Abstract
SummaryThe molecular basis of signal-dependent transcriptional activation has been extensively studied in macrophage polarization, but our understanding remains limited regarding the molecular determinants of repression. Here we show that IL-4-activated STAT6 transcription factor is required for the direct transcriptional repression of a large number of genes during in vitro and in vivo alternative macrophage polarization. Repression results in decreased lineage-determining transcription factor, p300, and RNA polymerase II binding followed by reduced enhancer RNA expression, H3K27 acetylation, and chromatin accessibility. The repressor function of STAT6 is HDAC3 dependent on a subset of IL-4-repressed genes. In addition, STAT6-repressed enhancers show extensive overlap with the NF-κB p65 cistrome and exhibit decreased responsiveness to lipopolysaccharide after IL-4 stimulus on a subset of genes. As a consequence, macrophages exhibit diminished inflammasome activation, decreased IL-1β production, and pyroptosis. Thus, the IL-4-STAT6 signaling pathway establishes an alternative polarization-specific epigenenomic signature resulting in dampened macrophage responsiveness to inflammatory stimuli.
Highlights
We found that a high proportion of IL-4-responsive genes (39%) were repressed
Our genome-wide analyses showed increased chromatin accessibility at the activator STAT6-bound sites (Figure 4A), while significant reduction was detected in chromatin accessibility at the repressor STAT6-bound genomic regions (Figure 4A). These results suggest that both STAT6-mediated enhancer activation and repression are associated with the modification of chromatin structure during alternative macrophage polarization
Applying K-mean clustering method, we found 1,628 IL4-repressed genes (p % 0.05) in WT bone marrow-derived macrophages (BMDMs) (Figure S4B) and identified an IL-4-repressed gene cluster that showed attenuated repression in Hdac3fl/fl Lyz2 Cre BMDMs following IL-4 treatment (Figures 4E and S4B)
Summary
RNA-seq cDNA library for RNA-Seq was generated from 1 mg total RNA using TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol. Poly-A tailed RNAs were purified by oligodT conjugated magnetic beads and fragmented on 94 C degree for 8 minutes, 1st strand cDNA was transcribed using random primers and SuperScript II reverse transcriptase (Lifetechnologies, Carslbad, CA, USA). Following this step second strand cDNA synthesized, double stranded cDNA end repaired and 30 ends adenylated Illumina index adapters were ligated. Paired read 100bp sequencing runs were performed on Illumina HiScan SQ instrument (Illumina, San Diego, CA, USA)
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