The glucocorticoid receptor recruits the COMPASS complex to regulate inflammatory transcription at macrophage enhancers

Abstract

Summary Glucocorticoids (GCs) are effective anti-inflammatory drugs; yet, their mechanisms of action are poorly understood. GCs bind to the glucocorticoid receptor (GR), a ligand-gated transcription factor controlling gene expression in numerous cell types. Here, we characterize GR’s protein interactome and find the SETD1A (SET domain containing 1A)/COMPASS (complex of proteins associated with Set1) histone H3 lysine 4 (H3K4) methyltransferase complex highly enriched in activated mouse macrophages. We show that SETD1A/COMPASS is recruited by GR to specific cis -regulatory elements, coinciding with H3K4 methylation dynamics at subsets of sites, upon treatment with lipopolysaccharide (LPS) and GCs. By chromatin immunoprecipitation sequencing (ChIP-seq) and RNA-seq, we identify subsets of GR target loci that display SETD1A occupancy, H3K4 mono-, di-, or tri-methylation patterns, and transcriptional changes. However, our data on methylation status and COMPASS recruitment suggest that SETD1A has additional transcriptional functions. Setd1a loss-of-function studies reveal that SETD1A/COMPASS is required for GR-controlled transcription of subsets of macrophage target genes. We demonstrate that the SETD1A/COMPASS complex cooperates with GR to mediate anti-inflammatory effects.

Protocol for using heterologous spike-ins to normalize for technical variation in chromatin immunoprecipitation

“…Our method is limited to the conservation of the protein of interest, but it is independent of the variability in IP efficiencies of the antibody. We successfully used this approach for profiling several histone modifications (H3K27ac, H3K4me1, H3K4me2 and H3K4me3) by ChIP-seq and ChIP-qPCR in murine macrophages with spike-ins from Drosophila S2 cells ( Greulich et al., 2021 ). We also performed spike-in normalization for transcription factors with limited conservation in murine cells using human spike-in chromatin (HEK293 cells) by ChIP-qPCR.…”

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Figure 2 Selection and testing of species-specific PCR primers (A) Drosophila melanogaster genome browser screen shot ( http://gbrowse.modencode.org/fgb2/gbrowse/fly/ ) showing publicly available data for H3K4me2 ChIP-Seq at the eRF3 (also known as Elf) locus. (B) UCSC genome browser track for H3K4me2 ChIP-Seq in murine bone marrow derived macrophages after 3 h 100 ng/mL LPS (purple, lower track) or 16 h 1 μM dexamethasone and 3 h 100 ng/mL LPS treatment (L+D, blue, upper track) ( Greulich et al., 2021 ). (C) ChIP-qPCR against H3K4me2 in either pure S2 cells (indicated by the fly), 25% S2 cells mixed with 75% murine macrophages treated with 100 ng/mL LPS for 3 h (marked by the fly + mouse symbol) or pure murine macrophages treated with LPS (marked by the mouse symbol).

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“…The choice of spike-in depends on the evolutionary conservation of the protein of interest and the antibody used. For complete details on the use and execution of this protocol, please refer to Greulich et al. (2021) .…”

“…For complete details on the use and execution of this protocol, please refer to Greulich et al. (2021) .…”

Glucocorticoids as Regulators of Macrophage-Mediated Tissue Homeostasis

“…It is expressed ubiquitously in almost every human cell, including all immune cells (2). When bound by its ligands, GR translocates to the nucleus and occupies specific palindromic DNA sequences within the open chromatin, called glucocorticoid response elements (GREs), to activate or repress gene expression (3)(4)(5)(6)(7)(8). GR activates gene expression through different mechanisms that involve direct binding of dimers to GREs within GR-binding sites or composite binding in which GR and another transcription factor interact with distinct response elements within the same genome location.…”