Deconvolution of multiplexed transcriptional responses to wood smoke particles defines rapid aryl hydrocarbon receptor signaling dynamics

Abstract

The heterogeneity of respirable particulates and compounds complicates our understanding of transcriptional responses to air pollution. Here, we address this by applying precision nuclear run-on sequencing and the assay for transposase-accessible chromatin sequencing to measure nascent transcription and chromatin accessibility in airway epithelial cells after wood smoke particle (WSP) exposure. We used transcription factor enrichment analysis to identify temporally distinct roles for ternary response factor–serum response factor complexes, the aryl hydrocarbon receptor (AHR), and NFκB in regulating transcriptional changes induced by WSP. Transcription of canonical targets of the AHR, such as CYP1A1 and AHRR , was robustly increased after just 30 min of WSP exposure, and we discovered novel AHR-regulated pathways and targets including the DNA methyltransferase, DNMT3L . Transcription of these genes and associated enhancers rapidly returned to near baseline by 120 min after exposure. The kinetics of AHR- and NFκB-regulated responses to WSP were distinguishable based on the timing of both transcriptional responses and chromatin remodeling, with induction of several cytokines implicated in maintaining NFκB-mediated responses through 120 min of exposure. In aggregate, our data establish a direct and primary role for AHR in mediating airway epithelial responses to WSP and identify crosstalk between AHR and NFκB signaling in controlling proinflammatory gene expression. This work also defines an integrated genomics-based strategy for deconvoluting multiplexed transcriptional responses to heterogeneous environmental exposures.

Integrated Genomics Approaches Identify Transcriptional Mediators and Epigenetic Responses to Afghan Desert Particulate Matter in Small Airway Epithelial Cells

“…We previously applied a combination of nascent transcript sequencing using Precision Run-on sequencing (PRO-seq) and chromatin profiling using the assay for transposase accessible chromatin with sequencing (ATAC-seq) to define the mechanistic basis for the immediate early effects of WSP on airway epithelial cells. Our data indicated that acute exposure to particulate matter under highly controlled experimental conditions results in broad transcriptional and epigenetic responses (17), including induction of NFkB activity. Furthermore, PRO-seq provides an indirect measure of the activity of transcriptional regulatory elements (TREs) based on associated enhancer RNA levels.…”

“…We first employed PRO-seq to characterize gene transcriptional responses to APM in SAECs. Our previous study demonstrated marked temporal variation in transcriptional responses within the first two hours of particulate exposure (26). Therefore, we anticipated nascent transcription after 20 hours of exposure would represent a steady-state response to APM.…”

“…Wood Smoke Particles (WSP) were generously gifted by Dr. Andrew Ghio at the Environmental Protection Agency Laboratory in North Carolina and generated as described previously (8). WSP composition, preparation and exposure were described previously by our laboratory (17). The mean diameter of the freshly generated wood smoke particles was 0.14 um.…”

“…TDF coverage files output by the pipeline, normalized by reads per million mapped, were visualized using the Integrative Genomics Viewer (IGV) genome browser (v. 2.8.0). FStitch (v. 1.0) and Tfit (v. 1.0) were used to identify regions with bidirectional transcriptional activity as described (17). Reference Sequences for hg38 were downloaded from the UCSC genome browser (May 18, 2018).…”

“…RNA purification and qRT-PCR was performed as described previously (17). Briefly, SAECs were grown to confluence in 12-well tissue culture dishes and treated with vehicle (PBS) or APM for 20 hours.…”