The Δ40p53 isoform inhibits p53-dependent eRNA transcription and enables regulation by signal-specific transcription factors during p53 activation.

The naturally occurring Δ40p53 isoform heterotetramerizes with wild-type p53 (WTp53) to regulate development, aging, and stress responses. How Δ40p53 alters WTp53 function remains enigmatic because their co-expression causes tetramer heterogeneity. We circumvented this issue with a well-tested strategy that expressed Δ40p53:WTp53 as a single transcript, ensuring a 2:2 tetramer stoichiometry. Human MCF10A cell lines expressing Δ40p53:WTp53, WTp53, or WTp53:WTp53 (as controls) from the native TP53 locus were examined with transcriptomics (precision nuclear run-on sequencing [PRO-seq] and RNA sequencing [RNA-seq]), metabolomics, and other methods. Δ40p53:WTp53 was transcriptionally active, and, although phenotypically similar to WTp53 under normal conditions, it failed to induce growth arrest upon Nutlin-induced p53 activation. This occurred via Δ40p53:WTp53-dependent inhibition of enhancer RNA (eRNA) transcription and subsequent failure to induce mRNA biogenesis, despite similar genomic occupancy to WTp53. A different stimulus (5-fluorouracil [5FU]) also showed Δ40p53:WTp53-specific changes in mRNA induction; however, other transcription factors (TFs; e.g., E2F2) could then drive the response, yielding similar outcomes vs. WTp53. Our results establish that Δ40p53 tempers WTp53 function to enable compensatory responses by other stimulus-specific TFs. Such modulation of WTp53 activity may be an essential physiological function for Δ40p53. Moreover, Δ40p53:WTp53 functional distinctions uncovered herein suggest an eRNA requirement for mRNA biogenesis and that human p53 evolved as a tetramer to support eRNA transcription.

Integrated genomics approaches identify transcriptional mediators and epigenetic responses to Afghan desert particulate matter in small airway epithelial cells.

Military Deployment to Southwest Asia and Afghanistan and exposure to toxic airborne particulates have been associated with an increased risk of developing respiratory disease, collectively termed deployment-related respiratory diseases (DRRDs). Our knowledge about how particulates mediate respiratory disease is limited, precluding the appropriate recognition or management. Central to this limitation is the lack of understanding of how exposures translate into dysregulated cell identity with dysregulated transcriptional programs. The small airway epithelium is involved in both the pathobiology of DRRD and fine particulate matter deposition. To characterize small airway epithelial cell epigenetic and transcriptional responses to Afghan desert particulate matter (APM) and investigate the functional interactions of transcription factors that mediate these responses, we applied two genomics assays, the assay for transposase accessible chromatin with sequencing (ATAC-seq) and Precision Run-on sequencing (PRO-seq). We identified activity changes in a series of transcriptional pathways as candidate regulators of susceptibility to subsequent insults, including signal-dependent pathways, such as loss of cytochrome P450 or P53/P63, and lineage-determining transcription factors, such as GRHL2 loss or TEAD3 activation. We further demonstrated that TEAD3 activation was unique to APM exposure despite similar inflammatory responses when compared with wood smoke particle exposure and that P53/P63 program loss was uniquely positioned at the intersection of signal-dependent and lineage-determining transcriptional programs. Our results establish the utility of an integrated genomics approach in characterizing responses to exposures and identifying genomic targets for the advanced investigation of the pathogenesis of DRRD.

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

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.

Nascent transcript analysis of glucocorticoid crosstalk with TNF defines primary and cooperative inflammatory repression.

The glucocorticoid receptor (NR3C1, also known as GR) binds to specific DNA sequences and directly induces transcription of anti-inflammatory genes that contribute to cytokine repression, frequently in cooperation with NF-kB. Whether inflammatory repression also occurs through local interactions between GR and inflammatory gene regulatory elements has been controversial. Here, using global run-on sequencing (GRO-seq) in human airway epithelial cells, we show that glucocorticoid signaling represses transcription within 10 min. Many repressed regulatory regions reside within "hyper-ChIPable" genomic regions that are subject to dynamic, yet nonspecific, interactions with some antibodies. When this artifact was accounted for, we determined that transcriptional repression does not require local GR occupancy. Instead, widespread transcriptional induction through canonical GR binding sites is associated with reciprocal repression of distal TNF-regulated enhancers through a chromatin-dependent process, as evidenced by chromatin accessibility and motif displacement analysis. Simultaneously, transcriptional induction of key anti-inflammatory effectors is decoupled from primary repression through cooperation between GR and NF-kB at a subset of regulatory regions. Thus, glucocorticoids exert bimodal restraints on inflammation characterized by rapid primary transcriptional repression without local GR occupancy and secondary anti-inflammatory effects resulting from transcriptional cooperation between GR and NF-kB.

The MUC5B-associated variant rs35705950 resides within an enhancer subject to lineage- and disease-dependent epigenetic remodeling.

The G/T transversion rs35705950, located approximately 3 kb upstream of the MUC5B start site, is the cardinal risk factor for idiopathic pulmonary fibrosis (IPF). Here, we investigate the function and chromatin structure of this -3 kb region and provide evidence that it functions as a classically defined enhancer subject to epigenetic programming. We use nascent transcript analysis to show that RNA polymerase II loads within 10 bp of the G/T transversion site, definitively establishing enhancer function for the region. By integrating Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) analysis of fresh and cultured human airway epithelial cells with nuclease sensitivity data, we demonstrate that this region is in accessible chromatin that affects the expression of MUC5B. Through applying paired single-nucleus RNA- and ATAC-seq to frozen tissue from IPF lungs, we extend these findings directly to disease, with results indicating that epigenetic programming of the -3 kb enhancer in IPF occurs in both MUC5B-expressing and nonexpressing lineages. In aggregate, our results indicate that the MUC5B-associated variant rs35705950 resides within an enhancer that is subject to epigenetic remodeling and contributes to pathologic misexpression in IPF.

Combining signal and sequence to detect RNA polymerase initiation in ATAC-seq data.

The assay for transposase-accessible chromatin followed by sequencing (ATAC-seq) is an inexpensive protocol for measuring open chromatin regions. ATAC-seq is also relatively simple and requires fewer cells than many other high-throughput sequencing protocols. Therefore, it is tractable in numerous settings where other high throughput assays are challenging to impossible. Hence it is important to understand the limits of what can be inferred from ATAC-seq data. In this work, we leverage ATAC-seq to predict the presence of nascent transcription. Nascent transcription assays are the current gold standard for identifying regions of active transcription, including markers for functional transcription factor (TF) binding. We combine mapped short reads from ATAC-seq with the underlying peak sequence, to determine regions of active transcription genome-wide. We show that a hybrid signal/sequence representation classified using recurrent neural networks (RNNs) can identify these regions across different cell types.

Distinct amino acid compositional requirements for formation and maintenance of the [PSI⁺] prion in yeast.

Multiple yeast prions have been identified that result from the structural conversion of proteins into a self-propagating amyloid form. Amyloid-based prion activity in yeast requires a series of discrete steps. First, the prion protein must form an amyloid nucleus that can recruit and structurally convert additional soluble proteins. Subsequently, maintenance of the prion during cell division requires fragmentation of these aggregates to create new heritable propagons. For the Saccharomyces cerevisiae prion protein Sup35, these different activities are encoded by different regions of the Sup35 prion domain. An N-terminal glutamine/asparagine-rich nucleation domain is required for nucleation and fiber growth, while an adjacent oligopeptide repeat domain is largely dispensable for prion nucleation and fiber growth but is required for chaperone-dependent prion maintenance. Although prion activity of glutamine/asparagine-rich proteins is predominantly determined by amino acid composition, the nucleation and oligopeptide repeat domains of Sup35 have distinct compositional requirements. Here, we quantitatively define these compositional requirements in vivo. We show that aromatic residues strongly promote both prion formation and chaperone-dependent prion maintenance. In contrast, nonaromatic hydrophobic residues strongly promote prion formation but inhibit prion propagation. These results provide insight into why some aggregation-prone proteins are unable to propagate as prions.