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

RNA Polymerase II Occupies the TNFα Proximal Promoter in MΦ Before Gene Activation.

TNFα gene expression is strongly induced in response to microbial products: LPS treatment of the murine RAW264.7 MΦ-like cell line and of primary bone marrow-derived MΦ (BMMΦ) led to a dramatic increase in TNFα transcript (Fig. 1 A and B, Left). Unexpectedly, chromatin immunoprecipitation (ChIP) analysis revealed a significant RNA Pol II occupancy near the TNFα promoter under basal conditions in both cell contexts (Fig. 1 A and B, Right). This high “basal” Pol II occupancy was focused in the promoter-proximal region; far less Pol II was detected upstream or farther downstream of the transcription start site (TSS). LPS treatment triggered a significant increase in Pol II signal particularly within the downstream region of TNFα, suggesting that LPS-mediated gene activation facilitates the release of Pol II from the promoter into the gene.

This Pol II binding pattern resembled that described for genes whose promoters are regulated at a step subsequent to Pol II recruitment and are constitutively occupied by “stalled” or “paused” Pol II. Pol II stalling, increasingly appreciated as a widespread regulatory checkpoint in transcription (19–21), occurs when Pol II initiates and produces a short RNA transcript but stalls during elongation through the promoter-proximal region.

LPS Target Genes in BMMΦ Display Different Patterns of Pol II Occupancy and Phosphorylation.

The C-terminal domain (CTD) of the Pol II largest subunit contains a series of heptapeptide repeats (YS₂PTS₅PS) that can be differentially phosphorylated during the transcription cycle. Interestingly, the stalled Poll II displays a characteristic phosphorylation pattern, whereby levels of Serine5 phosphorylation (P-S5), a marker for initiated Pol II, are high, whereas phosphorylation levels at Serine2 (P-S2), a marker for the transition to productive elongation, are low (23, 24). Hence, we examined CTD phosphorylation across the TNFα gene in resting and LPS-stimulated MΦ.

Consistent with polymerase stalling, Pol II adjacent to the TNFα TSS was phosphorylated on S5 in untreated BMMΦ [supporting information (SI) Fig. S1A], whereas P-S2 Pol II was undetectable (Fig. 2A Left). Upon LPS treatment, Pol II was strongly phosphorylated on S2 and associated with the downstream regions of TNFα. Hence CTD phosphorylation at the TNFα gene suggested that Pol II initiated transcription in resting cells but required an additional signal (e.g., LPS-induced transcription factor binding) to undergo S2 phosphorylation, be released from the promoter, and enter productive elongation.

Indeed, cyclin T1, the regulatory subunit of the positive transcription elongation factor (P-TEFb) kinase complex responsible for S2 phosphorylation and release of stalled Pol II into the gene, was recruited to the TNFα proximal promoter specifically in response to LPS (Fig. 2A Right). A modest LPS-induced increase in cyclin T1 occupancy was also seen downstream within the TNFα gene. ChIP analysis revealed a similar LPS-dependent increase in occupancy by Cdk9, the catalytic subunit of P-TEFb, at the TNFα promoter-proximal region (Fig. S1B).

In stark contrast to TNFα, a gene encoding an LPS-inducible chemokine IP-10 (Fig. 2B Right) was devoid of detectible Pol II in resting BMMΦ; Pol II recruitment to the IP-10 promoter-proximal region and CTD phosphorylation on S5 and S2 were strictly LPS dependent (Fig. 2B Right and Fig. S1). Consistent with the data at TNFα (Fig. 2A) and previous studies (24), levels of S2 phosphorylation were greater on the fully elongation-competent Pol II within the IP-10 gene than for the Pol II beginning productive elongation near the IP-10 promoter. Thus, IP-10 transcription appears to be controlled at the level of LPS-induced Pol II recruitment and preinitiation complex assembly.

The TNFα Promoter Is Occupied by the Negative Elongation Factor (NELF) Complex in MΦ.

It has been previously shown that the 4-subunit NELF complex plays a key role in Pol II stalling by inhibiting early transcription elongation (25–27). The negative effects of NELF are counteracted by phosphorylation of the CTD, and potentially NELF itself, by the P-TEFb kinase, whose recruitment is concomitant with release of stalled Pol II from the promoter region. To evaluate a possible role for NELF in regulating TNFα transcription in vivo, we first confirmed that NELF was expressed in MΦ (Fig. S2). Furthermore, immunohistochemical analysis revealed abundant NELF-E staining (Fig. 3 B and E) in the nuclei of the resident MΦ occupying the majority of the red pulp of the mouse spleen (Fig. 3 A and D; F4/80 staining); NELF-E expression appeared to be particularly high in MΦ around and immediately adjacent to the white pulp of the follicles (Fig. 3B; compare to Fig. 3 C and F; CD3 staining for T cells).

We next assessed NELF occupancy at several LPS target genes under basal and inducing conditions. Consistent with the idea that NELF inhibits early elongation, both NELF-E and NELF-A were present specifically in the promoter-proximal region of TNFα in resting RAW264.7 cells (Fig. 4A Right and Fig. S3A, respectively). Similarly, NELF-A associated with the TNFα promoter in untreated BMMΦ (Fig. 4B Right). Interestingly, in both cell contexts, NELF occupancy rapidly and transiently decreased upon addition of LPS, which inversely correlated with TNFα RNA induction. These data suggested that gene activation involves the temporary dissociation of NELF from the TNFα promoter, allowing Pol II to efficiently elongate into the gene. Strikingly, by 1–2 h of LPS treatment, NELF resumed occupancy at the TNFα promoter (Fig. 4A and Fig. S3).

In contrast, we observed no appreciable NELF-A occupancy above the background of control IgG at the IP-10 gene (promoter proximally or downstream) in either resting or LPS-treated BMMΦ whereas Pol II was, as expected, recruited in response to LPS (Fig. 4C). In the same experiment NELF occupied the promoter of Eif4a1, a control gene whose transcription is LPS unresponsive, and importantly this occupancy was not altered in LPS-stimulated BMMΦ (Fig. 4C).

Permanganate Footprinting in BMMΦ Reveals an Open Transcription Bubble in the TNFα Promoter-Proximal Region.

The above experiments suggested that Pol II recruited to the TNFα promoter in resting MΦ initiated transcription but was halted within the promoter-proximal region by NELF. To confirm the presence of an engaged but stalled Pol II at TNFα, we used in vivo permanganate footprinting, a technique that capitalizes on the ability of KMnO₄ to specifically interact with thymine bases in ssDNA, such as those in the open transcription bubble. When Pol II stalls near the promoter for an extended period, ss regions associated with an engaged Pol II can be readily detected by their KMnO₄ hyperreactivity. Fig. 5(lane “0”) illustrates strong permanganate reactivity in resting BMMΦ from near the TNFα TSS through the promoter-proximal region (dashes). This finding is consistent with the presence of an open transcription bubble at the uninduced TNFα gene generated by Pol II, which has produced a short transcript and stalled during early transcription elongation. LPS treatment both enhanced (particularly at 0.5 h) and extended KMnO₄ reactivity further downstream into the gene (bracket), suggesting that Pol II has been released from the promoter to enter productive transcription elongation. This downstream expansion of the KMnO₄-reactive region is reminiscent of the patterns seen at the induced heat-shock genes (28), as is the appearance of detectable reactivity at the TSS (asterisk), which indicates a very high level of transcription initiation upon gene activation.

Pol II Stalling at the Genes in the TNF Family Is Highly Evolutionarily Conserved.

Interestingly, recent studies identified eiger, the sole Drosophila member of the TNF family, as a gene whose expression was attenuated in NELF-depleted cells (19, 22). Eiger, like TNFα, is an essential, stress-inducible regulator of cell death pathways (29, 30), critical for protection against extracellular pathogens. We therefore examined whether eiger is also regulated by Pol II stalling. Our ChIP analysis in Drosophila S2 cells revealed a striking resemblance of the Pol II and NELF-E binding profiles at eiger to those of Pol II and the NELF complex at the mammalian TNFα gene in MΦ. Indeed, both Pol II and NELF-E were strongly enriched at the eiger promoter-proximal region, but not in the upstream or downstream regions (Fig. 6A). Furthermore, RNAi-mediated depletion of NELF significantly decreased Pol II occupancy at eiger (Fig. 6B), consistent with the role of the NELF complex in maintaining stalled Pol II at this promoter.

To obtain direct evidence for the transcriptional status of the Pol II bound near the eiger promoter, we performed permanganate footprinting of the gene. As shown in Fig. 6C, Pol II detected by ChIP is associated with a stalled, open transcription bubble in the eiger promoter-proximal region, as evidenced by KMnO₄-sensitive sites at positions between +34 and +41 relative to the TSS. Further corroborating the idea that Pol II stalling at the eiger promoter is mediated by the NELF complex (Fig. 6B), NELF-B knockdown by RNAi decreases KMnO₄ reactivity (Fig. 6C). Combined, these data demonstrate a remarkable level of evolutionary conservation of the transcriptional regulatory mechanisms governing TNF gene expression from Drosophila to mammals.

Pol II Stalling and the RNA Induction Profile in LPS-Inducible Genes in MΦ.

In mammals, MΦ represent an early line of defense against infection that respond to invading pathogens by producing a vast number of proinflammatory cytokines, chemokines, and factors that provide negative feedback loops limiting excessive inflammation. We quantified the expression of several such factors in LPS-treated MΦ over time. As shown in Fig. 7 A and B, TNFα nascent unprocessed transcript, as measured by the amplification of the intronic region, was induced with strikingly rapid kinetics, reaching a maximum in both RAW264.7 cells and BMMΦ by 0.5–1 h and dropping off precipitously thereafter. Interestingly, this down-regulation of TNFα transcription coincides with NELF reloading onto the gene (Fig. 4). Consistent with earlier observations (11), the TTP gene displayed a fast and transient induction profile similar to that of TNFα itself (Fig. 7 A and B). In contrast, IP-10 induction occurred much more gradually and was sustained for several hours; an even slower induction profile was characteristic of RANTES, another well-established LPS-inducible chemokine. Importantly, similarly “delayed” induction of IP-10 or RANTES was seen whether mRNA or nascent transcript was analyzed.

To begin to address the potential mechanisms underlying such dramatic differences in the transcriptional response of these 2 pairs of genes to a common stimulus, we assessed Pol II and NELF occupancy at TTP and RANTES. Pol II was significantly enriched at the TTP promoter-proximal region in untreated BMMΦ (Fig. 7C); LPS treatment promoted additional Pol II loading onto the promoter and, particularly, the downstream region of the TTP gene. In addition, NELF complex occupied the TTP promoter-proximal region in resting MΦ, was dismissed upon 0.5 h LPS exposure, coinciding with cycT1 recruitment, but resumed promoter occupancy by 1 h, as cycT1 occupancy began to decline (Fig. 7D). These data were consistent with promoter-proximal Pol II stalling and LPS-dependent release and elongation at TTP, similar to that occurring at TNFα. In contrast, RANTES was not occupied by Pol II or NELF in untreated BMMΦ (Fig. 7 C and D); Pol II loading onto the RANTES promoter was LPS dependent, indicating that, as with IP-10, Pol II recruitment is a rate-limiting step in RANTES transcription. Thus, in a limited subset of genes tested, Pol II stalling correlated with a rapid and transient expression profile.

Pol II Stalling and Not the Nature of an Inducer Determines the Induction Profile of LPS Targets.

Most cytokines and chemokines are induced by multiple PAMPs, which signal through specific TLRs. For example, bacterial lipopeptide Pam3Cys activates TLR2, dsRNA [and its synthetic analog poly(IC)] activates TLR3, and ssRNA signals through TLR7 (2). Signal transduction cascades initiated by these TLRs are partially overlapping yet distinct, creating a sophisticated regulatory matrix whereby, in principle, each PAMP may trigger a different transcriptional response of a given gene. We therefore examined the induction profile of TNFα, TTP, IP-10, and RANTES in BMMΦ treated with poly(IC), Pam3Cys, and ssRNA. We found that TNFα was induced by Pam3Cys and ssRNA and that TTP was highly ssRNA responsive, but, in all cases, their expression peaked by 0.5–1 h and subsided thereafter (Fig. 8A). In contrast, activation of IP-10 by poly(IC) or RANTES by all 3 PAMPs did not peak until 3–5 h irrespective of the specific TLR engaged (Fig. 8B). We conclude that for a given gene, the kinetics of induction are specific to the gene itself regardless of a signaling pathway responsible for activation and reflect a particular rate-limiting step in transcription (Pol II recruitment vs. release from promoter-proximal stalling).

Cell Culture and BMMΦ Preparation.

Mouse RAW264.7 cells were maintained in DMEM (Invitrogen) supplemented with 10% FBS (HyClone), treated with sterile PBS (vehicle) or LPS (1 μg/mL) as described in the figure legends, and harvested. BMMΦ were prepared from 8-wk-old C57BL/6 mice as described in ref. 41 except L-cell conditioned media were used for the 6-day MΦ expansion; cells were then scraped, incubated in RPMI-20% FBS overnight, and treated as described in the figure legends.

Measurement of RNA Levels.

Total RNA was isolated from RAW264.7 cells and BMMΦ and analyzed (41) for the expression of each gene, using β-actin as an internal control. Primer pairs are listed in Table S1.

Permanganate Footprinting.

In vivo permanganate footprinting in Drosophila was performed as in ref. 19. Day 7 BMMΦ (≈100 × 10⁶) were harvested in ice-cold PBS (400 × g; 5 min), washed in PBS, collected as above, and subjected to KMnO₄ probing as in ref. 19 using 1 μg DNA per reaction and 25 cycles of ligation-mediated PCR (see primer sequences in Table S1).

ChIP Experiments and Immunohistochemistry.

See SI Methods.