Biology subjects

Zacharewski, T. R.

Publications and source records attributed to Zacharewski, T. R..

4 recordsLinked to original sources

bioRxiv2021

Benchmarking of a Bayesian single cell RNAseq differential gene expression test for dose-response study designs.

The application of single-cell RNA sequencing (scRNAseq) for the evaluation of chemicals, drugs, and food contaminants presents the opportunity to consider cellular heterogeneity in pharmacological and toxicological responses. Current differential gene expression analysis (DGEA) methods focus primarily on two group comparisons, not multi-group dose-response study designs used in safety assessments. To benchmark DGEA methods for dose-response scRNAseq experiments, we proposed a multiplicity corrected Bayesian testing approach and compare it against 8 other methods including two frequentist fit-for-purpose tests using simulated and experimental data. Our Bayesian test method outperformed all other tests for a broad range of accuracy metrics including control of false positive error rates. Most notable, the fit-for-purpose and standard multiple group DGEA methods were superior to the two group scRNAseq methods for dose-response study designs. Collectively, our benchmarking of DGEA methods demonstrates the importance in considering study design when determining the most appropriate test methods.

genomics↗

bioRxiv2021

Genome-wide ChIPseq analysis of AhR, COUP-TF, and HNF4 enrichment in TCDD-treated mouse liver

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor known for mediating the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Although the canonical mechanism of AhR activation involves heterodimerization with the aryl hydrocarbon receptor nuclear translocator, other transcriptional regulators that interact with AhR have been identified. Enrichment analysis of motifs in AhR-bound genomic regions implicated co-operation with COUP transcription factor (COUP-TF) and hepatocyte nuclear factor 4 (HNF4). The present study investigated AhR, HNF4 and COUP-TFII genomic binding and effects on gene expression associated with liver-specific function and cell differentiation in response to TCDD. Hepatic ChIPseq data from male C57BL/6 mice at 2 hrs after oral gavage with 30 {micro}g/kg TCDD were integrated with bulk RNA-sequencing (RNAseq) time-course (2 - 72 hrs) and dose-response (0.01 - 30 {micro}g/kg) datasets to assess putative AhR, HNF4 and COUP-TFII interactions associated with differential gene expression. TCDD treatment resulted in the genomic enrichment of 23,701, 11,688, and 9,547 binding regions for AhR, COUP-TFII and HNF4, respectively, throughout the genome. Functional enrichment analysis of differentially expressed genes (DEGs) identified differential binding enrichment for AhR, COUP-TFII, and HNF4a to regions within liver-specific genes suggesting intersections associated with the loss of liver-specific functions and hepatocyte differentiation. Analysis found that the repression of liver-specific, HNF4 target and hepatocyte differentiation genes, involved increased AhR and HNF4 binding with decreased COUP-TFII binding. Collectively, these results suggested TCDD-elicited loss of liver-specific functions and markers of hepatocyte differentiation involved interactions between AhR, COUP-TFII and HNF4.

pharmacology and toxicology↗

bioRxiv2021

Thioesterase induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin results in a futile cycle that inhibits hepatic β-oxidation

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a persistent environmental contaminant, induces steatosis by increasing hepatic uptake of dietary and mobilized peripheral fats, inhibiting lipoprotein export, and repressing {beta}-oxidation. In this study, the mechanism of {beta}-oxidation inhibition was investigated by testing the hypothesis that TCDD dose-dependently repressed straight-chain fatty acid oxidation gene expression in mice following oral gavage every 4 days for 28 days. Untargeted metabolomic analysis revealed a dose-dependent decrease in hepatic acyl-CoA levels, while octenoyl-CoA and dicarboxylic acid levels increased. TCDD also dose-dependently repressed the hepatic gene expression associated with triacylglycerol and cholesterol ester hydrolysis, fatty acid binding proteins, fatty acid activation, and 3-ketoacyl-CoA thiolysis while inducing acyl-CoA hydrolysis. Moreover, octenoyl-CoA blocked the hydration of crotonyl-CoA suggesting short chain enoyl-CoA hydratase (ECHS1) activity was inhibited. Collectively, the integration of metabolomics and RNA-seq data suggested TCDD induced a futile cycle of fatty acid activation and acyl-CoA hydrolysis resulting in incomplete {beta}-oxidation, and the accumulation octenoyl-CoA levels that inhibited the activity of short chain enoyl-CoA hydratase (ECHS1).

pharmacology and toxicology↗

bioRxiv2018

Gene Coregulation and Coexpression in the Aryl Hydrocarbon Receptor-mediated Transcriptional Regulatory Network in the Mouse Liver

Tissue-specific network models of chemical-induced gene perturbation can improve our mechanistic understanding of the intracellular events leading to adverse health effects resulting from chemical exposure. The aryl hydrocarbon receptor (AHR) is a ligand-inducible transcription factor (TF) that activates a battery of genes and produces a variety of species-specific adverse effects in response to the potent and persistent environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Here we assemble a global map of the AHR gene regulatory network in TCDD-treated mouse liver from a combination of previously published gene expression and genome-wide TF binding data sets. Using Kohonen selforganizing maps and subspace clustering, we show that genes co-regulated by common upstream TFs in the AHR network exhibit a pattern of co-expression. Specifically, directly-bound, indirectly-bound and non-genomic AHR target genes exhibit distinct patterns of gene expression, with the directly bound targets generally associated with highest median expression. Further, among the directly bound AHR target genes, the expression level increases with the number of AHR binding sites in the proximal promoter regions. Finally, we show that co-regulated genes in the AHR network activate distinct groups of downstream biological processes, with AHR-bound target genes enriched for metabolic processes and enrichment of immune responses among AHR-unbound target genes, likely reflecting infiltration of immune cells into the mouse liver upon TCDD treatment. This work describes an approach to the reconstruction and analysis of transcriptional regulatory cascades underlying cellular stress response using bioinformatic and statistical tools.

genomics↗