Epigenetic silencing of tumor necrosis factor alpha (TNF-alpha) and interleukin 1beta (IL-1beta) transcription occurs in blood leukocytes of animals and humans after the initiation of severe systemic inflammation (SSI). We previously reported that the epigenetic signature requires induction of NF-kappaB factor RelB, which directs histone H3K9 dimethylation, disrupts assembly of transcription activator NF-kappaB p65, and induces a sustained switch from the euchromatin to heterochromatin. Here, we report the novel findings that intracellular high mobility group box 1 protein (HMGB1) and nucleosome linker histone H1 protein are necessary components of endotoxin-mediated silencing of TNF-alpha in THP-1 human promonocytes. HMGB1 binds the TNF-alpha promoter during transcription silencing and promotes assembly of the repressor RelB. Depletion of HMGB1 by small interfering RNA results in dissociation of RelB from the promoter and partially restores TNF-alpha transcription. Histone H1, which typically displaces HMGB1 from nucleosomal DNA, also binds concomitantly with HMGB1 to the heterochromatin of the silenced TNF-alpha promoter. Combined knockdown of HMGB1 and H1 restores binding of the transcriptionally active NF-kappaB p65 and reestablishes TNF-alpha mRNA levels. Chromatin reimmunoprecipitation experiments demonstrate that HMGB1 and H1 are likely recruited to TNF-alpha sequences independently and that their binding correlates with histone H3K9 dimethylation, as inhibition of histone methylation blocks HMGB1 and H1 binding. Moreover, HMGB1- and H1-mediated chromatin modifications are gene specific during endotoxin silencing in that they also bind and repress acute proinflammatory IL-1beta, while no binding nor repression of antiinflammatory IkappaBalpha is observed. Finally, we find that H1 and HMGB1 bind to the TNF-alpha a promoter in human leukocytes obtained from patients with SSI. We conclude proinflammatory HMGB1 and structural nucleosome linker H1 couple as a component of the epigenetic complex that silences acute proinflammatory TNF-alpha during the assembly of heterochromatin in the SSI phenotype.