Immediate mediators of the inflammatory response are poised for gene activation through RNA polymerase II stalling

Abstract

The kinetics and magnitude of cytokine gene expression are tightly regulated to elicit a balanced response to pathogens and result from integrated changes in transcription and mRNA stability. Yet, how a single microbial stimulus induces peak transcription of some genes (TNF␣) within minutes whereas others (IP-10) require hours remains unclear. Here, we dissect activation of several lipopolysaccharide (LPS)-inducible genes in macrophages, an essential cell type mediating inflammatory response in mammals. We show that a key difference between the genes is the step of the transcription cycle at which they are regulated. Specifically, at TNF␣, RNA Polymerase II initiates transcription in resting macrophages, but stalls near the promoter until LPS triggers rapid and transient release of the negative elongation factor (NELF) complex and productive elongation. In contrast, no NELF or polymerase is detectible near the IP-10 promoter before induction, and LPS-dependent polymerase recruitment is rate limiting for transcription. We further demonstrate that this strategy is shared by other immune mediators and is independent of the inducer and signaling pathway responsible for gene activation. Finally, as a striking example of evolutionary conservation, the Drosophila homolog of the TNF␣ gene, eiger, displayed all of the hallmarks of NELF-dependent polymerase stalling. We propose that polymerase stalling ensures the coordinated, timely activation the inflammatory gene expression program from Drosophila to mammals. cytokine gene expression ͉ inflammation ͉ TNF␣ ͉ transcription initiation and elongation ͉ NELF

dDsk2 regulates H2Bub1 and RNA polymerase II pausing at dHP1c complex target genes

“…Polyclonal rat aWOC (IF: 1/500) and rabbit aROW (WB: 1/10,000, ChIP/IP: 1 ml) antibodies were raised against recombinant proteins aa230-626 and aa588-956, respectively. Other antibodies used are described in the indicated references: rat aHP1c (WB: 1/10,000, IF: 1/500, ChIP/IP: 1 ml), aHP1b (WB: 1/ 10,000, ChIP/IP: 1 ml) and aROW (WB: 1/10,000, IF: 1/400, ChIP/IP: 1 ml) 18 , rabbit aWOC (WB: 1/10,000, ChIP/IP: 1 ml) 62 , rabbit and rat aDDP1 (ChIP/IP: 1 ml) 63 , rabbit adDsk2 (WB: 1/12,000, IF: 1/1,000, ChIP/IP: 1 ml) 16 , mouse ap54 (WB: 1/50) 64 , rabbit aNELF-E (WB: 1/500, ChIP/IP: 3 ml) 65 and rabbit aRPB3 (WB: 1/1,500, ChIP: 15 ml) 26 . Commercially available antibodies used were as follows: rabbit aH3K4me3 (Abcam, ab8580, ChIP: 1 ml), rabbit aIIoser5 (Abcam, ab5131, ChIP: 3 ml), rabbit aBre1 (NOVUS, 40280002, WB: 1/1,000, ChIP: 2 ml) and aRad6 (SANTA CRUZ, sc-30078, WB: 1/500, ChIP: 2 ml), mouse aGST (glutathione S-transferase; Novagen, 71097-3, WB: 1/10,000), mouse aGFP (Roche, 1814460, IF: 1/50), mouse aH2Bub1 (Millipore, 05-1312, WB: 1/4,000, ChIP/IP: 1 ml), mouse aTubulin (Millipore, MAB3408, WB: 1/2,000), rabbit aActin (Sigma, A2066, WB: 1/10,000), mouse aV5 (Invitrogen, 460705, WB: 1/5,000), mouse aUbiquitin (SANTA CRUZ, sc-8017, WB: 1/500), rabbit aPanAc (Abcam, Ab193-100, ChIP: 1 ml) and rabbit aH4Ac (Millipore, no.…”

Identification of a core TP53 transcriptional program with highly distributed tumor suppressive activity

“…Although the idea of RNA polymerase II holoenzyme pausing as a prerequisite for rapid gene activation is a subject of debate (Adelman et al 2009;Lin et al 2011), we tested for differences in pausing between early direct and late TP53 targets with proximal binding. Early direct targets tend to display higher "pausing indexes," indicative of greater occupancy by paused RNA polymerases at their promoters ( Fig.…”

Nup98 promotes antiviral gene expression to restrict RNA viral infection in Drosophila

“…This response is divided into two stages; within minutes, a rapid primary response independent of new protein synthesis is initiated, which instructs the downstream translationdependent secondary response (2,5). Many primary response genes have active chromatin marks and features of transcriptional pausing, including high occupancy of the initiating form of RNA polymerase II (RNAPII), S5 phosphorylated (S5P) (2,4,6), along with negative elongation factor complex (NELF) and DRB Sensitivity-Inducing Factor complex (DSIF), which prevent transcriptional elongation (4,(6)(7)(8)(9)(10). Paused RNAPII can be activated by the positive transcription elongation factor b (P-TEFb) in a stimulusdependent manner, which phosphorylates NELF, DSIF, and RNAPII to release the pause and promote transcriptional elongation and thus the production of mature mRNAs (9,(11)(12)(13).…”