SnapShot: Transcription Regulation: Pausing George Fromm, Daniel A. Gilchrist, and Karen Adelman Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA

Assembly of the preinitiation complex (PIC) and initiation

DNA-binding transcription factors (TFs) associate with cis-regulatory elements (CRE) to promote assembly of the PIC. TFs can stimulate PIC formation directly through interactions with the Mediator complex and help recruit the general transcription factors (GTFs). TFs also recruit coactivators that can remove nucleosomes from the promoter or modify histone proteins, for example by acetylation (Ac) or methylation (Me). Coactivator Transcription start site TF Mediator Pol II Me Ac Ac CRE CRE Nucleosome GTFs TFIIH CTD Initiation of RNA synthesis requires the ATP-dependent opening of the DNA strands by the TFIIH helicase. The Pol II C-terminal domain (CTD) is comprised of many repeats of the

sequence Y1S2P3T4S5P6S7 that undergo posttranslational modi cations during the transcription cycle but are generally unmodi ed in the PIC.

Pausing during early elongation

After a 20–60 nt RNA has been synthesized, pause-inducing factors DSIF and NELF bind the early elongation complex, inhibiting further transcription. Levels of promoter Pol II often The duration of pausing depends on the rate of recruitment of factors that trigger correlate with the amount of pause release, which is variable from gene to gene and under different cell conditions. trimethylation of histone H3

at the K4 position. TF Mediator Me Ac Ac CRE CRE P GTFs TFIIH CE 5´

The GTF component TFIIH contains the kinase Cdk7, which The capping enzyme complex (CE) adds a protective 5´ cap to the nascent RNA. CE recruitment is stimulated by its phosphorylates the Pol II CTD at the S5 and S7 residues. interactions with DSIF and S5-phosphorylated CTD.

Pause release

Release of paused Pol II is triggered by the kinase P-TEFb, Modes of P-TEFb Recruitment: which phosphorylates pause-inducing factors to dissociate TFs (e.g., MYC, NF-κB) NELF and promote productive elongation. Mediator (Med26 subunit or Cdk8 module) Acetylated histones and Brd4 Other elongation factors as part of the superelongation complex

NELF TF TF P-TEFb Brd4 Mediator Me P Ac Ac CRE DSIF P P GTFs TFIIH

5´

Productive elongation

Following phosphorylation by P-TEFb, DSIF helps recruit factors that stimulate productive elongation. Transcription through nucleosomes and their ef cient These factors (e.g., Paf1c) enhance the rate or processivity of RNA synthesis. reassembly in the wake of transcribing Pol II are facilitated by histone chaperones like FACT and Spt6.

TF TF FACT Mediator P Me Ac RNA Ac CRE CRE proc DSIF P P GTFs TFIIH P

5´

The Pol II CTD is also phosphorylated at S2 by P-TEFb and the kinase Cdk12/cyclinK, creating a binding platform for factors that aid in RNA processing and transcription through chromatin.

930 Cell 153, May 9, 2013 ©2013 Elsevier Inc. DOI http://dx.doi.org/10.1016/j.cell.2013.04.011 See online version for legend and references. SnapShot: Transcription Regulation: Pausing George Fromm, Daniel A. Gilchrist, and Karen Adelman Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA

This SnapShot highlights recent advances in our understanding of gene expression in higher eukaryotes, with an emphasis on the regulation of transcription elongation through the promoter-proximal pausing of RNA polymerase II (Pol II). We illustrate how Pol II pausing can facilitate coordination between transcription, chromatin features, and RNA- processing machineries to generate finely tuned gene expression programs. We outline the sequence of events leading from assembly of the Pol II transcription machinery at a gene promoter to productive RNA synthesis.

Assembly of a Preinitiation Complex and Initiation Recruitment of Pol II and the general transcription factors (GTFs) is regulated by the association of DNA-binding transcription factors (TFs) with specific DNA sequences, known as cis-regulatory elements (CRE). The transcription activation domains of TFs participate in a number of interactions with other proteins and bring them to the promoter region. These binding partners include coactivators that can remodel or modify nucleosomes to generate an accessible chromatin architecture around the promoter. Further, TFs often interact with the large Mediator complex that serves as a bridge between TFs and general transcription machinery, stabilizing the association of GTFs, Pol II, and several addi- tional coactivators. We note that TFs bound within the promoter-proximal region often work over long distances in conjunction with TFs located in enhancer regions. Once the preinitiation complex (PIC) has formed, a helicase component of the GTFs, TFIIH, uses the energy of ATP hydrolysis to separate the DNA strands around the transcription start site, giving Pol II access to the template DNA and enabling the initiation of RNA synthesis.

Pausing during Early Elongation When the nascent RNA reaches ~20 nucleotides (nt) in length, two pause-inducing factors, DRB-sensitivity-inducing factor (DSIF) and negative elongation factor (NELF), associ- ate with the early elongation complex and the RNA transcript, inhibiting further synthesis. Genomic location analysis of DSIF and NELF indicate that they are globally present with Pol II near promoters, suggesting that Pol II transitions through a paused phase at nearly all genes. The duration of pausing, however, is highly variable from gene to gene (from several seconds to many minutes) and depends upon the rate of recruitment of pause-release factors.

The early elongation complex undergoes phosphorylation on the Pol II C-terminal domain (CTD) by the Cdk7 component of TFIIH. Cdk7 targets both S5 and S7 residues on the CTD, and both modifications are enriched promoter proximally. Pausing of Pol II and CTD phosphorylation are both thought to enhance the efficiency of capping the′ 5 end

of the RNA because the capping enzyme complex (CE) interacts with DSIF as well as the S5-phosphorylated CTD.

Pause Release The transcriptional roadblock caused by DSIF and NELF is relieved through phosphorylation of the pause-inducing factors by the kinase P-TEFb. Phosphorylation is thought to dissociate NELF from Pol II and convert DSIF into a positive elongation factor that enhances productive RNA synthesis. Multiple mechanisms trigger recruitment of P-TEFb and release of the paused elongation complex, including TFs, chromatin modifications, and coregulatory complexes. Interestingly, P-TEFb is recruited to the HIV LTR through its interaction with the Tat protein and TAR RNA, but no cellular genes have been yet reported to employ a structured nascent RNA to attract P-TEFb. Importantly, the TFs that stimulate recruitment of P-TEFb can often be distinct from those that regulate assembly of the PIC, such that TFs that control initiation and elongation phases can work together to determine transcription output.

Productive Elongation As Pol II transitions to a fully elongation-competent form, it is bound by a number of factors that assist it in the journey downstream. Some of these factors may act directly upon Pol II to increase the rate or processivity of transcription elongation, whereas many others facilitate the disassembly and reassembly of nucleosomes in the path of transcribing

Pol II. A hallmark of the fully elongation-competent Pol II complex is phosphorylation of the CTD at S2. This modification is carried out by P-TEFb and is likely augmented by the kinase Cdk12/CyclinK as Pol II moves downstream into the gene. The S2-phosphorylated CTD provides a large binding surface for the entourage of factors that travel with the elongation complex through the gene body, including chromatin-modifying enzymes and RNA-processing machineries that carry out splicing and 3′ end formation. Thus, P-TEFb plays a key role in both pause release and the recruitment of factors that stimulate efficient production and maturation of RNA, enabling coordination of these processes.

Abbreviations Ac or Me, acetyl or methyl group; Brd4, bromodomain-containing protein 4; CE, capping enzyme complex; CRE, cis-regulatory element; CTD, Pol II Rpb1 subunit C-terminal domain; DSIF, DRB-sensitivity-inducing factor; FACT, facilitates chromatin transcription; GTF, general transcription factor; NELF, negative elongation factor; nt, nucleotides; P-TEFb, positive transcription elongation factor-b, comprised of Cdk9 and cyclin T; Mediator, large coregulatory complex comprised of multiple proteins and modules, including Med26 and Cdk8; P, phosphorylated serine residue on the Pol II CTD; Paf1c, polymerase-associated factor 1 complex; PIC, preinitiation complex; Pol II, RNA polymerase II; RNA proc, RNA-processing factors; TF, DNA-binding transcription factor; TFIIH, general transcription factor IIH.

Acknowledgments

We thank members of the Adelman lab for helpful comments. This research was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences to K.A. (Z01 ES101987).

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