Biology subjects

Newman, J. R. B.

Publications and source records attributed to Newman, J. R. B..

5 recordsLinked to original sources

bioRxiv2024

Manipulation of alternative splicing of IKZF1 elicits distinct gene regulatory responses in T cells

Genome-wide studies have identified significant allelic associations between genetic variants in or near the IKZF1 gene and multiple autoimmune disorders. IKZF1, encoding the transcription factor IKAROS, produces at least 10 distinct transcripts. To explore the impact of alternative splicing of IKZF1 on the function of mature T cells, we generated a panel of human T-cell clones with truncating mutations in IKZF1 exons 4, 6 or both. Differences in gene expression, chromatin accessibility, and protein abundance among clones were assessed by RNA-seq, ATAC-seq and immunoblotting. Clones with single targeting events clustered separately from double-targeted clones on multiple parameters, but overall, clone responses were highly heterogeneous. Perturbation of IKZF1 splicing resulted in significant differences in expression and chromatin accessibility of other autoimmunity-associated genes and elicited compensatory expression changes in other IKAROS family members. Our results suggest that even modest alterations of IKZF1 splicing can have significant effects on gene expression and function in mature T cells, potentially contributing to autoimmunity in susceptible individuals. Author SummaryRNA sequencing has revealed an unexpectedly large population of alternative transcripts produced by most human genes, but the functional significance of such transcripts has been debated, with some authors arguing that they represent non-functional "noise" and others arguing that they are largely functional and greatly expand the potential human proteome. Here, we explore these issues for the transcription factor IKZF1, which produces numerous alternative transcripts in human T cells and is significantly associated with risk for type 1 diabetes and other autoimmune disorders. We introduce stop codons at multiple sites in alternatively spliced exons and evaluate transcript levels and chromatin accessibility in individually targeted clones, allowing us to assay the broad impact of alternative splicing of IKZF1 in human T cells. Our studies reveal that perturbation of IKZF1 splicing results in significant differences in gene expression, chromatin accessibility and protein production of other autoimmunity-associated genes and elicits compensatory expression changes in other IKAROS family members. Even modest alterations in IKZF1 splicing had significant effects on gene expression and function in mature T cells, potentially contributing to autoimmunity in susceptible individuals and suggesting that transcript isoforms produced by alternative splicing can and do have functional impacts.

genomics↗

bioRxiv2023

Multi-omics profiling reveals ethylene signalling as a key pathway underlying both genetic and epigenetic responses to low-dose ionizing radiation in Arabidopsis

There is increasing interest in the effects of low-dose ionizing radiation (IR) on plants as might occur during spaceflight, or as a consequence of human activities, such as nuclear power generation, that may result in the release of radioactive materials into the environment. High IR doses have long been used for the induction of mutations in plants with the goal of generating desirable traits for agribusiness. Less is known about the responses of plants to acute low doses of IR exposure. Here, we take a multi-omics approach to characterize the response to low dose IR in Arabidopsis thaliana. We adapt the Methyltransferase Accessibility Protocol for individual templates (MAPit) technique for use in plants allowing us to assay the epigenetic response to acute low-dose IR (10 cGy and 100 cGy) 72 hr after exposure, and, in parallel, use RNA sequencing to profile the transcription response at 1, 3, 24 and 72 hr after exposure. IR exposures as low as 10 cGy elicit robust genetic responses in A. thaliana detectable as early as 1 hr after exposure. Further examination revealed dose-dependent changes in gene expression, chromatin accessibility and DNA methylation that implicate the ethylene signalling pathway and abiotic stress response as underlying the transcriptional and epigenetic changes associated with IR. These changes are observable up to 72 hr post-exposure, suggesting that they are maintained well after the initial acute exposure. Our findings indicate that A. thaliana executes a multi-modal response to low-dose IR through induction and regulation of the ethylene response pathway.

genomics↗

bioRxiv2023

Sex-biased expression is associated with chromatin state in D. melanogaster and D. simulans

We propose a new model for the association of chromatin state and sex-bias in expression. We hypothesize enrichment of open chromatin in the sex where we see expression bias (OS) and closed chromatin in the opposite sex (CO). In this study of D. melanogaster and D. simulans head tissue, sex-bias in expression is associated with H3K4me3 (open mark) in males for male-biased genes and in females for female-biased genes in both species. Sex-bias in expression is also largely conserved in direction and magnitude between the two species on the X and autosomes. In male-biased orthologs, the sex-bias ratio is more divergent between species if both species have H3K27me2me3 marks in females compared to when either or neither species has H3K27me2me3 in females. H3K27me2me3 marks in females are associated with male-bias in expression on the autosomes in both species, but on the X only in D. melanogaster. In female-biased orthologs the relationship between the species for the sex-bias ratio is similar regardless of the H3K27me2me3 marks in males. Female-biased orthologs are more similar in the ratio of sex-bias than male-biased orthologs and there is an excess of male-bias in expression in orthologs that gain/lose sex-bias. There is an excess of male-bias in sex-limited expression in both species suggesting excess male-bias is due to rapid evolution between the species. The X chromosome has an enrichment in male-limited H3K4me3 in both species and an enrichment of sex-bias in expression compared to the autosomes.

evolutionary biology↗

bioRxiv2022

Flap-enabled next-generation capture (FENGC): precision targeted single-molecule profiling of epigenetic heterogeneity, chromatin dynamics, and genetic variation

Targeted sequencing is an increasingly sought technology. Available methods, however, are often costly and yield high proportions of off-target reads. Here, we present FENGC, a scalable, multiplexed method in which target sequences are assembled into 5' flaps for precise excision by flap endonuclease. Recovery of length-matched sequences, amplification with universal primers, and exonucleolytic removal of non-targeted genomic regions mitigate amplification biases and consistently yield [≥] 80% on-target sequencing. Furthermore, optimized sequential reagent addition and purifications minimize sample loss and facilitate rapid processing of sub-microgram quantities of DNA for detection of genetic variants and DNA methylation. Treatment of cultured human glioblastoma cells and primary murine monocytes with GC methyltransferase followed by FENGC and high-coverage enzymatic methyl sequencing provides single-molecule, long-read detection of differential endogenous CG methylation, dynamic nucleosome repositioning, and transcription factor binding. FENGC provides a versatile and cost-effective platform for targeted sequence enrichment for analysis of genetic and/or epigenetic heterogeneity.

cancer biology↗

bioRxiv2022

Shifts in isoform usage underlie transcriptional differences in regulatory T cells in type 1 diabetes

Genome-wide association studies have identified numerous loci with allelic associations to Type 1 Diabetes (T1D) risk. Most disease-associated variants are enriched in regulatory sequences active in lymphoid cell types, suggesting that lymphocyte gene expression is altered in T1D. We assayed gene expression between T1D cases and healthy controls in two autoimmunity-relevant lymphocyte cell types, memory CD4+/CD25+ T-regulatory cells (Treg) and memory CD4+/CD25- T-cells, using a splicing event-based approach to characterize tissue-specific transcriptomes. Limited differences in isoform usage between T1D cases and controls were observed in memory CD4+/CD25- T-cells. In Tregs, 553 genes demonstrated differences in isoform usage between cases and controls, particularly RNA recognition and splicing factor genes. Many of these genes are regulated by the variable inclusion of exons that can trigger nonsense mediated decay. Our results suggest that dysregulation of gene expression, through shifts in alternative splicing in Tregs, contributes to T1D etiology.

genetics↗