G9a and HP1 Couple Histone and DNA Methylation to TNFα Transcription Silencing during Endotoxin Tolerance

Abstract

TNF␣ gene expression is silenced in the endotoxin tolerant phenotype that develops in blood leukocytes after the initial activation phase of severe systemic inflammation or sepsis. The silencing phase can be mimicked in vitro by LPS stimulation. We reported that the TNF␣ transcription is disrupted in endotoxin tolerant THP-1 human promonocyte due to changes in transcription factor binding and enrichment with histone H3 dimethylated on lysine 9 (H3K9). Here we show that the TNF␣ promoter is hypermethylated during endotoxin tolerance and that H3K9 methylation and DNA methylation interact to silence TNF␣ expression. Chromatin immunoprecipitation and RNA interference analysis demonstrated that, in tolerant cells, TNF␣ promoter is bound by the H3K9 histone methyltransferase G9a which dimethylates H3K9 and creates a platform for HP1 binding, leading to the recruitment of the DNA methyltransferase Dnmt3a/b and an increase in promoter CpG methylation. Knockdown of HP1 resulted in a decreased Dnmt3a/b binding, sustained G9a binding, and a modest increase in TNF␣ transcription, but had no effect on H3K9 dimethylation. In contrast, G9a knockdown-disrupted promoter silencing and restored TNF␣ transcription in tolerant cells. This correlated with a near loss of H3K9 dimethylation, a significant decrease in HP1 and Dnmt3a/b binding and promoter CpG methylation. Our results demonstrate a central role for G9a in this process and suggest that histone methylation and DNA methylation cooperatively interact via HP1 to silence TNF␣ expression during endotoxin tolerance and may have implication for proinflammatory gene silencing associated with severe systemic inflammation.Epigenetic mechanisms generate heritable marks on DNA and N-terminal tails of histones that maintain stable patterns of gene expression and are crucial in regulating gene activity as they impact chromatin structure and dynamics. These chromatin-based modifications control the recruitment of specific transcription factors and/or chromatin effectors, thereby providing a mechanism by which histones and DNA modifications regulate gene transcription (reviewed in Refs. 1-3). Methylation of histone H3 on lysine 9 (H3K9) and DNA on 5-cytosine bases, within the context of CpG dinucleotides, are two epigenetic marks whose increased levels are associated with heterochromatin formation and transcriptional silencing of several gene promoters (4).H3K9 can exist in mono-, di-, or trimethylated state. Mono-and dimethylation are catalyzed by the histone methyltransferase G9a, whereas trimethylation is catalyzed by the methyltransferase SUV39h and is predominant in pericentric (constitutive) heterochromatin domains (1, 5). While G9a can also trimethylate H3K9 in vitro (6), it is a major histone methyltransferase for mono-and dimethylation of H3K9 in euchromatic (regulated) domains (7). Methylated H3K9 serves as a docking site for chromatin modifiers such as the heterochromatin-binding protein 1 (HP1), 2 which mediates heterochromatin formation and is implicated in gene silencing (4,...

Dynamic and Selective Nucleosome Repositioning during Endotoxin Tolerance

“…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. 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.…”

“…proximal promoter nucleosome and induces DNA and histone methylation in tolerant cells (32). To further characterize the remodeled nucleosome and examine whether histone modifications associate with nucleosome repositioning, we compared the extent of histone methylation of the repositioned nucleosomes, specifically H3K4me3 (which marks actively transcribed genes) and H3k9me2 (which correlates with silenced genes) by chromatin immunoprecipitation analysis.…”

“…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).…”

The expanding role of the Ehmt2/G9a complex in neurodevelopment

“…9 The molecular mechanisms that target the Ehmt1/2 complex to unmethylated chromatin sites is not well defined, but may ultimately rely on physical association with auxiliary cofactor proteins such as the zinc finger-containing proteins Wiz 10,11 and Znf518, 12 or in some cases non-coding RNA. 13 Once recruited, to exert its repressive effects the Ehmt1/2 complex functions with co-repressors such as H3K4 demethylase Jarid1a, 14 the histone deacetylase Hdac1, [15][16][17] the heterochromatin promoting HP1 18,19 and various DNMTs. [20][21][22] We, and others, have recently identified an additional cofactor necessary for proper ehmt1/2 recruitment to target genes: znf644.…”