Our data support a general model for gene regulation wherein the underlying DNA sequence around promoters directly influences both chromatin architecture and the step in the transcription cycle that is rate-limiting for gene expression. We find that genes with high levels of Pol II pausing () inherently favor the formation of nucleosomes over the promoter, establishing an active competition between Pol II and nucleosomes for promoter occupancy. We propose that this intrinsically repressive chromatin structure prevents aberrant expression of paused genes, which are often components of highly-regulated pathways. Nucleosome remodeling, likely initiated by proteins such as GAGA factor, would be required to disassemble nucleosomes at these promoters and allow for gene activity (small red arrow). Nucleosome removal would uncover strong promoter motifs that facilitate efficient, stable recruitment of the transcription machinery (large green arrow). Extended NELF-mediated pausing of polymerase at these promoters makes the transition to productive elongation slow (small red arrow). However, upon pause release, low levels of downstream nucleosomes would minimize barriers to transcription elongation and additional Pol II molecules would be rapidly recruited to maintain high Pol II occupancy and prevent nucleosome formation.
Different Default Chromatin Architectures Specify Distinct Gene Regulatory Strategies
In contrast, genes that lack extended pausing () appear to disfavor promoter nucleosome assembly and instead harbor nucleosomes flanking the nucleosome-deprived promoter region. Localized DNA accessibility near TSSs could both help target the transcription machinery to the promoter region and diminish the requirement for nucleosome remodeling to allow gene activity. The dearth of core promoter elements could make these genes more reliant on activator binding for recruitment of the transcription machinery, and Pol II recruitment would be the rate-limiting step for expression of these genes (small red arrow). Pausing would be short-lived at these genes, and despite higher downstream nucleosome occupancy, polymerase escapes efficiently into productive synthesis.
Importantly, these two strategies present different opportunities for gene regulation. Highly paused genes present two distinct steps at which they can be regulated: promoter accessibility and release of Pol II from pausing. We propose that this two-step mechanism facilitates precise control of gene expression. We envision that the first step, nucleosome remodeling, functions as a molecular switch that relieves repression by chromatin to permit expression. This step can be temporally uncoupled from gene activation and could potentiate genes for future activation rather than prompting their immediate expression. The second step, release of paused Pol II, might be analogous to a volume dial, which permits fine-tuning of expression levels in response to changing conditions. Transcription levels could be rapidly regulated solely by manipulating the efficiency of P-TEFb recruitment through its interactions with DNA-binding transcription activators and histone modifications (Peterlin and Price, 2006
; Rahl et al., 2010
). This idea is supported by observations that activation of highly paused genes is both fast and synchronous (Lis, 1998
; Boettiger and Levine, 2009
). In contrast, genes that lack promoter-proximal pausing and nucleosome occupancy rely chiefly on a single-step mechanism to alter gene expression: regulated, step-wise recruitment of the transcription machinery. This mode of regulation has been suggested to be inherently more stochastic and prone to transcriptional noise (Boettiger and Levine, 2009
), which may explain why many genes regulated by recruitment are constitutively active housekeeping genes.
We provide evidence that NELF-mediated pausing during early elongation is a general feature of the transcription cycle that is exploited at some genes to regulate transcription output. We propose that each round of transcription entails pausing, perhaps serving as an early “checkpoint” to ensure proper maturation of the elongation complex before release into productive elongation. At some genes, this halt in elongation may be transient, whereas at others it may involve a long-lived paused complex that becomes rate-limiting for gene expression. Importantly, these results imply that the release from pausing through P-TEFb recruitment is an important, regulated step that broadly impacts gene expression, in agreement with recent work (Peterlin and Price, 2006
; Rahl et al., 2010
). We note that general recruitment of NELF during early elongation likely explains the seemingly paradoxical observation made in several systems that NELF levels increase at activated genes that experience robust recruitment of additional Pol II.
Our data also reveal that the inherent preference towards repression of highly-regulated promoters by nucleosome occlusion is an evolutionarily conserved phenomenon (Tirosh and Barkai, 2008
). Moreover, our results are in agreement with recent work in yeast which reveals that Pol II plays a role in displacing nucleosomes from promoter regions (Weiner et al., 2009
). However, in yeast, nucleosome disassembly is coupled directly to gene activation, whereas in Drosophila
nucleosome disassembly is coupled to Pol II pausing. Perhaps Drosophila
and other metazoans have evolved promoter-proximal pausing as an additional layer of regulation to accommodate increased demands for precise and rapid gene regulation during development and organismal responses to stress. In addition, it might be beneficial to maintain highly-regulated promoters poised in an open chromatin state, to prevent their incorporation into the more inaccessible, condensed heterochromatin that exists in metazoans.
In summary, we report that a primary function of paused Pol II is to prevent promoter-proximal nucleosome formation. This represents a fundamental shift in our thinking about the role of Pol II pausing, which has long been thought to simply repress gene expression. Instead, we argue that pausing should be viewed as a mechanism to fine-tune gene expression, and to potentiate genes for further or future activation. In addition, we have shown that sequence-specified “default” nucleosome architecture instructs the regulatory properties of Drosophila promoters. We propose that metazoans have evolved a gene regulatory strategy in which nucleosomes and paused Pol II compete for promoter occupancy, affording multiple opportunities for regulation of gene expression.