Polarization of Primary Human Monocytes by IFN-γ Induces Chromatin Changes and Recruits RNA Pol II to the TNF-α Promoter

Abstract

Monocyte polarization by IFN-γ or IL-4 drives a complex series of cellular responses leading to increased intracellular killing (IFN-γ) or enhanced healing (IL-4) among other functional responses. We studied the effect of IL-4 and IFN-γ polarization on histone modifications at the TNF-α locus in human primary monocytes. IFN-γ polarization markedly increased the expression of TNF-α, whereas IL-4 treatment decreased the expression. We found that IFN-γ alone increased histone H4 acetylation at the TNF-α promoter. The effect of IFN-γ on TNF-α expression was durable upon cytokine washout and even repolarization with IL-4. Concordantly, IFN-γ-mediated H4 acetylation was also durable. IFN-γ recruited activating transcription factor-2 via p38 to the TNF-α promoter, but inhibition of p38 had minimal effect on H4 acetylation. In a novel finding, we found that IFN-γ recruited RNA Pol II to the human TNF-α promoter via ERK signaling, but did so without initiating transcription, leading to a poised condition. These studies provide an important perspective on monocyte polarization. Polarization by IFN-γ has a durable effect on TNF-α expression, and histone acetylation may provide a mechanism for persistence of the effect.

The SLE Transcriptome Exhibits Evidence of Chronic Endotoxin Exposure and Has Widespread Dysregulation of Non-Coding and Coding RNAs

“…Primary human monocytes were purified using elutriation and adherence from eight healthy controls and nine SLE patients with no other autoimmunity, as previously described [22], [23], [24], [25]. The purity of monocytes was more than 90% by flow cytometry for CD14 staining.…”

Transcriptional Control of the TNF Gene

“…Increased histone H3 and H4 acetylation at the TNF promoter region (and, in some cases, at the third intron of TNF) in primary human monocytes or human monocytic cell lines (e.g. THP-1) has been associated with induction of TNF transcription by LPS [186, 187] and high glucose concentrations [181], maturation of monocytes to macrophages [188], diabetes [181], and systemic lupus erythematosus [189]. Moreover, IFN-γ treatment of primary human monocytes led to increased, persistent H4 acetylation along with recruitment of ATF-2 and RNA Pol II, yielding a ‘poised’ pretranscription state and, subsequently, elevated LPS-induced levels of both TNF transcription and histone H3 and H4 acetylation at the TNF promoter [187].…”

Monocyte Enhancers are Highly Altered in Systemic Lupus Erythematosus

“…Monocytes were purified from healthy female controls (with no medical conditions other than inactive allergies) and female SLE patients by adherence as previously described [19][20][21][22][23]. CD14 staining confirmed >90% purity.…”

“…Immunoprecipiation of histones was carried out as previously described [19][20][25][26] and utilized H3K4me3 (Active Motif, Carlsbad, CA, USA) and H3K27me3 (Millipore, Temecula, CA, USA). Immunoprecipitation with anti-GST (Invitrogen, Camarillo, CA, USA) and input were used to define background.…”