Class-specific Regulation of Pro-inflammatory Genes by MyD88 Pathways and IκBζ

Hideo Iba,Vladimir R Ramirez-Carrozzi,Kiyoshi Takeda,Stephen T Smale,Hirotaka Kuwata,Masahiro Yamamoto,Hisako Kayama,Kenya Honda,Makoto Matsumoto,Taketoshi Mizutani

doi:10.1074/jbc.m709965200

Abstract

Toll-like receptors trigger the induction of primary response genes via MyD88-mediated activation of NF-kappaB and other transcription factors. These factors then act in concert with primary response gene products to induce secondary response genes. Although the MyD88 pathway is important for the expression of both primary and secondary response genes, we show that the recruitment of NF-kappaB, RNA polymerase, and the TATA-binding protein is MyD88-dependent only at secondary response genes. This selective dependence correlates with the fact that MyD88 is required for nucleosome remodeling and histone H3K4 trimethylation at secondary response promoters, whereas rapidly induced primary response promoters are assembled into poised MyD88-independent chromatin structures. At a subset of secondary response promoters, IkappaBzeta was identified as a selective regulator of H3K4 trimethylation and preinitiation complex assembly after nucleosome remodeling. These mechanistic distinctions advance our understanding of the diverse molecular cascades that underlie the differential regulation of pro-inflammatory genes.

Highlights

Toll-like receptor (TLR)4-dependent recognition of microbial components controls immune responses through the activation of innate immunity and the subsequent development of antigen-specific adaptive immunity [1,2,3]
Initial evidence that chromatin structure may be critical for the differential regulation of primary and secondary response genes following TLR stimulation was provided in an influential study by Saccani et al [26]
Chromatin immunoprecipitation (ChIP) experiments revealed that NF-␬B associates rapidly with rapidly induced primary response genes but much more slowly with genes induced with delayed kinetics

Summary

EXPERIMENTAL PROCEDURES

Antibodies and Mice—Antibodies against NF-␬B p65 (C-20) (sc-372), pol II (H-224) (sc-9001), and TFIID (TBP) (Sl-1) (sc273) were purchased from Santa Cruz Biotechnology. The cells were scraped after washing with PBS and centrifuging at 3000 rpm, and the pellet was resuspended in SDS buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 0.5% SDS). Chromatin was sonicated eight times with 30-s pulses, centrifuged at 14,000 rpm to remove debris, diluted 5-fold with ChIP dilution buffer (16.7 mM Tris-HCl, 167 mM NaCl, 1.2 mM EDTA, 1.1% X-100) supplemented with protease inhibitor, and precleared with salmon sperm DNA/protein A-agarose (Upstate). BRG1/BRM RNA interference-depleted cells and control cells were stimulated and crosslinked 5 days after the first spin infection. Nuclei were pelleted at 1000 rpm, followed by washing with RE buffer (10 mM Tris-HCl (pH 7.4), 50 mM NaCl, 10 mM MgCl2, 0.2 mM EDTA, 0.2 mM EGTA, 1 mM ␤-mercaptoethanol, 0.15 mM spermine, 0.5 mM spermidine). Samples were analyzed by Southern blotting with 32P-labeled gene-specific probes designed at the following regions, Il12b promoter (ϩ64 to ϩ437), Il12b enhancer (Ϫ8711 to Ϫ9113), and Il6 promoter (Ϫ544 to Ϫ1043)

RESULTS

TBP Input

Remodeling at Secondary Response

TBP input

To further evaluate the role of

DISCUSSION

TBP pol II