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A genetic tool that converts bacterial regulators of translational initiation into regulators of transcriptional elongation is described. This adaptor is used to engineer several transcriptional attenuators and activators that can be predictably assembled into higher-order gene regulatory functions. Bacterial regulators of transcriptional elongation are versatile units for building custom genetic switches, as they control the expression of both coding and noncoding RNAs, act on multigene operons and can be predictably tethered into higher-order regulatory functions (a property called composability). Yet the less versatile bacterial regulators of translational initiation are substantially easier to engineer. To bypass this tradeoff, we have developed an adaptor that converts regulators of translational initiation into regulators of transcriptional elongation in Escherichia coli . We applied this adaptor to the construction of several transcriptional attenuators and activators, including a small molecule–triggered attenuator and a group of five mutually orthogonal riboregulators that we assembled into NOR gates of two, three or four RNA inputs. Continued application of our adaptor should produce large collections of transcriptional regulators whose inherent composability can facilitate the predictable engineering of complex synthetic circuits.

The regulated transcription of genes determines cell identity and function. Recent structural studies have elucidated mechanisms that govern the regulation of transcription by RNA polymerases during the initiation and elongation phases. Microscopy studies have revealed that transcription involves the condensation of factors in the cell nucleus. A model is emerging for the transcription of protein-coding genes in which distinct transient condensates form at gene promoters and in gene bodies to concentrate the factors required for transcription initiation and elongation, respectively. The transcribing enzyme RNA polymerase II may shuttle between these condensates in a phosphorylation-dependent manner. Molecular principles are being defined that rationalize transcriptional organization and regulation, and that will guide future investigations. Structural and microscopy studies of gene transcription underpin a model in which phosphorylation controls the shuttling of RNA polymerase II between promoter and gene-body condensates to regulate transcription initiation and elongation.

Key Points RNA polymerase II (Pol II) elongation is a highly regulated process. Regulation of transcription is often mediated at the level of promoter-proximal pausing of Pol II, in which Pol II is paused approximately 30–60 nucleotides downstream of the transcription start site (TSS) and awaits recruitment of kinase positive transcription elongation factor-b (P-TEFb). P-TEFb is the main factor required to release paused Pol II from the promoter-proximal region, and can directly or indirectly be recruited by many factors, including bromodomain-containing protein 4 (BRD4) and the super elongation complex (SEC). Elongation rates throughout the gene body are not uniform but vary between, and within genes, and can range from ∼1 to 6 kb per minute. Transient slowdown of Pol II is observed up to 15 kb downstream of the TSS, at exons and near the poly(A) cleavage site. Elongation rates can affect co-transcriptional RNA processes such as splicing and termination, as well as genome stability. Pausing of RNA polymerase II (Pol II) in promoter-proximal regions and its release to initiate productive elongation are key steps in the regulation of transcription, and involve many factors. Evidence is now emerging that transcriptional elongation is highly dynamic. Elongation rates vary between genes and across the length of a gene, affecting splicing, termination and genome stability. Recent advances in sequencing techniques that measure nascent transcripts and that reveal the positioning of RNA polymerase II (Pol II) have shown that the pausing of Pol II in promoter-proximal regions and its release to initiate a phase of productive elongation are key steps in transcription regulation. Moreover, after the release of Pol II from the promoter-proximal region, elongation rates are highly dynamic throughout the transcription of a gene, and vary on a gene-by-gene basis. Interestingly, Pol II elongation rates affect co-transcriptional processes such as splicing, termination and genome stability. Increasing numbers of factors and regulatory mechanisms have been associated with the steps of transcription elongation by Pol II, revealing that elongation is a highly complex process. Elongation is thus now recognized as a key phase in the regulation of transcription by Pol II.

Trimethylation of histone H3 lysine 4 (H3K4me3) is associated with transcriptional start sites and has been proposed to regulate transcription initiation 1 , 2 . However, redundant functions of the H3K4 SET1/COMPASS methyltransferase complexes complicate the elucidation of the specific role of H3K4me3 in transcriptional regulation 3 , 4 . Here, using mouse embryonic stem cells as a model system, we show that acute ablation of shared subunits of the SET1/COMPASS complexes leads to a complete loss of all H3K4 methylation. Turnover of H3K4me3 occurs more rapidly than that of H3K4me1 and H3K4me2 and is dependent on KDM5 demethylases. Notably, acute loss of H3K4me3 does not have detectable effects on transcriptional initiation but leads to a widespread decrease in transcriptional output, an increase in RNA polymerase II (RNAPII) pausing and slower elongation. We show that H3K4me3 is required for the recruitment of the integrator complex subunit 11 (INTS11), which is essential for the eviction of paused RNAPII and transcriptional elongation. Thus, our study demonstrates a distinct role for H3K4me3 in transcriptional pause-release and elongation rather than transcriptional initiation. Acute loss of H3K4me3 does not have detectable effects on transcriptional initiation, but leads to a widespread decrease in transcriptional output, an increase in RNA polymerase II pausing and slower elongation

The journey of RNA polymerase II (Pol II) as it transcribes a gene is anything but a smooth ride. Transcript elongation is discontinuous and can be perturbed by intrinsic regulatory barriers, such as promoter-proximal pausing, nucleosomes, RNA secondary structures and the underlying DNA sequence. More substantial blocking of Pol II translocation can be caused by other physiological circumstances and extrinsic obstacles, including other transcribing polymerases, the replication machinery and several types of DNA damage, such as bulky lesions and DNA double-strand breaks. Although numerous different obstacles cause Pol II stalling or arrest, the cell somehow distinguishes between them and invokes different mechanisms to resolve each roadblock. Resolution of Pol II blocking can be as straightforward as temporary backtracking and transcription elongation factor S-II (TFIIS)-dependent RNA cleavage, or as drastic as premature transcription termination or degradation of polyubiquitylated Pol II and its associated nascent RNA. In this Review, we discuss the current knowledge of how these different Pol II stalling contexts are distinguished by the cell, how they overlap with each other, how they are resolved and how, when unresolved, they can cause genome instability.Transcript elongation by RNA polymerase II can be perturbed by barriers such as promoter-proximal pausing and nucleosomes and by obstacles such as the replication machinery and DNA lesions. Recent studies revealed how different contexts of RNA polymerase II stalling are distinguished and resolved, and how unresolved stalling can cause genome instability.

Metazoan gene regulation often involves the pausing of RNA polymerase II (Pol II) in the promoter-proximal region. Paused Pol II is stabilized by the protein complexes DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF). Here we report the cryo-electron microscopy structure of a paused transcription elongation complex containing Sus scrofa Pol II and Homo sapiens DSIF and NELF at 3.2 Å resolution. The structure reveals a tilted DNA–RNA hybrid that impairs binding of the nucleoside triphosphate substrate. NELF binds the polymerase funnel, bridges two mobile polymerase modules, and contacts the trigger loop, thereby restraining Pol II mobility that is required for pause release. NELF prevents binding of the anti-pausing transcription elongation factor IIS (TFIIS). Additionally, NELF possesses two flexible ‘tentacles’ that can contact DSIF and exiting RNA. These results define the paused state of Pol II and provide the molecular basis for understanding the function of NELF during promoter-proximal gene regulation. The cryo-electron microscopy structure of a paused transcription elongation complex of RNA polymerase II bound to DRB sensitivity-inducing factor and negative elongation factor is reported at 3.2 Å resolution.

Gene regulation involves activation of RNA polymerase II (Pol II) that is paused and bound by the protein complexes DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF). Here we show that formation of an activated Pol II elongation complex in vitro requires the kinase function of the positive transcription elongation factor b (P-TEFb) and the elongation factors PAF1 complex (PAF) and SPT6. The cryo-EM structure of an activated elongation complex of Sus scrofa Pol II and Homo sapiens DSIF, PAF and SPT6 was determined at 3.1 Å resolution and compared to the structure of the paused elongation complex formed by Pol II, DSIF and NELF. PAF displaces NELF from the Pol II funnel for pause release. P-TEFb phosphorylates the Pol II linker to the C-terminal domain. SPT6 binds to the phosphorylated C-terminal-domain linker and opens the RNA clamp formed by DSIF. These results provide the molecular basis for Pol II pause release and elongation activation. The cryo-electron microscopy structure of an activated transcription elongation complex of RNA polymerase II bound to DRB sensitivity-inducing factor and the elongation factors PAF1 complex and SPT6 is reported at 3.1 Å resolution.

ENL, identified in a genome-scale loss-of-function screen as a crucial requirement for proliferation of acute leukaemia, is required for leukaemic gene expression, and its YEATS chromatin-reader domain is essential for leukaemic growth. Gene control in acute leukaemia Recurrent chromosomal translocations involving the mixed lineage leukaemia (MLL) gene give rise to acute myeloid leukaemia (AML). Here, James Bradner and colleagues perform a genome-scale loss-of-function screen using CRISPR–Cas9 technology in MLL-AF4 AML cells. They find that the ENL gene is critical for cell proliferation and use a chemical genetic strategy of targeted protein degradation to show that loss of ENL suppresses transcriptional activation as well as leukaemic growth. ENL-dependent leukaemic growth depends on its YEATS chromatin reader domain, indicating that competitive antagonists of the YEATS domain could be potential therapeutics for AML. A related paper in this week's issue of Nature from Xiaobing Shi and colleagues provides insights into the function of the ENL YEATS domain in recognizing acetylated histones. Recurrent chromosomal translocations producing a chimaeric MLL oncogene give rise to a highly aggressive acute leukaemia associated with poor clinical outcome 1 . The preferential involvement of chromatin-associated factors as MLL fusion partners belies a dependency on transcription control 2 . Despite recent progress made in targeting chromatin regulators in cancer 3 , available therapies for this well-characterized disease remain inadequate, prompting the need to identify new targets for therapeutic intervention. Here, using unbiased CRISPR–Cas9 technology to perform a genome-scale loss-of-function screen in an MLL-AF4-positive acute leukaemia cell line, we identify ENL as an unrecognized gene that is specifically required for proliferation in vitro and in vivo . To explain the mechanistic role of ENL in leukaemia pathogenesis and dynamic transcription control, a chemical genetic strategy was developed to achieve targeted protein degradation. Acute loss of ENL suppressed the initiation and elongation of RNA polymerase II at active genes genome-wide, with pronounced effects at genes featuring a disproportionate ENL load. Notably, an intact YEATS chromatin-reader domain was essential for ENL-dependent leukaemic growth. Overall, these findings identify a dependency factor in acute leukaemia and suggest a mechanistic rationale for disrupting the YEATS domain in disease.

Physiological homeostasis becomes compromised during ageing, as a result of impairment of cellular processes, including transcription and RNA splicing 1 – 4 . However, the molecular mechanisms leading to the loss of transcriptional fidelity are so far elusive, as are ways of preventing it. Here we profiled and analysed genome-wide, ageing-related changes in transcriptional processes across different organisms: nematodes, fruitflies, mice, rats and humans. The average transcriptional elongation speed (RNA polymerase II speed) increased with age in all five species. Along with these changes in elongation speed, we observed changes in splicing, including a reduction of unspliced transcripts and the formation of more circular RNAs. Two lifespan-extending interventions, dietary restriction and lowered insulin–IGF signalling, both reversed most of these ageing-related changes. Genetic variants in RNA polymerase II that reduced its speed in worms 5 and flies 6 increased their lifespan. Similarly, reducing the speed of RNA polymerase II by overexpressing histone components, to counter age-associated changes in nucleosome positioning, also extended lifespan in flies and the division potential of human cells. Our findings uncover fundamental molecular mechanisms underlying animal ageing and lifespan-extending interventions, and point to possible preventive measures. Increases in transcriptional elongation speed with age affect organismal lifespan and ageing-related changes could be reversed with lifespan-extending interventions.

Transcription of a long non-coding RNA, known as upperhand ( Uph ) located upstream of the HAND2 transcription factor is required to maintain transcription of the Hand2 gene by RNA polymerase, and blockade of Uph expression leads to heart defects and embryonic lethality in mice. upperhand regulates Hand2 expression in early cardiogenesis The expression of the transcription factor HAND2 is controlled by several upstream enhancer elements, confined in a region delimited by the presence of the chromatin mark H3K27Ac. Eric Olson and colleagues have found that the transcription of long non-coding RNA located upstream of HAND2 is required to maintained these chromatin marks and let the RNA polymerase transcribe the Hand2 gene. Preventing the expression of this long non-coding RNA with a termination cassette leads to defects in heart development in mice. HAND2 is an ancestral regulator of heart development and one of four transcription factors that control the reprogramming of fibroblasts into cardiomyocytes 1 , 2 , 3 , 4 . Deletion of Hand2 in mice results in right ventricle hypoplasia and embryonic lethality 1 , 5 . Hand2 expression is tightly regulated by upstream enhancers 6 , 7 that reside within a super-enhancer delineated by histone H3 acetyl Lys27 (H3K27ac) modifications 8 . Here we show that transcription of a Hand2- associated long non-coding RNA, which we named upperhand ( Uph ), is required to maintain the super-enhancer signature and elongation of RNA polymerase II through the Hand2 enhancer locus. Blockade of Uph transcription, but not knockdown of the mature transcript, abolished Hand2 expression, causing right ventricular hypoplasia and embryonic lethality in mice. Given the substantial number of uncharacterized promoter-associated long non-coding RNAs encoded by the mammalian genome 9 , the Uph – Hand2 regulatory partnership offers a mechanism by which divergent non-coding transcription can establish a permissive chromatin environment.