The NF-κB Factor RelB and Histone H3 Lysine Methyltransferase G9a Directly Interact to Generate Epigenetic Silencing in Endotoxin Tolerance

Abstract

The interplay of transcription factors, histone modifiers, and DNA modification can alter chromatin structure that epigenetically controls gene transcription. During severe systemic inflammatory (SSI), the generation of facultative heterochromatin from euchromatin reversibly silences transcription of a set of acute proinflammatory genes. This gene-specific silencing is a salient feature of the endotoxin tolerant phenotype that is found in blood leukocytes of SSI patients and in a human THP-1 cell model of SSI. We previously reported that de novo induction of the NF-B transcription factor RelB by endotoxin activation is necessary and sufficient for silencing transcription of acute proinflammatory genes in the endotoxin tolerant SSI phenotype. Here, we examined how RelB silences gene expression and found that RelB induces facultative heterochromatin formation by directly interacting with the histone H3 lysine 9 methyltransferase G9a. We found that heterochromatin protein 1 (HP1) and G9a formed a complex at the interleukin-1␤ promoter that is dependent on the Rel homology domain (RHD) of RelB. RelB knockdown disassociated the complex and reversed transcription silencing. We also observed that whereas RelB chromatin binding was independent of G9a, RelB transcriptional silencing required G9a accumulation at the silenced promoter. Binding between RelB and G9a was confirmed by glutathione S-transferase pulldown in vitro and coimmunoprecipitation in vivo. These data provide novel insight into how RelB is required to initiate silencing in the phenotype associated with severe systemic inflammation in humans, a disease with major morbidity and mortality.Inflammation is an evolutionarily conserved stereotypic stress response primarily orchestrated by temporal alterations in gene expression, with important contributions from complement, coagulation, and neurogenic processes (1, 2). The genetic information encoded to generate inflammation regulates distinct functional sets of genes, including pro-and anti-inflammatory modifiers, directors of cell death, and mediators of cell respiration and metabolism (3). The initiating stage of virtually all inflammation depends on sensory receptors coupled to intracellular signals that activate the immunity master regulator NF-B to generate p65 and p50 transactivating heterodimers at euchromatin promoters of a set of early response proinflammatory genes. When spread throughout the circulation, this early stage may precipitate the extreme stress response of severe systemic inflammation (SSI). 4 Later stages of inflammation reprogramming often require protein synthesis and induce expression of distinct sets of genes with anti-inflammatory, survival, and energy regulation (4, 5). Recent data in humans from our laboratory and in animals from others highlight the role of epigenetics in regulating gene expression in the SSI phenotype (6 -11). These epigenetic events provide specificity and plasticity among distinct sets of genes and depend on varied chromatin structure and modifications rather t...

Dynamic and Selective Nucleosome Repositioning during Endotoxin Tolerance

“…RelB is essential initiator of silencing by directly interacting with and recruiting G9a to promote and maintain a silent heterochromatin structure (27,32). Our finding that inhibiting RelB expression in tolerant cells reactivated TNF␣ and IL-1␤ transcription (26,27,31) raised the possibility that nucleosome remodeling physically contributes to transcriptional silencing of proinflammatory genes in SSI.…”

“…DNA was transfected by electroporation as described above. This truncation disrupts the Rel homology domain of RelB and therefore affects its dimerization and interaction with G9a (26).…”

“…The transcription silencing phase is initiated and maintained through a combinatorial silencing mechanism that involves interactions between the transcription repressor RelB and chromatin-associated proteins (26,31,32). The silencing mechanism requires dimethylation on histone H3 lysine 9 (H3K9me2) by G9a, increased binding of heterochromatin protein HP1, and formation of silent facultative heterochromatin structure (26,31,32). This process correlates with diminished binding of the active NF-B factor p65 and increased binding of feedback repressor transcription factor RelB to the proximal promoters.…”

“…This gene reprogramming event can be remodeled in vitro by generating a state of lipopolysaccharide (LPS) tolerance in cultured cell lines by the prolonged stimulation with LPS (28 -30). The transcription silencing phase is initiated and maintained through a combinatorial silencing mechanism that involves interactions between the transcription repressor RelB and chromatin-associated proteins (26,31,32). The silencing mechanism requires dimethylation on histone H3 lysine 9 (H3K9me2) by G9a, increased binding of heterochromatin protein HP1, and formation of silent facultative heterochromatin structure (26,31,32).…”

Epigenetic Mechanisms in Inflammation

“…2D) for more efficient repression. Another pathway leading to gene repression was recently described for NF-κB/RelB-dependent silencing in severe systemic inflammation (SSI) caused by sepsis and other acute inflammatory processes (Chen et al, 2009). Induction of RelB by endotoxin activation is necessary and sufficient to repress acute pro-inflammatory genes.…”