Altered regulation and expression of genes by BET family of proteins in COPD patients

Correction: PLoS One 2018 12 (4): 0175997Background BET proteins (BRD2, BRD3, BRDT and BRD4) belong to the family of bromodomain containing proteins, which form a class of transcriptional co-regulators. BET proteins bind to acetylated lysine residues in the histones of nucleosomal chromatin and function either as co-activators or co-repressors of gene expression. An imbalance between HAT and HDAC activities resulting in hyperacetylation of histones has been identified in COPD. We hypothesized that pan-BET inhibitor (JQ1) treatment of BET protein interactions with hyperacety-lated sites in the chromatin will regulate excessive activation of pro-inflammatory genes in key inflammatory drivers of alveolar macrophages (AM) in COPD. Methods and findings Transcriptome analysis of AM from COPD patients indicated up-regulation of macrophage M1 type genes upon LPS stimulation. Pan-BET inhibitor JQ1 treatment attenuated expression of multiple genes, including pro-inflammatory cytokines and regulators of innate and adaptive immune cells. We demonstrated for the first time that JQ1 differentially modulated LPS-induced cytokine release from AM or peripheral blood mononuclear cells (PBMC) of COPD patients compared to PBMC of healthy controls. Using the BET regulated gene signature, we identified a subset of COPD patients, which we propose to benefit from BET inhibition. Conclusions This work demonstrates that the effects of pan-BET inhibition through JQ1 treatment of inflammatory cells differs between COPD patients and healthy controls, and the expression of BET protein regulated genes is altered in COPD. These findings provide evidence of histone hyperacetylation as a mechanism driving chronic inflammatory changes in COPD.Peer reviewe

Correction: Altered regulation and expression of genes by BET family of proteins in COPD patients.

[This corrects the article DOI: 10.1371/journal.pone.0173115.]

Categorisation of genes.

<p>(A) Venn diagram showing the four comparisons and the overlaps. Group 1 (red) represents the 212 genes induced by LPS at 6 hours. Group 2 (green) represents the 87 genes induced by LPS at 6 hours and inhibited following pan-BET inhibitor JQ1 treatment at 6 hours. Group 3 (blue) represents the 23 genes induced by LPS at both 6 and 24 hours and inhibited following JQ1 treatment at only 24 hours. (B) A table showing the identity (Gene Symbols) of genes in the three groups 1, 2 and 3.</p

Quantitative PCR validation of selected genes identified by NGS.

<p>Effect of JQ1 treatment on LPS induced expression of signature genes and cytokine control genes in AM from COPD patients. JQ1 treatment lead to inhibited expression of 8 signature genes (CMPK2, EPSTI1, IFI44, IFI44L, IFIT1, IFIT3, MX1 and RSAD2) but had a partial effect on cytokines (IL1a, IL1b, IL6 and IL8) not inhibited at 6h in NGS studies.</p

Expression profile boxplot of the 23 late inhibition genes (Group 3).

<p>Expression levels are shown in the form of transcripts per million (TPM). Each donor is represented as a different color in duplicate. Treatment groups are represented at the bottom of the plots with t = time (6; 6hours, 24; 24 hours. LPS and JQ1 indicates if these are present (+) or absent (-). Gene symbols are shown at the top of each plot.</p

Signature distribution in ECLIPSE sputum samples.

<p>Heat map of the ECLIPSE sputum data for the 10 signature genes. Samples are ordered according to the enrichment scores generated by GSVA with the 10 gene signature. The value distribution is shown in the top panel. Data was normalized by subtracting the row median then scaling by the row median absolute deviation. Red color denotes high expression; blue color denotes low expression. Bar below indicates GSVA enrichment score for these samples as either negative (blue) or positive (orange).</p

M1/M2 polarisation of LPS stimulated genes.

<p>Volcano plot of M1 (red) and M2 (blue) genes at 6 hours (A) and 24 hours (B) after LPS induction. Differential expression of unstimulated vs. LPS stimulated cells was tested using DESeq2 and the Log2 fold change (x-axis) is plotted against the–Log10 adjusted p-values (y-axis). Genes with LFC > 1.5 are labelled by name. The vertical grey lines denotes no LPS induced expression change. The horizontal grey lines shows the 0.05 adjusted p-value cut-off.</p

Subgroups of ECLIPSE WB COPD patients characterised by the 10 gene signature.

<p>A: Plots of gene set enrichment scores generated using either the entire set of early response genes (left) or the 10 gene signature (right) with p-values (t-test, COPD vs control). B Heat maps showing the distribution of the 10 gene signature across samples split into 2 groups based on enrichment score for either the COPD samples (B) or Control samples (C). Numbers and percentage of samples in each group are below the heat maps. Blue bar; negative enrichment score, orange bar; positive enrichment score.</p