2: The P-TEFb-dependent gene coding for IL-1β is more sensitive to cellular metabolism than that of the BRD4-dependent TNFα-coding gene

Abstract

The induction of the immediate-early genes coding for Interleukin 1β (IL1B) and Tumor Necrosis Factor α (TNF) is dependent upon Toll-Like receptor 4 (TLR4) detection of bacterial lipopolysaccharide (LPS). We have observed various distinctions for these genes that correlate with RNA Polymerase II (Pol II) pausing, specific transcription factor involvement, promoter architecture and histone epigenetics. Distinct from human TNF, which possesses paused Pol II and pre-bound TATA Binding Protein (TBP), human IL1B requires lineage-specific Spi-1/PU.1 as an anchor for induction-dependent interaction with C/EBPβ and NF-kB, resulting in rapid LPS-dependent de novo recruitment of TBP and Pol II in concert with a permissive state for elongation mediated by the rapid recruitment of the Cdk9 kinase-containing elongation factor P-TEFb. In contrast, we observed that LPS-inducible binding of NF-κB to TNF facilitates immediate recruitment of the atypical bromodomain kinase, BRD4. Both of these kinases have been implicated in Pol II elongation via phosphorylation of the polymerase carboxy-terminal domain (CTD). Treatment of murine macrophages with the metabolic inhibitor 2-deoxyglucose reduced Pol II levels at the Il1b gene, while Tnf remained unaffected. P-TEFb recruitment, Pol II serine 2 CTD phosphorylation and histone H3K36me3 modification were reduced more significantly on Il1b than Tnf. A similar effect is observed in the presence of a PI3-kinase inhibitor. Consequently, IL-1β gene transcription is both more sensitive to the metabolic state of the cell and a dependence upon P-TEFb than the gene coding for TNFα.