Distinct Mechanisms for Induction and Tolerance Regulate the Immediate Early Genes Encoding Interleukin 1β and Tumor Necrosis Factor α

Abstract

Interleukin-1β and Tumor Necrosis Factor α play related, but distinct, roles in immunity and disease. Our study revealed major mechanistic distinctions in the Toll-like receptor (TLR) signaling-dependent induction for the rapidly expressed genes (IL1B and TNF) coding for these two cytokines. Prior to induction, TNF exhibited pre-bound TATA Binding Protein (TBP) and paused RNA Polymerase II (Pol II), hallmarks of poised immediate-early (IE) genes. In contrast, unstimulated IL1B displayed very low levels of both TBP and paused Pol II, requiring the lineage-specific Spi-1/PU.1 (Spi1) transcription factor as an anchor for induction-dependent interaction with two TLR-activated transcription factors, C/EBPβ and NF-κB. Activation and DNA binding of these two pre-expressed factors resulted in de novo recruitment of TBP and Pol II to IL1B in concert with a permissive state for elongation mediated by the recruitment of elongation factor P-TEFb. This Spi1-dependent mechanism for IL1B transcription, which is unique for a rapidly-induced/poised IE gene, was more dependent upon P-TEFb than was the case for the TNF gene. Furthermore, the dependence on phosphoinositide 3-kinase for P-TEFb recruitment to IL1B paralleled a greater sensitivity to the metabolic state of the cell and a lower sensitivity to the phenomenon of endotoxin tolerance than was evident for TNF. Such differences in induction mechanisms argue against the prevailing paradigm that all IE genes possess paused Pol II and may further delineate the specific roles played by each of these rapidly expressed immune modulators.

EZH2 or HDAC1 Inhibition Reverses Multiple Myeloma–Induced Epigenetic Suppression of Osteoblast Differentiation

“…Chromatin from MC4 cells, MM-BMSC, and HD-BMSC was analyzed using a modification of the ChIP Millipore/Upstate protocol (MCPROTO407) as described (29) using Magna ChIP Protein A+G Beads (16-663, Millipore). In brief, a total of 2x10 7 cells were fixed in 1% formaldehyde (F79-500, Fisher) for 10 min at room temperature.…”

Distinct mechanisms regulate IL1B gene transcription in lymphoid CD4 T cells and monocytes

“…Spi1 often associates with inducible transcription factors, such as NFκB and C/EBPβ, on lipopolysaccharide (LPS)-responsive promoters and enhancers in human and murine macrophages[7, 8]. In the monocyte/macrophage lineage, Spi1 constitutively binds to the IL1B promoter at two distinct sites located between −50 to −39 and −115 to −97 relative to the transcription start site[9, 10]. In non-myeloid cells, its ectopic expression can result in IL1B transcription in the presence of an activation signal for NF-κB and C/EBPβ[9–11].…”

“…In the monocyte/macrophage lineage, Spi1 constitutively binds to the IL1B promoter at two distinct sites located between −50 to −39 and −115 to −97 relative to the transcription start site[9, 10]. In non-myeloid cells, its ectopic expression can result in IL1B transcription in the presence of an activation signal for NF-κB and C/EBPβ[9–11]. Additionally, Spi1 can act as a “pioneer factor”, binding nucleosome-occluded DNA and facilitating chromatin accessibility for LPS-responsive transcription factors in activated monocytes[12, 13].…”

“…Additionally, Spi1 can act as a “pioneer factor”, binding nucleosome-occluded DNA and facilitating chromatin accessibility for LPS-responsive transcription factors in activated monocytes[12, 13]. Further, it directly recruits TATA-binding protein (TBP), which is involved in forming the pre-initiation complex (PIC) that helps to recruit RNA Polymerase II (Pol II) to gene promoters[9, 14]. …”

“…IL-1β is expressed at extremely high levels in myeloid-derived cells in response to microbial invasion and tissue injury[9]. Although activated monocytes are a major source of IL-1β; NK cells, B cells, dendritic cells, fibroblasts, and epithelial cells also express this protein, but at much lower levels[15].…”