SemOpenAlex |

SemOpenAlex

Matches in SemOpenAlex for { <https://semopenalex.org/work/W4243486360> ?p ?o ?g. }

Showing items 1 to 73 of 73 with 100 items per page.

W4243486360 abstract "Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The glucocorticoid receptor (GR) potently represses macrophage-elicited inflammation, however, the underlying mechanisms remain obscure. Our genome-wide analysis in mouse macrophages reveals that pro-inflammatory paused genes, activated via global negative elongation factor (NELF) dissociation and RNA Polymerase (Pol)2 release from early elongation arrest, and non-paused genes, induced by de novo Pol2 recruitment, are equally susceptible to acute glucocorticoid repression. Moreover, in both cases the dominant mechanism involves rapid GR tethering to p65 at NF-kB-binding sites. Yet, specifically at paused genes, GR activation triggers widespread promoter accumulation of NELF, with myeloid cell-specific NELF deletion conferring glucocorticoid resistance. Conversely, at non-paused genes, GR attenuates the recruitment of p300 and histone acetylation, leading to a failure to assemble BRD4 and Mediator at promoters and enhancers, ultimately blocking Pol2 initiation. Thus, GR displays no preference for a specific pro-inflammatory gene class; however, it effects repression by targeting distinct temporal events and components of transcriptional machinery. https://doi.org/10.7554/eLife.34864.001 eLife digest Inflammation is one of the body’s responses to fight infection and heal tissue damage. The response is controlled by hundreds of genes, which fall into two classes. In the first class, an injury or infection triggers the enzyme RNA Polymerase to bind to and transcribe the gene into long RNA strands, which are then translated into the proteins that play a role in the inflammation response. The second class has a more quick-fire response. RNA Polymerase binds to these genes even without an injury or infection to serve as a trigger. But most of the time the enzyme only transcribes the beginning of these genes. This is because it is inhibited by a so-called negative elongation factor, which acts like a brake. For this second class of genes, an infection or injury triggers the release of the negative elongation factor from the enzyme, and allows RNA Polymerase to transcribe the full RNA strand. In excess, inflammation can be dangerous. The body’s way of limiting or controlling inflammation is via steroid hormones called glucocorticoids. These bind to the glucocorticoid receptor, which acts to switch off the inflammatory genes. But exactly how the receptor does this has not been fully understood. Sacta et al. investigated how the glucocorticoid receptor turns off these gene complexes. Experiments looking at white blood cells in mice found that the receptor can switch off both groups of inflammatory genes, but by a different mechanism for each class. Sacta et al. discovered that in the first gene class, the receptor blocks proteins that open up the DNA for RNA Polymerase, so it could not bind to the gene. In the second class, the receptor stops the release of the brake-like negative elongation factor from RNA Polymerase. As a result, the enzyme stalls at the beginning of the gene and fails to make a full-length transcript required to make the necessary protein. Glucocorticoids are often used as drugs to treat chronic inflammation, but they can have debilitating side effects. Understanding how the glucocorticoid receptor switches off inflammatory genes could help to design drugs with fewer side effects to treat chronic inflammation, and diseases caused by specific inflammatory genes. https://doi.org/10.7554/eLife.34864.002 Introduction Inflammation is an innate immune response to tissue injury or infection. It relies on macrophages, which recognize pathogen-associated molecular patterns and other ‘danger’ signals via their toll-like receptors (TLRs) (Glass and Saijo, 2010). This initiates a signaling cascade that leads to the activation and DNA binding of the effector transcription factors NF-kB and AP1 (O'Neill et al., 2013) which recruit coregulators, and, ultimately, the basal transcription machinery that together alter the chromatin state in the vicinity of many pro-inflammatory genes and enable their transcription (Smale and Natoli, 2014; Glass and Natoli, 2015). Acute transcriptional activation of pro-inflammatory genes is, therefore, critical for overriding the homeostatic set-point and producing a robust immune response that helps to resolve infection or tissue injury (Kotas and Medzhitov, 2015). Although the magnitude and dynamics of inflammation is affected at multiple levels, the temporal coordination of cytokine gene transcription by RNA Polymerase (Pol) 2 is a key mechanism that defines acute inflammatory response. The Pol 2 transcription cycle has been divided into three phases: initiation, elongation and termination. Initiation involves the recruitment of Pol 2 to the promoter, histone modifications and changes in histone occupancy. In addition, the C-terminal domain (CTD) of Pol 2, which contains multiple heptad repeats (YS2PTS5PS), is phosphorylated at S5, and Pol 2 synthesizes short (20–60 nt) RNA transcripts. During the elongation step, Pol 2 is further phosphorylated at S2 by the cyclin T1/CDK9 positive transcription elongation factor (P-TEFb) and synthesizes the full length RNA transcript, which is followed by the termination step and RNA transcript dissociation from the DNA (Nechaev and Adelman, 2011). Although Pol 2 recruitment and initiation has been historically considered the rate-limiting step in signal-dependent transcription, numerous recent studies revealed that transcriptionally engaged Pol 2 often remains paused near promoters in the absence of activating signal and that entry into productive elongation is rate-limiting for activation of up to 40% of inducible genes (Core et al., 2012). The paused Pol 2 is in a complex with the 4-subunit negative elongation factor (NELF); NELF phosphorylation by P-TEFb leads to its release and Pol 2 entry into productive elongation (Chiba et al., 2010; Nechaev and Adelman, 2011). A subset of cytokine genes in macrophages is controlled at the level of Pol 2 pausing. Indeed, while for genes such as Il1a and Il1b, signal-dependent Pol 2 recruitment to their transcription start sites (TSS) and transcription initiation are rate-limiting, other genes, exemplified by Tnf, are occupied by Pol 2 even under resting conditions (Adelman et al., 2009; Hargreaves et al., 2009; Gupte et al., 2013). At Tnf, Pol 2 is S5-phosphorylated, bound by NELF and paused ~50 bp downstream of the TSS. Pause release following S2 and NELF phosphorylation by P-TEFb occurs in response to inflammatory signal. Aside from Pol 2 occupancy, the chromatin state plays an integral part in the regulation of transcription (Smale et al., 2014). In particular, histone code ‘writers’ such as acetyltransferases (HATs) GCN5 and p300 have been implicated in modifying H3K9/14 and H4K5/8/12 at inflammatory genes in macrophages following treatment with TLR4 ligands (Hargreaves et al., 2009; Escoubet-Lozach et al., 2011). Both HATs are recruited by the NF-kB subunit p65 to regulatory regions in a stimulus-dependent manner (Hargreaves et al., 2009; Ghisletti et al., 2010). Histone modifications are then bound by ‘readers’ such as BRD4, a protein containing two conserved N-terminal bromodomains (BD1 and BD2), which associates with most active promoters and some active enhancers, and has been proposed to couple the acetylation state at enhancers and promoters with Pol 2 elongation (Lovén et al., 2013; Brown et al., 2014). BRD4 occupancy correlates with acetylation marks at H4K5/8/12, H3K9/27 (Lovén et al., 2013; Kanno et al., 2014; Nagarajan et al., 2014) and with gene activation, whereas chemical inhibition of BRD4 binding abrogates the induction of a subset of genes (Nicodeme et al., 2010). Furthermore, BRD4 has been shown to associate with P-TEFb, affecting Pol 2 CTD phosphorylation, and hence, transcription elongation (Itzen et al., 2014). These events coalesce ensuring a rapid remodeling of the inflammatory transcriptome, with hundreds of genes undergoing a dramatic upregulation (Escoubet-Lozach et al., 2011; Chinenov et al., 2012; Gupte et al., 2013; Uhlenhaut et al., 2013; Tong et al., 2016). Although essential for host defense, unabated inflammation imposes a threat to the host and can result in tissue damage and autoimmunity. One systemic mechanism that controls acute inflammatory response is a feedback loop whereby inflammatory cytokines trigger the production of steroid hormones known as glucocorticoids (GCs) (reviewed in [Sacta et al., 2016]). Lipophilic GCs diffuse through the cell membrane and bind the intracellular glucocorticoid receptor (GR), a transcription factor (TF), which then translocates to the nucleus and regulates gene expression. The transcriptional outcomes of GR activation are context-specific and are determined by the genomic GC response elements (GRE) to which the receptor binds. GR can bind directly to specific, usually pseudopalindromic, DNA sequences either as a homodimer or complexed with other TFs such as AP1 and STAT3 (Biddie et al., 2011; Langlais et al., 2012). In this context, GR recruits various coregulators such as members of the p160 family, HATs, the Mediator complex and ATP-dependent chromatin remodelers (Weikum et al., 2017b), ultimately leading to the activation of numerous genes including the anti-inflammatory genes, such as Dusp1 and Tsc22d3 (GILZ). At other sites, known as ‘tethering’ GREs, GR does not directly bind DNA but interacts with other DNA-bound TFs such as pro-inflammatory AP1 and NF-kB and usually represses their activity (reviewed in [Chinenov et al., 2013]) – a property fundamental to the ability of GCs to dramatically attenuate inflammation. In contrast to GR-mediated activation, the mechanisms of transcriptional repression by GR remain poorly understood. Strikingly, however, in a few cases analyzed, genes activated through Pol 2 recruitment and those induced by signal-dependent Pol 2 pause release were both susceptible to GR-mediated repression (Gupte et al., 2013). Here, we use a combination of cell-based and genome-wide approaches to elucidate the mechanisms by which GR represses pro-inflammatory genes in primary macrophages challenged acutely with the TLR4 agonist lipopolysaccharide (LPS) and GCs. We present evidence of ‘tethering’ as a prevalent mechanism of repression among p65/GR co-regulated genes. We further demonstrate a widespread yet gene class-specific role of NELF in glucocorticoid-mediated repression of early elongation. Conversely, at other genes, GR precludes the ordered assembly of HATs, Brd4 and the Mediator complex which ultimately blocks Pol 2 recruitment and transcription initiation. Results Genomic binding of GR and p65 upon inflammatory and anti-inflammatory stimulation To understand the mechanisms by which GR elicits its repressive effects, we first assessed by RNA-seq the global transcriptional changes upon acute activation of primary mouse bone-marrow-derived macrophages (BMDM) with LPS or LPS together with a synthetic GC dexamethasone (Dex) for 1 hr. At FDR < 0.1 we found that, compared to vehicle-treated BMDM, 597 genes were induced by LPS >1.5 fold. Of these, the induction of 201 genes was attenuated >1.3 fold by Dex co-treatment (Figure 1A and Supplementary file 1). As expected, GO analysis of acutely GR-repressed genes revealed predominantly those involved in cytokine signaling (Figure 1A). Figure 1 with 2 supplements see all Download asset Open asset GR represses LPS-induced genes via p65-assisted tethering. (A) Over 30% of LPS-induced genes (597) in BMDM are repressed by Dex (201; Venn diagram and normalized expression values) and show a pro-inflammatory gene signature (GO analysis). BMDM were untreated (U) or treated with 10 ng/ml LPS ±100 nM Dex (L and LD) for 1 hr, and gene expression levels were determined by RNA-seq (n = 2). (B) The overlap between ChIP-seq peak calls for GR and p65 in LPS + Dex-treated BMDM (Venn diagram) was determined using subsetByOverlap function from GenomicRanges package (Bioconductor) with the minimum overlap of 1 nt (see Materials and methods). Ab initio sequence motif discovery and over-representation in each subset of GR or p65 binding peaks was determined using MEME-ChIP (Ma et al., 2014). E-values for the enrichment of the motif are shown. (C) Dex- and LPS + Dex-induced GR ChIP-seq peaks are shown (Venn diagram). LPS + Dex unique peaks are enriched for NF-kB-binding sites as indicated by MEME-ChIP analysis as in B. (D) Genomic location of p65 and GR binding sites relative to known genomic features is determined by ChIPpeakAnno (Bioconductor) (Zhu et al., 2010). (E) The distribution of GR-binding sites located in a 200 Kb region centered on LPS-induced Dex-repressed genes in BMDM treated with Dex or LPS + Dex (left). Pie-charts show the % of LD-unique GR peaks either genome-wide (center) or those associated with LPS-induced Dex-repressed genes only (right). (F) GR and p65 ChIP-seq read density profiles of representative LPS-induced Dex-repressed genes are shown for untreated (U), LPS (L) or LPS + Dex (L + D) treated BMDM. Also see Figure 1—figure supplements 1–2 and Supplementary files 1 and 2. https://doi.org/10.7554/eLife.34864.003 Despite rapid remodeling of the macrophage LPS-induced transcriptome in response to Dex observed by us and others (Figure 1, [Rao et al., 2011; Chinenov et al., 2012; Uhlenhaut et al., 2013; Chinenov et al., 2014]), no comprehensive analysis of the GR and p65 genome-wide occupancy under acutely repressing conditions has been reported. Therefore, we determined the distribution of p65 and GR genomic binding sites in BMDM pulsed with LPS, Dex or LPS + Dex for 45 min (see Figure 1—figure supplements 1–2 and Supplementary file 2 for quality metrics and comparison of replicates). Following LPS + Dex exposure, we detected 9987 GR peaks (union of two replicates) 5397 (54.1%) of which did not overlap with p65 peaks at the same conditions (Figure 1B, top, Figure 1—figure supplement 1A). Motif overrepresentation analysis in these GR unique peaks revealed predominance of centrally enriched NR3C-binding motifs, which represent GREs and highly related NR-binding sites, those for ETS family members, such as the macrophage lineage-determining TF SPI1 (PU.1 and SPIB), and AP1 family members (Figure 1B, Figure 1—figure supplement 1B, left panel). The analysis of p65 binding after LPS + Dex treatment revealed 7052 peaks (union of two replicates) of which 2344 (33.8%) were uniquely bound by p65 (Figure 1B, Figure 1—figure supplement 2A). Motif analysis indicated an enrichment of NF-kB/Rela binding motifs, as well as ETS and AP1 motifs (Figure 1B). Importantly, the GR and p65 cistromes shared 4589 peaks, which corresponds to nearly half of all GR- and 2/3 of all p65-binding peaks. Motif analysis of these peaks showed a predominance for NR3C/GRE, ETS family, NF-kB/Rela and AP1 binding motifs that were enriched near the peak summits (Figure 1B, bottom, Figure 1—figure supplement 1B, middle panel). Because of the significant enrichment of peaks with NF-kB elements (especially among those overlapping p65-binding peaks) in the GR cistrome under repressing conditions, we performed GR ChIP-seq in BMDM treated with Dex only for 45 min to compare the two GR cistromes. In Dex-treated BMDM, GR-binding sites formed 3377 peaks. Of those, 3165 also appeared in the GR LPS + Dex cistrome (with only 212 peaks unique to Dex-treated BMDM), whereas 6817 were gained in the GR LPS + Dex cistrome (Figure 1—figure supplement 1A, right panel). ETS and NR3C-binding motifs were over-represented in both Dex-unique and Dex – LPS + Dex shared subsets of GR peaks and trended toward the peak summit (Figure 1C, Figure 1—figure supplement 1B, right panel). We did not detect NF-kB/Rela motif enrichment in these two subsets of GR-binding peaks. However, among 6817 peaks unique to the GR LPS + Dex cistrome we readily observed an overrepresentation of NF-kB and AP1 motifs while NR3C motifs were no longer enriched (Figure 1C, compare top/middle vs. bottom motif enrichment panels) indicating that inflammatory signaling and p65/NF-kB activation was driving GR recruitment to such sites specifically under repressing LPS + Dex conditions. The majority of GR and p65-binding sites were located in distal intergenic (~39–47% of peaks) and intronic (~40% on average) regions (Figure 1D), similar to previously reported GR and p65 cistromes in various cell lines (Reddy et al., 2009; Barish et al., 2010). To correlate GR binding with transcriptional outcomes, we focused on our subset of 201 LPS-induced Dex-repressed genes as determined by RNA-seq (Figure 1A, Supplementary file 1) and evaluated GR peak localization within these genes and 100 Kb of their 5’- and 3’-flanking regions in Dex- and LPS + Dex-treated BMDM. In this subset, a somewhat larger fraction (~52%, compared to 39–47% genome-wide) of GR-binding peaks were located in distal intergenic regions, whereas the fraction of peaks in the introns dropped from 40% to 24% compared to whole-genome GR cistrome (Figure 1D). This shift was not due to a preponderance of shorter introns or genes in Dex-repressed subset (Figure 1—figure supplement 1C). Comparison of GR binding near the 201 Dex-repressed genes with an entire GR cistrome shows that a greater fraction of binding sites was unique to the LPS + Dex condition (81% vs. 68%, Figure 1E) consistent with a disproportional increase in unique binding site utilization among this functionally constrained set of genes. Several representative examples of GR and p65 co-binding near GR-sensitive genes are shown in Figure 1F: at each gene, GR binding occurred at sites matching those of p65, but only in LPS + Dex and not LPS- or Dex-alone treated BMDM. Importantly, LPS-dependent p65 binding fully persisted in the presence of Dex. In fact, the total number of p65 binding peaks in the presence of LPS and LPS + Dex was comparable both genome-wide, and in the vicinity of our GR-repressed genes (Figure 1—figure supplement 2A, right and 2B). In each case, ~2/3 of the LPS-induced p65 peaks persisted in LPS + Dex-treated BMDM. Moreover, among p65 LPS + Dex peaks functionally constrained to Dex-repressed genes, 80% (up from 68% genome-wide) overlapped LPS-induced peaks (Figure 1—figure supplement 2C). Interestingly, of the 201 genes repressed by Dex in the context of LPS-mediated macrophage activation, only 56 were repressed ≥1.3 fold (and only 16 of those ≥2 fold) upon treatment with Dex alone (Supplementary file 1; RNA-seq dataset from [Chinenov et al., 2014]) – further supporting a requirement for NF-kB activation for GR recruitment to the majority of genes Dex-sensitive genes. Combined, these results further corroborate a tethering model in which p65 is a central component of repression complexes in GC-treated BMDM. NELF mediates repressive effects of GR at paused genes We have reported that at several pro-inflammatory genes in unstimulated BMDM, promoter-proximally paused Pol 2 is in a complex with NELF and enters productive elongation following LPS treatment (Adelman et al., 2009; Gupte et al., 2013). To assess how common this type of Pol 2 dynamics is among inflammatory genes, we performed Pol 2 ChIP-seq in untreated, LPS- or LPS + Dex treated BMDM. Figure 2A shows Pol 2 tracks for six genes all of which were among 201 that were rapidly induced by LPS and repressed by Dex as established by RNA-seq (Figure 1A). Of those, Tnf, Hilpda and Btg2, all display accumulation of Pol 2 near the TSS in untreated BMDM. Upon a 45-min LPS treatment, we detect additional Pol 2 loading and, notably, its redistribution into the body of the gene; conversely, upon LPS + Dex treatment, Pol 2 largely remains near the TSS resembling a ‘paused’ pattern seen in the unstimulated BMDM (Figure 2A, left). In contrast, non-paused genes Il1a, Il1b and Cd83 display no substantial Pol 2 occupancy in the unstimulated BMDM, and a dramatic and uniform increase in Pol 2 occupancy throughout the gene in response to LPS, which is nearly abrogated by co-treatment with Dex (Figure 2A, right). Figure 2 Download asset Open asset Pol 2 and NELF dynamics at different classes of GR-sensitive genes. (A) Pol 2 ChIP-seq read density profiles and pausing indexes (PI) for representative paused and non-paused genes in the untreated (U), LPS (L) and LPS +Dex (LD) treated BMDM. (B) PI (a ratio of Log-transformed Pol 2 counts at the promoter and gene body in untreated BMDM) was calculated for all LPS-induced Dex-repressed transcript variants with unique 5’ ends (see Materials and methods). Genes with PI >1 were considered paused and those with a PI <0.8 non-paused. (C) Pol 2 ChIP-seq heat maps of paused (n = 62) and non-paused (n = 82) transcripts sorted by the PI indexes corresponding to 198 Dex-repressed genes (see Materials and methods) are shown for the U, L and L + D conditions for each individual replica. Only transcripts overlapping Pol 2 peaks in LPS-treated BMDMs as determined by MACS2 are shown. NELF-E heat maps from U BMDM ChIP-seq for the same transcript classes are shown on the right. Heat maps scales are equalized to visualize Pol 2 and NELF distribution across the genes; color scale bars are shown below corresponding maps. (D) Average Pol 2 (in each treatment condition) and NELF-E (untreated BMDM) occupancy for each gene class defined in C. The confidence band shows the SEM and 95% confidence interval. Also see Supplementary files 2 and 3. https://doi.org/10.7554/eLife.34864.006 These findings raised the possibility that GR mediates its repressive effects genome-wide by regulating distinct steps of Pol 2 transcription cycle depending on the rate-limiting step for gene activation. To address this possibility, we first calculated Pol 2 pausing indexes (PI) for approximately 300 transcripts corresponding to our 198 LPS-induced Dex-repressed genes (three genes were excluded due to the conflict of annotation). As described in Nechaev et al. (2010), we defined PI as the ratio of log-transformed normalized Pol 2 counts around the promoter (−200/+500 bp relative to the annotated TSS) to those within the gene body downstream of +500 bp (Figure 2B, Supplementary file 3). Based on the PI in untreated BMDM, we classified GC-repressed genes into two groups: 61 transcripts had a PI >1 and were considered to be paused (twice as much of Pol2 at the promoter region versus gene body), whereas 82 had a PI <0.8 and were considered non-paused (see Materials and methods and [Nechaev et al., 2010]). Figure 2C shows Pol 2 distribution within the −200/+1500 region for individual transcripts of both classes in unstimulated BMDM, as well as BMDM exposed for 45 min to LPS or LPS + Dex. The read density distribution for 61 paused and 82 non-paused transcripts in differentially treated BMDM (Figure 2D) reveals a peak of Pol 2 occupancy in the promoters of the paused genes, additional Pol 2 loading, and, importantly, its entry into gene bodies in response to LPS. Co-treatment with Dex decreases Pol 2 occupancy in gene body with most Pol 2 remaining near the TSS (Figure 2C and D). Conversely, little Pol 2 is seen in the non-paused genes in untreated BMDM; Pol 2 occupancy increases dramatically throughout the genes in LPS-treated BMDM and this loading is largely abrogated by Dex (Figure 2C and D), consistent with the pattern shown in Figure 2A for representative genes. Because Pol 2 pausing within the first 100 nt of a gene is mediated by NELF (Adelman and Lis, 2012), we assessed genome-wide NELF distribution by ChIP-seq using antibodies to the NELF-E subunit of the complex. Aligned with Pol 2 PI heat maps, NELF-E occupancy matched closely Pol 2 distribution in untreated BMDM with striking accumulation immediately downstream of TSS of paused genes and little to no NELF-E seen in non-paused genes (Figure 2C and D, far right). Indeed, read density distribution in NELF-E ChIP-seq shows highly gene class-specific NELF-E occupancy at paused (PI >1) promoters (Figure 2D, right). As reported previously for a few individual genes (Adelman et al., 2009; Schaukowitch et al., 2014), following LPS stimulation, NELF-E was broadly evicted from promoters of LPS-induced genes with little occupancy detected at 1 hr (Figure 3A). Interestingly, however, this dismissal was transient, as despite continued LPS exposure, NELF reloaded onto promoters reaching widespread occupancy by 3 hr (Figure 3A, also see average occupancy graphed for all paused transcripts). This release and reloading can be seen at specific paused GC-sensitive genes, Tnf, Hilpda, and Btg2 (Figure 3A, right), which show substantial NELF-E occupancy at the TSS co-localizing with Pol 2 peaks in resting BMDM, its dissociation following a 1 hr LPS induction, and re-establishment of the TSS-associated NELF-E peaks by 3 hr. Figure 3 with 1 supplement see all Download asset Open asset Gene-class-specific contribution of NELF to GR-mediated repression. (A) Heat maps show NELF-E occupancy in the U (from Figure 2C), 1 and 3 hr L-treated BMDM for paused and non-paused transcripts. Average occupancy for the paused genes in each condition is graphed as in Figure 2D. Representative examples of Pol 2 and NELF ChIP-seq read density profiles are shown on the right. (B) Pie charts show the percentage of all paused (24%) and non-paused (66%) LPS-induced Dex-repressed genes that exhibit promoter-proximal NELF-E binding in the UNT (86.3% and 31.7%, respectively) and L + D conditions (83.6% and 33.2%, respectively). NELF-E ChIP-seq read density profiles for the L + D condition are shown for a set of representative genes. Red rectangles in Tnf, Myc, Errfi1 and Ccl2 profiles indicate MACS2 NELF-E peaks in the L + D condition. (C) NELF-B KO mice were generated as described in Methods. NELF-B RNA in WT and KO BMDM was quantified by RT-qPCR and normalized to Actb (n = 5, p<0.0001, two-tailed Student’s t-test; error bars are SEM). For western blots, three mice per genotype were used to visualize NELF-B, NELF-E and HSP90 as a loading control (top). Bottom: WT and NELF-B KO BMDM were U or treated with L-/+D for 30 min (Tnf) or 1 hr (all others) and the expression of indicated genes (matching those in B) was assessed by RT-qPCR, normalized to Actb, and shown as ‘fold activation by LPS’ over basal levels (=1) and ‘fold repression by Dex’ (a ratio of L over L + D level of each transcript). *p<0.05, **p<0.01 (Two-tailed Student’s t-test). Error bars are SEM. (D) The volcano plot comparing gene expression in L + D (1 hr) treated BMDM from the WT vs. NELF-B KO mice (n = 3) (fold change = 1.5, FDR p<0.05). Pausing indices (PI) of 201 LPS-induced Dex-repressed genes from Figure 1A are shown in color. (E) CDK9 occupancy at selected genes in BMDM treated for 1 hr as indicated. n = 4–9. **p<0.01, ****p<0.001 (two-tailed Student’s t-test). Error bars are SEM. Also see Figure 3—figure supplement 1 and Supplementary file 2. https://doi.org/10.7554/eLife.34864.007 Figure 3—source data 1 Source raw data for Fig. 3C (RT-qPCR in WT and NELF-B KO) and 3E (ChIP-qPCR for CDK9). https://doi.org/10.7554/eLife.34864.009 Download elife-34864-fig3-data1-v2.xlsx To directly assess whether NELF occupancy in GC-sensitive genes in BMDM correlates with Pol 2 pausing in early elongation, we compared the NELF-E and Pol 2 cistromes in the unstimulated BMDM. Among the LPS-induced Dex-sensitive genes with PI >1 (approximately 24% of 300 Dex-repressed transcripts), 86.3% displayed promoter-associated NELF-E peaks, compared to only 31.7% in genes with PI <0.8 (which comprised approximately 66% of 300 transcripts; Figure 3B, left). Importantly, similar relative numbers of paused and non-paused genes (23% and 70%, respectively; Figure 3—figure supplement 1B) were found among LPS-induced Dex-insensitive genes from RNA-seq (Figure 1A). In this group, NELF-E occupancy in untreated BMDM was again much more prevalent in paused genes (81.1%) than in non-paused ones (44.2%). Thus, GR does not preferentially repress genes in one class vs. the other, and high levels of TSS-associated NELF in a basal state is a common feature of paused genes irrespective of their sensitivity to GC. Given that NELF and Pol 2 co-localize at the TSS of the paused genes in unstimulated BMDM, that activation of such genes by LPS coincides with NELF dismissal, and that Pol 2 remains near the promoters of these genes under repressing conditions consistent with their early elongation arrest, we questioned whether GR-mediated repression was globally mediated by NELF. We first evaluated NELF-E occupancy in BMDM co-treated with LPS + Dex by ChIP-seq and found the relative distribution of peaks among paused (PI >1) and non-paused (PI <0.8) repressed genes to be indistinguishable from NELF-E distribution in resting BMDM (83.6% and 33.2%, respectively; Figure 3B, right – compare to left). We then evaluated NELF-E distribution across several of our target genes in the presence of LPS + Dex and detected striking promoter-proximal NELF peaks in paused Tnf, Myc, Errfi1 and Ccl2, but not in non-paused Il1b or Lif (Figure 3B). To address directly whether NELF is necessary for GR-mediated repression, we used a new mouse strain conditionally lacking the NELF-B subunit and, hence, the functional NELF complex in myeloid cells (see Materials and methods). BMDM from NELF-B LysM-Cre mice (NELF-B KO) show a dramatic reduction in NEL" @default.
W4243486360 created "2022-05-12" @default.
W4243486360 creator A5012536712 @default.
W4243486360 creator A5021643471 @default.
W4243486360 creator A5032725070 @default.
W4243486360 creator A5036111899 @default.
W4243486360 creator A5048946642 @default.
W4243486360 creator A5052902433 @default.
W4243486360 creator A5059905381 @default.
W4243486360 creator A5065731483 @default.
W4243486360 creator A5068438737 @default.
W4243486360 creator A5070160160 @default.
W4243486360 creator A5078809930 @default.
W4243486360 creator A5091201954 @default.
W4243486360 date "2018-01-29" @default.
W4243486360 modified "2023-09-26" @default.
W4243486360 title "Author response: Gene-specific mechanisms direct glucocorticoid-receptor-driven repression of inflammatory response genes in macrophages" @default.
W4243486360 doi "https://doi.org/10.7554/elife.34864.026" @default.
W4243486360 hasPublicationYear "2018" @default.
W4243486360 type Work @default.
W4243486360 citedByCount "0" @default.
W4243486360 crossrefType "peer-review" @default.
W4243486360 hasAuthorship W4243486360A5012536712 @default.
W4243486360 hasAuthorship W4243486360A5021643471 @default.
W4243486360 hasAuthorship W4243486360A5032725070 @default.
W4243486360 hasAuthorship W4243486360A5036111899 @default.
W4243486360 hasAuthorship W4243486360A5048946642 @default.
W4243486360 hasAuthorship W4243486360A5052902433 @default.
W4243486360 hasAuthorship W4243486360A5059905381 @default.
W4243486360 hasAuthorship W4243486360A5065731483 @default.
W4243486360 hasAuthorship W4243486360A5068438737 @default.
W4243486360 hasAuthorship W4243486360A5070160160 @default.
W4243486360 hasAuthorship W4243486360A5078809930 @default.
W4243486360 hasAuthorship W4243486360A5091201954 @default.
W4243486360 hasBestOaLocation W42434863601 @default.
W4243486360 hasConcept C104317684 @default.
W4243486360 hasConcept C150194340 @default.
W4243486360 hasConcept C186310378 @default.
W4243486360 hasConcept C203014093 @default.
W4243486360 hasConcept C2776914184 @default.
W4243486360 hasConcept C2780841215 @default.
W4243486360 hasConcept C3020196126 @default.
W4243486360 hasConcept C54355233 @default.
W4243486360 hasConcept C59493245 @default.
W4243486360 hasConcept C86803240 @default.
W4243486360 hasConcept C95444343 @default.
W4243486360 hasConceptScore W4243486360C104317684 @default.
W4243486360 hasConceptScore W4243486360C150194340 @default.
W4243486360 hasConceptScore W4243486360C186310378 @default.
W4243486360 hasConceptScore W4243486360C203014093 @default.
W4243486360 hasConceptScore W4243486360C2776914184 @default.
W4243486360 hasConceptScore W4243486360C2780841215 @default.
W4243486360 hasConceptScore W4243486360C3020196126 @default.
W4243486360 hasConceptScore W4243486360C54355233 @default.
W4243486360 hasConceptScore W4243486360C59493245 @default.
W4243486360 hasConceptScore W4243486360C86803240 @default.
W4243486360 hasConceptScore W4243486360C95444343 @default.
W4243486360 hasLocation W42434863601 @default.
W4243486360 hasOpenAccess W4243486360 @default.
W4243486360 hasPrimaryLocation W42434863601 @default.
W4243486360 hasRelatedWork W14335379 @default.
W4243486360 hasRelatedWork W20911189 @default.
W4243486360 hasRelatedWork W25048727 @default.
W4243486360 hasRelatedWork W2554471 @default.
W4243486360 hasRelatedWork W2899880 @default.
W4243486360 hasRelatedWork W44003308 @default.
W4243486360 hasRelatedWork W53374033 @default.
W4243486360 hasRelatedWork W5360043 @default.
W4243486360 hasRelatedWork W55327338 @default.
W4243486360 hasRelatedWork W58136152 @default.
W4243486360 isParatext "false" @default.
W4243486360 isRetracted "false" @default.
W4243486360 workType "peer-review" @default.