Adam Burkholder

Stable Pausing by RNA Polymerase II Provides an Opportunity to Target and Integrate Regulatory Signals

Metazoan gene expression is often regulated after the recruitment of RNA polymerase II (Pol II) to promoters, through the controlled release of promoter-proximally paused Pol II into productive RNA synthesis. Despite the prevalence of paused Pol II, very little is known about the dynamics of these early elongation complexes or the fate of the short transcription start site-associated (tss) RNAs they produce. Here, we demonstrate that paused elongation complexes can be remarkably stable, with half-lives exceeding 15 min at genes with inefficient pause release. Promoter-proximal termination by Pol II is infrequent, and released tssRNAs are targeted for rapid degradation. Further, we provide evidence that the predominant tssRNA species observed are nascent RNAs held within early elongation complexes. We propose that stable pausing of polymerase provides a temporal window of opportunity for recruitment of factors to modulate gene expression and that the nascent tssRNA represents an appealing target for these interactions.

Regulating the regulators: the pervasive effects of Pol II pausing on stimulus-responsive gene networks

The expression of many metazoan genes is regulated through controlled release of RNA polymerase II (Pol II) that has paused during early transcription elongation. Pausing is highly enriched at genes in stimulus-responsive pathways, where it has been proposed to poise downstream targets for rapid gene activation. However, whether this represents the major function of pausing in these pathways remains to be determined. To address this question, we analyzed pausing within several stimulus-responsive networks in Drosophila and discovered that paused Pol II is much more prevalent at genes encoding components and regulators of signal transduction cascades than at inducible downstream targets. Within immune-responsive pathways, we found that pausing maintains basal expression of critical network hubs, including the key NF-κB transcription factor that triggers gene activation. Accordingly, loss of pausing through knockdown of the pause-inducing factor NELF leads to broadly attenuated immune gene activation. Investigation of murine embryonic stem cells revealed that pausing is similarly widespread at genes encoding signaling components that regulate self-renewal, particularly within the MAPK/ERK pathway. We conclude that the role of pausing goes well beyond poising-inducible genes for activation and propose that the primary function of paused Pol II is to establish basal activity of signal-responsive networks.

Tracking replication enzymology in vivo by genome-wide mapping of ribonucleotide incorporation.

Ribonucleotides are frequently incorporated into DNA during replication in eukaryotes. Here we map genome-wide distribution of these ribonucleotides as markers of replication enzymology in budding yeast, using a new 5′ DNA end–mapping method, hydrolytic end sequencing (HydEn-seq). HydEn-seq of DNA from ribonucleotide excision repair–deficient strains reveals replicase- and strand-specific patterns of ribonucleotides in the nuclear genome. These patterns support the roles of DNA polymerases α and δ in lagging-strand replication and of DNA polymerase ɛ in leading-strand replication. They identify replication origins, termination zones and variations in ribonucleotide incorporation frequency across the genome that exceed three orders of magnitude. HydEn-seq also reveals strand-specific 5′ DNA ends at mitochondrial replication origins, thus suggesting unidirectional replication of a circular genome. Given the conservation of enzymes that incorporate and process ribonucleotides in DNA, HydEn-seq can be used to track replication enzymology in other organisms.

Heterogeneous polymerase fidelity and mismatch repair bias genome variation and composition

Mutational heterogeneity must be taken into account when reconstructing evolutionary histories, calibrating molecular clocks, and predicting links between genes and disease. Selective pressures and various DNA transactions have been invoked to explain the heterogeneous distribution of genetic variation between species, within populations, and in tissue-specific tumors. To examine relationships between such heterogeneity and variations in leading- and lagging-strand replication fidelity and mismatch repair, we accumulated 40,000 spontaneous mutations in eight diploid yeast strains in the absence of selective pressure. We found that replicase error rates vary by fork direction, coding state, nucleosome proximity, and sequence context. Further, error rates and DNA mismatch repair efficiency both vary by mismatch type, responsible polymerase, replication time, and replication origin proximity. Mutation patterns implicate replication infidelity as one driver of variation in somatic and germline evolution, suggest mechanisms of mutual modulation of genome stability and composition, and predict future observations in specific cancers.

Pausing of RNA polymerase II Regulates Mammalian Developmental Potential Through Control of Signaling Networks

The remarkable capacity for pluripotency and self-renewal in embryonic stem cells (ESCs) requires a finely tuned transcriptional circuitry wherein the pathways and genes that initiate differentiation are suppressed, but poised to respond rapidly to developmental signals. To elucidate transcriptional control in mouse ESCs in the naive, ground state, we defined the distribution of engaged RNA polymerase II (Pol II) at high resolution. We find that promoter-proximal pausing of Pol II is most enriched at genes regulating cell cycle and signal transduction and not, as expected, at developmental or bivalent genes. Accordingly, ablation of the primary pause-inducing factor NELF does not increase expression of lineage markers, but instead causes proliferation defects, embryonic lethality, and dysregulation of ESC signaling pathways. Indeed, ESCs lacking NELF have dramatically attenuated FGF/ERK activity, rendering them resistant to differentiation. This work thus uncovers a key role for NELF-mediated pausing in establishing the responsiveness of stem cells to developmental cues.

The THO complex regulates pluripotency gene mRNA export and controls embryonic stem cell self-renewal and somatic cell reprogramming.

Embryonic stem cell (ESC) self-renewal and differentiation are governed by a broad-ranging regulatory network. Although the transcriptional regulatory mechanisms involved have been investigated extensively, posttranscriptional regulation is still poorly understood. Here we describe a critical role of the THO complex in ESC self-renewal and differentiation. We show that THO preferentially interacts with pluripotency gene transcripts through Thoc5 and is required for self-renewal at least in part by regulating their export and expression. During differentiation, THO loses its interaction with those transcripts due to reduced Thoc5 expression, leading to decreased expression of pluripotency proteins that facilitates exit from self-renewal. THO is also important for the establishment of pluripotency, because its depletion inhibits somatic cell reprogramming and blastocyst development. Together, our data indicate that THO regulates pluripotency gene mRNA export to control ESC self-renewal and differentiation, and therefore uncover a role for this aspect of posttranscriptional regulation in stem cell fate specification.

TRIM28 regulates RNA polymerase II promoter-proximal pausing and pause release.

Promoter-proximal pausing of RNA polymerase II (Pol II) is a major checkpoint in transcription. An unbiased search for new human proteins that could regulate paused Pol II at the HSPA1B gene identified TRIM28. In vitro analyses indicated HSF1-dependent attenuation of Pol II pausing upon TRIM28 depletion, whereas in vivo data revealed de novo expression of HSPA1B and other known genes regulated by paused Pol II upon TRIM28 knockdown. These results were supported by genome-wide ChIP-sequencing analyses of Pol II occupancy that revealed a global role for TRIM28 in regulating Pol II pausing and pause release. Furthermore, in vivo and in vitro mechanistic studies suggest that transcription-coupled phosphorylation regulates Pol II pause release by TRIM28. Collectively, our findings identify TRIM28 as a new factor that modulates Pol II pausing and transcriptional elongation at a large number of mammalian genes.

Widespread transcriptional pausing and elongation control at enhancers

Regulation by gene-distal enhancers is critical for cell type-specific and condition-specific patterns of gene expression. Thus, to understand the basis of gene activity in a given cell type or tissue, we must identify the precise locations of enhancers and functionally characterize their behaviors. Here, we demonstrate that transcription is a nearly universal feature of enhancers in Drosophila and mammalian cells and that nascent RNA sequencing strategies are optimal for identification of both enhancers and superenhancers. We dissect the mechanisms governing enhancer transcription and discover remarkable similarities to transcription at protein-coding genes. We show that RNA polymerase II (RNAPII) undergoes regulated pausing and release at enhancers. However, as compared with mRNA genes, RNAPII at enhancers is less stable and more prone to early termination. Furthermore, we found that the level of histone H3 Lys4 (H3K4) methylation at enhancers corresponds to transcriptional activity such that highly active enhancers display H3K4 trimethylation rather than the H3K4 monomethylation considered a hallmark of enhancers. Finally, our work provides insights into the unique characteristics of superenhancers, which stimulate high-level gene expression through rapid pause release; interestingly, this property renders associated genes resistant to the loss of factors that stabilize paused RNAPII.

Obesity, rather than diet, drives epigenomic alterations in colonic epithelium resembling cancer progression.

While obesity represents one of several risk factors for colorectal cancer in humans, the mechanistic underpinnings of this association remain unresolved. Environmental stimuli, including diet, can alter the epigenetic landscape of DNA cis-regulatory elements affecting gene expression and phenotype. Here, we explored the impact of diet and obesity on gene expression and the enhancer landscape in murine colonic epithelium. Obesity led to the accumulation of histone modifications associated with active enhancers at genomic loci downstream of signaling pathways integral to the initiation and progression of colon cancer. Meanwhile, colon-specific enhancers lost the same histone mark, poising cells for loss of differentiation. These alterations reflect a transcriptional program with many features shared with the program driving colon cancer progression. The interrogation of enhancer alterations by diet in colonic epithelium provides insights into the biology underlying high-fat diet and obesity as risk factors for colon cancer.

Life without TTP: apparent absence of an important anti-inflammatory protein in birds

Both innate and adaptive immunity in birds are different from their mammalian counterparts. Understanding bird immunity is important because of the enormous potential impact of avian infectious diseases, both in their role as food animals and as potential carriers of zoonotic diseases in man. The anti-inflammatory protein tristetraprolin (TTP) is an important component of the mammalian innate immune response, in that it binds to and destabilizes key cytokine mRNAs. TTP knockout mice exhibit a severe systemic inflammatory syndrome, and they are abnormally sensitive to innate immune stimuli such as LPS. TTP orthologs have been found in most vertebrates studied, including frogs. Here, we attempted to identify TTP orthologs in chicken and other birds, using database searches and deep mRNA sequencing. Although sequences encoding the two other widely expressed TTP family members, ZFP36L1 and ZFP36L2, were identified, we did not find sequences corresponding to TTP in any bird species. Sequences corresponding to TTP were identified in both lizards and alligators, close evolutionary relatives of birds. The induction kinetics of Zfp36l1 and Zfp36l2 mRNAs in LPS-stimulated chicken macrophages or serum-stimulated chick embryo fibroblasts did not resemble the normal mammalian TTP response to these stimuli, suggesting that the other two family members might not compensate for the TTP deficiency in regulating rapidly induced mRNA targets. Several mammalian TTP target transcripts have chicken counterparts that contain one or more potential TTP binding sites, raising the possibility that birds express other proteins that subsume TTPs function as a rapidly inducible regulator of AU-rich element (ARE)-dependent mRNA turnover.