Signal-dependent recruitment of Pol II to promoters of target genes is one of the key regulated steps in inducible gene expression. However, detailed analyses of several model genes and genome-wide studies of Pol II occupancy have demonstrated signal-independent Pol II recruitment in the absence of gene expression. Given the prevalence of this phenomenon, occurring at many genes and in at least a few cell types, several fundamental questions regarding signal-dependent gene expression emerge: What is the nature of the genes that are pre-associated with Pol II prior to expression? How is inducible transcription of these genes regulated? What are the roles of inducible transcription factors in the induction of these genes? We addressed these questions using LPS-inducible inflammatory gene expression in macrophages, to make the following findings. First, we find that genes pre-associated with Pol II are induced uniquely in the primary response. Second, we show that the induction of these genes is regulated at post-initiation steps, specifically by signal-dependent P-TEFb recruitment via Brd4 binding to H4K5/8/12Ac. We demonstrate that S5-P Pol II at PRG-Is constitutively produces unspliced transcripts, while signal-induced S2 Pol II phosphorylation results in productive elongation that generates mature, protein coding transcripts. Finally, we show that PRGs are uniquely associated with corepressor complexes that presumably prevent their constitutive, signal-independent expression.
We find a dramatic difference in the chromatin configuration of PRG-I and PRG-II/SRG promoters with respect to basal levels of H3K4me3, H3Ac, and promoter-bound Pol II. Interestingly, the status of PRGs correlated closely with the GC content of their promoters, PRG-Is having abundant levels of pre-associated Pol II, H3K4me3, and H3K9Ac and PRG-II/SRGs having little to none. In addition, the levels of positive histone modifications and Pol II at PRG-Is ranged from very high, comparable to that of transcriptionally active HKGs, to very low levels, comparable to PRG-II/SRGs. Thus, GC content may account for the qualitative differences between GC-rich PRG-Is and GC-poor PRG-IIs/SRGs, as well as the quantitative differences between different PRG-Is. Importantly, GC-rich PRG-I promoters have intrinsically lower affinity for nucleosomes, a property that contributes to their inducible expression in the absence of remodeling (S. Smale, accompanying manuscript). Moreover, we found that Sp1 was required for Pol II recruitment to PRG-I promoters in the basal state (). Constitutive transcription driven by Sp1-recruited Pol II was required to maintain the permissive status of PRG-I promoters (). Thus, the number and distribution of constitutive transcription factor binding sites within promoters presumably also contributes to the quantitative differences between individual PRG-Is.
Multiple lines of evidence suggest that PRG-I transcription is regulated at post-initiation steps. For example, despite similar levels of S5-P Pol II at HKGs and PRG-Is in unstimulated macrophages, PRG-Is were not expressed while HKGs were. This is reminiscent of Drosophila
heat-shock genes, which undergo abortive initiation due to Pol II pausing (Saunders et al., 2006
). However, PRG-Is are regulated differently from heat-shock genes. Specifically, full length, unspliced transcripts of many PRG-Is were detectable at basal state, while the production of mature, processed transcripts was strictly signal-dependent (). Unspliced transcripts were not generated by low levels of S2-P Pol II, undetectable in our assay, as they were insensitive to DRB treatment (). Thus, although S2 phosphorylation is required for productive elongation and mRNA processing, S5-P Pol II can elongate in the absence of S2 phosphorylation, albeit with low efficiency, to generate unspliced transcripts. These data are in agreement with isolated examples of DRB-insensitive transcription of intron-less genes and histone genes, which are processed by a distinct mechanism (Medlin et al., 2005
). Moreover, they are consistent with the role of S2 phosphorylation in the recruitment of splicing factors to Pol II (Sims et al., 2004
Our data suggest a critical post-initiation checkpoint in the induction of PRG-Is. This is in contrast to SRGs, for which the key regulatory step is the recruitment of Pol II prior to initiation. LPS stimulation most likely amplifies both pre- and post-initiation steps at PRG-Is, as exemplified by the additional recruitment of Pol II following stimulation (), to allow for multiple rounds of transcription at these highly inducible genes. However, Pol II complexes recruited by constitutive (Sp1) versus inducible (NF-κB) transcription factors play distinct roles in PRG-I regulation: the former generates unspliced transcripts and maintains PRG-I chromatin in an active state, while the latter results in gene expression.
We demonstrate that P-TEFb engagement is a key regulatory step in PRG induction, and that Brd4 is essential for P-TEFb recruitment and CTD S2 phosphorylation at PRGs. These results are consistent with a recent report showing a requirement for Brd4 in the recruitment of P-TEFb to NF-κB-inducible genes following stimulation with LPS or TNFα(Huang et al., 2009
). However, these authors describe a gene-specific requirement for Brd4 based on the recruitment of Brd4 to acetylated p65, while our study suggests that Brd4 is likely to be a general regulator of inducible gene expression through binding to H4K5/8/12Ac. Consistent with an essential role for these histone modifications in gene induction, a prior study showed that acetylation of H4K5/8 correlates strongly with gene expression genome-wide (Wang et al., 2008
). Interestingly, mutation of any one of the lysines 5, 8, or 12 of H4 to arginines resulted in a similar change in gene expression in yeast, suggesting that these residues are interchangeable (at least in the context of transcription) (Dion et al., 2005
). This is consistent with our finding that H4K5, K8 and K12 are all involved in Brd4 recruitment and thus individual mutations at these residues should have the same effect on transcription.
Other histone modifications have been associated with transcriptional elongation, including H2BK123Ub (K120 in humans), H3K36me3, H3K79me3, H2AK119Ub, and H3S10P, either because they map to coding regions, and/or because they are associated with gene expression (Kouzarides, 2007
; Pokholok et al., 2005
; Schubeler et al., 2004
). However, for some of the modifications (H3K36me3, H3K79me3), there is little evidence to suggest a causal role in transcription elongation, especially because they occur downstream of Pol II S2 phosphorylation (Kouzarides, 2007
). Other modifications (H2BK123Ub, H2AK119Ub, and H3S10) may be permissive for, or enhance the rate and efficiency of transcriptional elongation, but have not been directly linked to the recruitment of the essential elongation factor P-TEFb (Ivaldi et al., 2007
; Pavri et al., 2006
; Stock et al., 2007
). Unlike these histone modifications, H4K5/8/12Ac has a unique role in inducible recruitment of Brd4 and P-TEFb and thus appears to be a key switch regulating productive elongation and subsequent transcript processing.
To address this possibility further, we examined the constitutive and LPS-induced recruitment of several HATs. We found that p300/CBP were present at many PRG-Is in unstimulated cells, while PCAF and GCN5 were inducibly recruited to PRGs, suggesting that they may be responsible for the signal-dependent acetylation of H4K5/8/12 (). Consistent with this model, the acetylation of H4K8 at the IFN-β promoter was found to be inhibited by the depletion of PCAF/GCN5, and not p300/CBP (Agalioti et al., 2002
). In addition, p300 interacts with S5-P Pol II, consistent with its constitutive recruitment to PRG-Is in unstimulated macrophages, while PCAF associates with S2-P Pol II (Cho et al., 1998
To account for promoter specificity of PRG-I induction, we hypothesized that inducible DNA-binding transcription factors must contribute, directly or indirectly, to the recruitment of P-TEFb. We found that NF-κB initiates a cascade of events that ultimately leads to the signal-dependent and promoter-specific recruitment of P-TEFb. These data are consistent with reports showing that p65 knockdown inhibits the recruitment of PCAF and cdk9 to the initiation-competent CD80 promoter following stimulation with anti-CD40 (Sharma et al., 2007
). Whether NF-κB directly recruits P-TEFb to PRGs is not clear. Addressing this question may require the generation of NF-κB mutants deficient in P-TEFb binding but not in any other function. However, it should be noted that any role for NF-κB in P-TEFb recruitment is clearly not redundant with the essential activity of Brd4. An important difference between NF-κB-mediated and Brd4-mediated recruitment of P-TEFb is that the former can only recruit P-TEFb to promoters of target genes, whereas the latter may function to recruit and maintain P-TEFb throughout the transcribed region, in proximity to elongating Pol II.
Sp1 recruits Pol II to both PRG-Is and HKGs, yet expression of HKGs is constitutive, while expression of PRG-Is is signal-dependent. What keeps PRG-Is inactive in unstimulated cells? We hypothesized that HDAC-containing corepressors would be constitutively present at the promoters of PRG-Is, but not HKGs, to maintain H4K5/8/12 in a deacetylated form thus preventing PRG-I transcription driven by Sp1. We found that NCoR/HDAC3 and CoREST/HDAC1 complexes are bound to PRGs, but not HKGs, in unstimulated cells and dismissed following stimulation (). These corepressors are most likely recruited by p50 homodimers () or c-Jun/corepressors (Ogawa et al., 2004
), which may serve as ‘placeholders’ in the absence of stimulation to ensure the inducible expression of PRGs following exchange with active p65:p50 and AP-1 heterodimers. Thus, PRGs may have evolved from constitutive genes by acquiring binding sites for inducible transcription factors, which account for both their signal-dependent expression and basal repression. Previous studies have identified several NF-κB-dependent genes that are regulated by NCoR derepression, but the features that stipulate this regulation were unknown (Baek et al., 2002
; Perissi et al., 2004
). Here we show that many PRGs are uniquely regulated by corepressor/HDAC complexes, while most SRGs employ other mechanisms, such as the requirement for nucleosome remodeling, to limit their transcription in the basal state. These findings also emphasize the very distinct roles of constitutive and inducible transcription factors, represented by Sp1 and NF-κB in our system, in controlling PRG induction.
The permissive features of PRG-Is appear to be largely independent of cell-type given that they are shared between macrophages, MEFs, and ES cells (Guenther et al., 2007
). We hypothesized that this would enable their inducibility in a variety of cell types, and indeed, PRG-Is were more likely than PRG-IIs or SRGs to be induced in different cell-types by NF-κB-inducing stimuli (). This is consistent with the role of the ubiquitous transcription factor Sp1 in the regulation of PRG-I expression. Interestingly, PRG-Is were also generally inducible by a range of stimuli, including TLR ligands, TNFα, serum, and TPA (S. Smale, accompanying manuscript). In contrast to PRGs, expression of SRGs is cell type specific, consistent with the fact that cell type specific genes are commonly regulated by lineage specific transcription factors, such as PU.1 and C/EBP in myeloid cells (Feng et al., 2008
). Thus, the distinct regulation of inducible transcription at PRG-Is and SRGs has important implications for their cell type- and signal-specific expression.
Collectively, our results suggest the following model of inducible transcription (). We propose that the model presented here is not restricted to LPS-inducible gene expression; rather, PRGs in a variety of signal-dependent transcriptional programs may be maintained in a permissive state by constitutive transcription factors and regulated by Brd4- and H4K5/8/12Ac-mediated recruitment of P-TEFb initiated by inducible transcription factors. The utilization of this step allows inducibility in multiple cell types by a variety of signals that converge on the signal-dependent transcription factors utilized by a particular gene. Collectively, these data highlight the biological rationale for the regulatory design of inducible transcription.