No abstract

Summary The inflammatory response requires coordinated activation of both transcription factors and chromatin to induce transcription for defense against pathogens and environmental insults. We sought to elucidate connections between inflammatory signaling pathways and chromatin through genomic foot-printing of kinase activity and unbiased identification of prominent histone phosphorylation events. We identified H3 serine 28 phosphorylation (H3S28ph) as the principal stimulation-dependent histone modification and observed its enrichment at induced genes in mouse macrophages stimulated with bacterial lipopolysaccharide. Using pharmacological and genetic approaches, we identified mitogen- and stress-activated protein kinases (MSKs) as primary mediators of H3S28ph in macrophages. Cell-free transcription assays demonstrated that H3S28ph directly promotes p300/CBP-dependent transcription. Further, MSKs can activate both signal-responsive transcription factors and the chromatin template with additive effects on transcription. Specific inhibition of MSKs in macrophages selectively reduced transcription of stimulation-induced genes. Our results suggest that MSKs incorporate upstream signaling inputs and control multiple downstream regulators of inducible transcription.

115In vitro kinase assay and dot blot: Recombinant CHK1 kinase (Sigma) was incubated with kinase buffer (40mM 116 HEPES pH7.4, 20mM MgCl2), Magnesium/ATP cocktail (EMD) and histone tail peptides for overnight at 37C (Total 117 reaction 15ul, 2ug Peptide, Mg(4.5mM)/ATP(30uM) cocktail and 4ng Enzyme). The samples were then added with 118 5ul of 0.5%SDS followed by boiling for 5min at 95C. The samples were dropped on a dry nitrocellulose membrane 119 and probed with a-H3S31ph antibody. 121CRISPR targeting of H3.3: CRISPR targeting H3f3b and H3f3a was performed in RAW264.7 cells using methods 122 described in Ran et al. 2013 18 . Targeting was done consecutively first targeting H3f3b, then using H3f3b mutants 123 to target H3f3a. 124The gRNAs (Primers caccTAGAAATACCTGTAACGATG forward aaacCATCGTTACAGGTATTTCTA reverse for 125 H3f3a and caccGAAAGCCCCCCGCAAACAGC forward aaacGCTGTTTGCGGGGGGCTTTC reverse for H3f3b)

The inflammatory response requires selective, rapid regulation of chromatin and induction of transcription for defense against pathogens and environmental insult, and also features abundant kinase activity. We sought to identify the connections between kinase pathway activity and regulation of chromatin through unbiased identification and genomic foot printing of prominent histone phosphorylation events. We identified histone H3 serine 28 phosphorylation (H3S28p) as the principal stimulation-dependent histone modification with enrichment at induced inflammatory genes in mouse macrophages stimulated with bacterial lipopolysaccharide (LPS). Use of chemical inhibitors and genetic rescue identified mitogen- and stress-activated protein kinases (MSK) as the primary mediators of H3S28p in macrophages. Cell-free transcription assays demonstrated that H3S28p directly promotes transcription. Further, we show that MSK can activate both signal-responsive transcription factors and the chromatin template with additive effects on transcription. Indeed, inhibition of MSK in macrophages selectively reduced transcription of stimulation-induced genes. Our results indicate MSK as a nexus of signaling inputs controlling multiple downstream regulators of inducible transcription.