878 and primary B cell datasets. We observed <1% of loops that demonstrated significantly differential looping between peaks called within the pediatric samples themselves and when called using ENCODE GM12878 peaks. Significant looping differences were even less when comparing loops of the pediatric called peaks to those of the ENCODE primary B cell peaks. When querying loops found in juvenile idiopathic arthritis, type 1 diabetes, or systemic lupus erythematosus risk haplotypes, we observed significant differences in only 2.2%, 1.0%, and 1.3% loops, respectively, when comparing peaks called within the pediatric samples and ENCODE GM12878 dataset. The differences were even less apparent when comparing loops called with the pediatric vs ENCODE adult primary B cell peak datasets.The 3D chromatin architecture in B cells is similar across pediatric, adult, and EBVtransformed cell lines. This conservation of 3D structure includes regions encompassing autoimmune risk haplotypes. ConclusionThus, even for pediatric autoimmune diseases, publicly available adult B cell and cell line datasets may be sufficient for assessing effects exerted in the 3D genomic space.

genomics↗

bioRxiv2023

Massively Parallel Reporter Assay Confirms Regulatory Potential of hQTLs and Reveals Important Variants in Lupus and Other Autoimmune Diseases

ObjectiveTo systematically characterize the potential for histone post-translational modifications, i.e., histone quantitative trait loci (hQTLs), expression QTLs (eQTLs), and variants on systemic lupus erythematosus (SLE) and autoimmune (AI) disease risk haplotypes to modulate gene expression in an allele dependent manner. MethodsWe designed a massively parallel reporter assay (MPRA) containing [~]32K variants and transfected it into an Epstein-Barr virus transformed B cell line generated from an SLE case. ResultsOur study expands our understanding of hQTLs, illustrating that epigenetic QTLs are more likely to contribute to functional mechanisms than eQTLs and other variant types, and a large proportion of hQTLs overlap transcription start sites (TSS) of noncoding RNAs. In addition, we nominate 17 variants (including 11 novel) as putative causal variants for SLE and another 14 for various other AI diseases, prioritizing these variants for future functional studies primary and immortalized B cells. ConclusionWe uncover important insights into the mechanistic relationships between genotype, epigenetics, gene expression, and SLE and AI disease phenotypes.

genomics↗

bioRxiv2021

Small-molecule ligands can inhibit -1 programmed ribosomal frameshifting in a broad spectrum of coronaviruses

Recurrent outbreaks of novel zoonotic coronavirus (CoV) diseases since 2000 have high-lighted the importance of developing therapeutics with broad-spectrum activity against CoVs. Because all CoVs use -1 programmed ribosomal frameshifting (-1 PRF) to control expression of key viral proteins, the frameshift signal in viral mRNA that stimulates -1 PRF provides a promising potential target for such therapeutics. To test the viability of this strategy, we explored a group of 6 small-molecule ligands, evaluating their activity against the frameshift signals from a panel of representative bat CoVs--the most likely source of future zoonoses--as well as SARS-CoV-2 and MERS-CoV. We found that whereas some ligands had notable activity against only a few of the frameshift signals, the serine protease inhibitor nafamostat suppressed -1 PRF significantly in several of them, while having limited to no effect on -1 PRF caused by frameshift signals from other viruses used as negative controls. These results suggest it is possible to find small-molecule ligands that inhibit -1 PRF specifically in a broad spectrum of CoVs, establishing the frameshift signal as a viable target for developing pan-coronaviral therapeutics.

biochemistry↗

bioRxiv2020

Structural and functional conservation of the programmed -1 ribosomal frameshift signal of SARS-CoV-2

17 years after the SARS-CoV epidemic, the world is facing the COVID-19 pandemic. COVID-19 is caused by a coronavirus named SARS-CoV-2. Given the most optimistic projections estimating that it will take over a year to develop a vaccine, the best short-term strategy may lie in identifying virus-specific targets for small molecule interventions. All coronaviruses utilize a molecular mechanism called -1 PRF to control the relative expression of their proteins. Prior analyses of SARS-CoV revealed that it employs a structurally unique three-stemmed mRNA pseudoknot to stimulate high rates of -1 PRF, and that it also harbors a -1 PRF attenuation element. Altering -1 PRF activity negatively impacts virus replication, suggesting that this molecular mechanism may be therapeutically targeted. Here we present a comparative analysis of the original SARS-CoV and SARS-CoV-2 frameshift signals. Structural and functional analyses revealed that both elements promote similar rates of -1 PRF and that silent coding mutations in the slippery sites and in all three stems of the pseudoknot strongly ablated -1 PRF activity. The upstream attenuator hairpin activity has also been functionally retained. Small-angle x-ray scattering indicated that the pseudoknots in SARS-CoV and SARS-CoV-2 had the same conformation. Finally, a small molecule previously shown to bind the SARS-CoV pseudoknot and inhibit -1 PRF was similarly effective against -1 PRF in SARS-CoV-2, suggesting that such frameshift inhibitors may provide promising lead compounds to counter the current pandemic.

genetics↗

bioRxiv2018

Composition of the Survival Motor Neuron (SMN) complex in Drosophila melanogaster

Spinal Muscular Atrophy (SMA) is caused by homozygous mutations in the human survival motor neuron 1 (SMN1) gene. SMN protein has a well-characterized role in the biogenesis of small nuclear ribonucleoproteins (snRNPs), core components of the spliceosome. SMN is part of an oligomeric complex with core binding partners, collectively called Gemins. Biochemical and cell biological studies demonstrate that certain Gemins are required for proper snRNP assembly and transport. However, the precise functions of most Gemins are unknown. To gain a deeper understanding of the SMN complex in the context of metazoan evolution, we investigated the composition of the SMN complex in Drosophila melanogaster. Using a stable transgenic line that exclusively expresses Flag-tagged SMN from its native promoter, we previously found that Gemin2, Gemin3, Gemin5, and all nine classical Sm proteins, including Lsm10 and Lsm11, co-purify with SMN. Here, we show that CG2941 is also highly enriched in the pulldown. Reciprocal co-immunoprecipitation reveals that epitope-tagged CG2941 interacts with endogenous SMN in Schneider2 cells. Bioinformatic comparisons show that CG2941 shares sequence and structural similarity with metazoan Gemin4. Additional analysis shows that three other genes (CG14164, CG31950 and CG2371) are not orthologous to Gemins 6-7-8, respectively, as previously suggested. In D.melanogaster, CG2941 is located within an evolutionarily recent genomic triplication with two other nearly identical paralogous genes (CG32783 and CG32786). RNAi-mediated knockdown of CG2941 and its two close paralogs reveals that Gemin4 is essential for organismal viability.

genetics↗

bioRxiv2018

Bulked-Segregant Analysis Coupled to Whole Genome Sequencing (BSA-Seq) for Rapid Gene Cloning in Maize

Forward genetics remains a powerful method for revealing the genes underpinning organismal form and function, and for revealing how these genes are tied together in gene networks. In maize, forward genetics has been tremendously successful, but the size and complexity of the maize genome made identifying mutant genes an often arduous process with traditional methods. The next generation sequencing revolution has allowed for the gene cloning process to be significantly accelerated in many organisms, even when genomes are large and complex. Here, we describe a bulked-segregant analysis sequencing (BSA-Seq) protocol for cloning mutant genes in maize. Our simple strategy can be used to quickly identify a mapping interval and candidate single nucleotide polymorphisms (SNPs) from whole genome sequencing of pooled F2 individuals. We employed this strategy to identify narrow odd dwarf as an enhancer of teosinte branched1, and to identify a new allele of defective kernel1. Our method provides a quick, simple way to clone genes in maize.\n\nReferences numbers for supporting data:figshare and NCBI SRA links to follow

plant biology↗