Genomic determination of the glucocorticoid response reveals unexpected mechanisms of gene regulation

Reddy, Timothy E.; Pauli, Florencia; Sprouse, Rebekka O.; Neff, Norma; Newberry, Kimberly M.; Garabedian, Michael J.; Myers, R

doi:10.1101/gr.097022.109

Cited by 481 publications

(564 citation statements)

References 44 publications

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“…To test the feasibility of the approach, we monitored the dynamics of intron and exon levels of single genes under well-characterized transcriptional and post-transcriptional perturbations. We first calculated expression changes of glucocorticoid receptor target genes upon stimulation of human A549 cells with glucocorticoid dexamethasone 27 . From a set of genes shown previously to be transcriptionally induced in a receptor-dependent manner 27 , we selected a subset of five genes and determined their RNA levels by counting either reads mapping to exons (which is the common approach) or only reads mapping to introns.…”

Section: Quantifying Expression Changes In Exons and Intronsmentioning

confidence: 99%

Analysis of intronic and exonic reads in RNA-seq data characterizes transcriptional and post-transcriptional regulation

et al. 2015

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A n A ly s i s RNA-seq experiments generate reads derived not only from mature RNA transcripts but also from pre-mRNA. Here we present a computational approach called exon-intron split analysis (EISA) that measures changes in mature RNA and pre-mRNA reads across different experimental conditions to quantify transcriptional and post-transcriptional regulation of gene expression. We apply EISA to 17 diverse data sets to show that most intronic reads arise from nuclear RNA and changes in intronic read counts accurately predict changes in transcriptional activity. Furthermore, changes in posttranscriptional regulation can be predicted from differences between exonic and intronic changes. EISA reveals both transcriptional and post-transcriptional contributions to expression changes, increasing the amount of information that can be gained from RNA-seq data sets.Cellular RNAs are regulated at multiple stages, including transcription, RNA maturation and degradation. Several analytic methods have been developed to measure these processes on a transcriptomewide scale. For example, global run-on sequencing (GRO-seq) 1 uses incorporation of a nucleotide analog to enrich for nascent RNA. In Nascent-seq 2,3 , newly transcribed RNAs are isolated by purification of their complex with proteins and the DNA template. Cellular fractionation techniques 4 have also been adapted to measure nascent transcripts, which are enriched in the nucleus. mRNA half-lives have been determined, for example, by blockage of transcription followed by transcriptional profiling 5 . RNA sequencing, the most widely used method for transcriptome analysis, has been applied in numerous studies to determine steady-state mRNA levels 6,7 and alternative splicing events 8 and to identify previously unknown transcripts and noncoding RNAs 9-11 . In general, these protocols aim to enrich for mature mRNA by selection of polyadenylated RNA or by depletion of ribosomal RNA.Many computational methods (reviewed in refs. 12,13) have been developed for the analysis of RNA-seq data, to enable spliced alignment 14,15 , transcript assembly 16,17 , transcript quantification 14,18 and differential expression analysis [19][20][21] . Although RNA-seq mostly generates reads that map to exons, it also captures less abundant intronic sequences 6 . However, their interpretation has remained controversial. Some have suggested that they originate from DNA contamination and can thus be used as a quality metric for RNA-seq data 22 (see also RNA-seq guidelines of the Roadmap Epigenomics Consortium, http://www.roadmapepigenomics.org/), whereas others have hypothesized that they stem from unknown exons or intronic enhancers 6,7 . In a study based on exon arrays, probes mapping to introns were used to investigate pre-mRNA dynamics 23 . Three recent studies based on RNA-seq provided evidence that intronic reads might correlate with transcriptional activity. In two of these, the read coverage along introns was related to nascent transcription in combination with co-transcriptional splicing e...

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Section: Quantifying Expression Changes In Exons and Intronsmentioning

confidence: 99%

Analysis of intronic and exonic reads in RNA-seq data characterizes transcriptional and post-transcriptional regulation

et al. 2015

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“…We focused our attention on the intron-11 nGRE, because this site was conserved across species and resided within a ∼800-bp region of DNA that was recently identified by ChIP-seq analysis as the only significant region of GR binding in the β-arrestin-2 gene (Fig. 4A) (18). We performed a ChIP assay on A549 cells treated with Dex for 90 min to verify recruitment of GRs to this intron-11 nGRE.…”

Section: Resultsmentioning

confidence: 99%

Glucocorticoids regulate arrestin gene expression and redirect the signaling profile of G protein-coupled receptors

Oakley

Revollo

Cidlowski

2012

Proc. Natl. Acad. Sci. U.S.A.

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G protein-coupled receptors (GPCRs) compose the largest family of cell surface receptors and are the most common target of therapeutic drugs. The nonvisual arrestins, β-arrestin-1 and β-arrestin-2, are multifunctional scaffolding proteins that play critical roles in GPCR signaling. On binding of activated GPCRs at the plasma membrane, β-arrestins terminate G protein-dependent responses (desensitization) and stimulate β-arrestin-dependent signaling pathways. Alterations in the cellular complement of β-arrestin-1 and β-arrestin-2 occur in many human diseases, and their genetic ablation in mice has severe consequences. Surprisingly, however, the factors that control β-arrestin gene expression are poorly understood. We demonstrate that glucocorticoids differentially regulate β-arrestin-1 and β-arrestin-2 gene expression in multiple cell types. Glucocorticoids act via the glucocorticoid receptor (GR) to induce the synthesis of β-arrestin-1 and repress the expression of β-arrestin-2. Glucocorticoid-dependent regulation involves the recruitment of ligand-activated glucocorticoid receptors to conserved and functional glucocorticoid response elements in intron-1 of the β-arrestin-1 gene and intron-11 of the β-arrestin-2 gene. In human lung adenocarcinoma cells, the increased expression of β-arrestin-1 after glucocorticoid treatment impairs G protein-dependent activation of inositol phosphate signaling while enhancing β-arrestin-1-dependent stimulation of the MAPK pathway by protease activated receptor 1. These studies demonstrate that glucocorticoids redirect the signaling profile of GPCRs via alterations in β-arrestin gene expression, revealing a paradigm for cross-talk between nuclear and cell surface receptors and a mechanism by which glucocorticoids alter the clinical efficacy of GPCR-based drugs.

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“…This suggests that a fourth class of GRregulated genes exist, possibly overlapping with GABP target genes. ChIP-seq analysis of GR binding sites in A549 lung cells has revealed some 2,600 genes that are bound by unliganded GR, though with a much lower signal than liganded GR targets (50), suggesting this may be a widespread mechanism for gene regulation. The regulation of basal expression of target genes by unliganded GR suggests that in some contexts the endogenous levels of GR may be significant for gene regulation.…”

Section: -Hc +Hcmentioning

confidence: 99%

The Unliganded Glucocorticoid Receptor Positively Regulates the Tumor Suppressor GeneBRCA1through GABP Beta

Ritter

Antonova

Mueller

2012

Molecular Cancer Research

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Loss of BRCA1 tumor suppressor function is a critical event in breast tumorigenesis. We have previously identified the stress hormone hydrocortisone as a negative regulator of BRCA1 expression in nonmalignant mammary cells. Here, we have identified a direct role for the unliganded glucocorticoid receptor (GR) in BRCA1 upregulation in the absence of hydrocortisone. The positive regulatory effect of GR is lost upon the addition of hydrocortisone. We have shown that GR interacts with the BRCA1 promoter only in the absence of hydrocortisone, and that this interaction is mediated through the b-subunit of the ets transcription factor GA-binding protein (GABP) at the RIBS promoter element. GR and GABPb interact in both coimmunoprecipitation and mammalian two-hybrid assays, and this interaction involves the N-terminal to central regions of both proteins. This work presents the first evidence of a ligand-independent role for GR as a positive regulator of gene expression, and loss of GR from the BRCA1 promoter in response to stress hormones leads to decreased BRCA1 expression. Because low levels of BRCA1 have been implicated in the development of sporadic breast cancer, this may represent a novel mechanism through which prolonged stress signaling increases breast cancer risk. Mol Cancer Res; 10(4); 558-69. Ó2012 AACR. IntroductionGerm line mutations in the BRCA1 tumor suppressor contribute to familial breast tumor formation but BRCA1 mutations do not seem to occur in sporadic breast cancer tumors (1). Instead, sporadic breast tumors exhibit decreased levels of BRCA1 expression, and the degree of downregulation seems to be correlated with tumor grade, rate of tumor progression, and risk of metastasis (2-6). This implies that loss of BRCA1 function, either through decreased activity or downregulation of expression, is a critical event in the etiology of breast cancer. Phenotypically normal breast epithelial cells from individuals harboring a BRCA1 germ line mutation (a so called "one-hit" mutation accompanied by a 50% decrease in BRCA1 function) express an altered mRNA profile compared with normal cells from individuals without a BRCA1 mutation (7). This suggests that decreases in functional BRCA1

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Genomic determination of the glucocorticoid response reveals unexpected mechanisms of gene regulation

Cited by 481 publications

References 44 publications

Analysis of intronic and exonic reads in RNA-seq data characterizes transcriptional and post-transcriptional regulation

Analysis of intronic and exonic reads in RNA-seq data characterizes transcriptional and post-transcriptional regulation

Glucocorticoids regulate arrestin gene expression and redirect the signaling profile of G protein-coupled receptors

The Unliganded Glucocorticoid Receptor Positively Regulates the Tumor Suppressor GeneBRCA1through GABP Beta

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