title: Dynamic and selective nucleosome repositioning during endotoxin tolerance.
abstract: Sepsis is encoded by a sequel of transcription activation and repression events that initiate, sustain, and resolve severe systemic inflammation. The repression/silencing phase occurs in blood leukocytes of animals and humans following the initiation of systemic inflammation due to developing endotoxin tolerance. We previously reported that NF-kappaB transcription factor RelB and histone H3 lysine methyltransferase G9a directly interact to induce facultative heterochromatin assembly and regulate epigenetic silencing during endotoxin tolerance, which is a major feature of sepsis. The general objective of this study was to assess whether dynamic temporal, structural, and positional changes of nucleosomes influence the sepsis phenotype. We used the THP-1 sepsis cell model to isolate mononucleosomes by rapid cell permeabilization and digestion of chromatin with micrococcal nuclease and then compared tumor necrosis factor alpha (TNFalpha) proximal promoter nucleosome alignment in endotoxin-responsive and -tolerant phenotypes. We found differential and dynamic repositioning of nucleosomes from permissive to repressive locations during the activation and silencing phases of transcription reprogramming and identified the following mechanisms that may participate in the process. 1) Two proximal nucleosomes repositioned to expose the primary NF-kappaB DNA binding site in endotoxin-responsive cells, and this "promoter opening" required the ATP-independent chaperone NAP1 to replace the core histone H2A with the H2A.Z variant. 2) During RelB-dependent endotoxin tolerance, the two nucleosomes repositioned and masked the primary NF-kappaB DNA binding site. 3) Small interfering RNA-mediated inhibition of RelB expression prevented repressive nucleosome repositioning and tolerance induction, but the "open" promoter required endotoxin-induced NF-kappaB p65 promoter binding to initiate transcription, supporting the known requirement of p65 posttranslational modifications for transactivation. 4) Sustaining the permissive promoter state after RelB knockdown required ATP-dependent nucleosome remodeler BAF complex. Moreover, we found that forced expression of RelB in responsive cells induced repressive nucleosome positioning and silenced TNFalpha transcription, demonstrating the plasticity of nucleosome remodeling and its dependence on RelB. Our data suggest that nucleosome repositioning controls both the induction and epigenetic silencing phases of TNFalpha transcription associated with sepsis.
authors.affiliation: Department of Internal Medicine, Section of Molecular Medicine, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA. melgazza@wfubmc.edu
authors.forename: Mohamed
authors.initials: M
authors.lastname: El Gazzar
authors.forename: Tiefu
authors.initials: T
authors.lastname: Liu
authors.forename: Barbara K
authors.initials: BK
authors.lastname: Yoza
authors.forename: Charles E
authors.initials: CE
authors.lastname: McCall
mesh_terms: D000818:Animals; D002460:Cell Line; D004351:Drug Resistance / Q000187:drug effects*; D004731:Endotoxins / Q000494:pharmacology*; D020868:Gene Silencing / Q000187:drug effects; D006570:Heterochromatin / Q000378:metabolism*; D006649:Histocompatibility Antigens / Q000378:metabolism; D011495:Histone-Lysine N-Methyltransferase / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D006801:Humans; D007962:Leukocytes / Q000378:metabolism*; D008954:Models, Biological; D009707:Nucleosomes / Q000235:genetics / Q000378:metabolism*; D020218:Response Elements; D018805:Sepsis / Q000378:metabolism; D051996:Transcription Factor RelA / Q000378:metabolism; D051841:Transcription Factor RelB / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*
chemical_list: D004731:Endotoxins; D006570:Heterochromatin; D006649:Histocompatibility Antigens; D006657:Histones; D009707:Nucleosomes; C498143:RELA protein, human; C497593:RELB protein, human; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; D051841:Transcription Factor RelB; C445405:EHMT2 protein, human; D011495:Histone-Lysine N-Methyltransferase