Pol II CTD Phosphorylation by P‐TEFb Influences Endotoxin Tolerance on Paused Genes

Endotoxin tolerance is a decreased responsiveness of immune cells to repeated lipopolysaccharide (LPS) stimulation. IL1B and TNF are immediate‐early genes rapidly induced in LPS‐treated monocytes. Following induction, TNF transcription is completely shut down within a few hours. In contrast, IL1B is not completely inhibited and continues for many hours post‐stimulation as revealed by nuclear run‐on and steady‐state mRNA kinetic analyses. Chromatin immuno‐precipitation (ChIP) studies on IL1B and TNF reveal spatial and kinetic differences in RNA Polymerase II (Pol II) occupancy and the state of Pol II carboxy‐terminal domain (CTD) Ser 5 and 2 phosphorylation for resting, activated and re‐stimulated monocytes. We observe that Pol II is pre‐loaded and paused at promoter proximal regions of both genes prior to stimulation. Differences are also observed for a specific set of histone marks contributing to the induction of these two genes. The interplay of Positive Elongation Factor b (P‐TEFb) and Negative Elongation Factor (NELF) appears to be responsible for maintaining and liberating Pol II from pausing on these genes. Our data reveal that upon secondary stimulus, P‐TEFb is re‐recruited to the IL1B promoter, resulting in the resumption and maintenance of transcriptional elongation by Pol II. This is in contrast to tolerized TNF , in which P‐TEFb and CTD Ser 2 are absent. Analysis of transcript polyadenylation for primary and secondary stimulation suggests that P‐TEFb contributes to proper IL1B mRNA polyadenylation and processing, which is deficient during re‐stimulation of the TNF gene.