Integrator-mediated clustering of poised RNA polymerase II synchronizes histone transcription.

Numerous components of the transcription machinery, including RNA polymerase II (Pol II), accumulate in regions of high local concentration known as clusters, which are thought to facilitate transcription. Using the histone locus of Drosophila nurse cells as a model, we find that Pol II forms long-lived, transcriptionally poised clusters distinct from liquid droplets, which contain unbound and paused Pol II. Depletion of the Integrator complex endonuclease module, but not its phosphatase module or Pol II pausing factors disperses these Pol II clusters. Consequently, histone transcription fails to reach peak levels during S-phase and aberrantly continues throughout the cell cycle. We propose that Pol II clustering is a regulatory step occurring near promoters that limits rapid gene activation to defined times. One Sentence Summary: Using the Drosophila histone locus as a model, we show that clustered RNA polymerase II is poised for synchronous activation.

Assessment of the roles of Spt5-nucleic acid contacts in promoter proximal pausing of RNA polymerase II.

Promoter proximal pausing of RNA polymerase II (Pol II) is a critical transcriptional regulatory mechanism in metazoans that requires the transcription factor DRB sensitivity-inducing factor (DSIF) and the inhibitory negative elongation factor (NELF). DSIF, composed of Spt4 and Spt5, establishes the pause by recruiting NELF to the elongation complex. However, the role of DSIF in pausing beyond NELF recruitment remains unclear. We used a highly purified in vitro system and Drosophila nuclear extract to investigate the role of DSIF in promoter proximal pausing. We identified two domains of Spt5, the KOW4 and NGN domains, that facilitate Pol II pausing. The KOW4 domain promotes pausing through its interaction with the nascent RNA while the NGN domain does so through a short helical motif that is in close proximity to the non-transcribed DNA template strand. Removal of this sequence in Drosophila has a male-specific dominant negative effect. The alpha-helical motif is also needed to support fly viability. We also show that the interaction between the Spt5 KOW1 domain and the upstream DNA helix is required for DSIF association with the Pol II elongation complex. Disruption of the KOW1-DNA interaction is dominant lethal in vivo. Finally, we show that the KOW2-3 domain of Spt5 mediates the recruitment of NELF to the elongation complex. In summary, our results reveal additional roles for DSIF in transcription regulation and identify specific domains important for facilitating Pol II pausing.

Regulation of Promoter Proximal Pausing of RNA Polymerase II in Metazoans.

Regulation of transcription is a tightly choreographed process. The establishment of RNA polymerase II promoter proximal pausing soon after transcription initiation and the release of Pol II into productive elongation are key regulatory processes that occur in early elongation. We describe the techniques and tools that have become available for the study of promoter proximal pausing and their utility for future experiments. We then provide an overview of the factors and interactions that govern a multipartite pausing process and address emerging questions surrounding the mechanism of RNA polymerase II's subsequent advancement into the gene body. Finally, we address remaining controversies and future areas of study.

The C-Terminal Domain of RNA Polymerase II Is a Multivalent Targeting Sequence that Supports Drosophila Development with Only Consensus Heptads.

The C-terminal domain (CTD) of RNA polymerase II (Pol II) is composed of repeats of the consensus YSPTSPS and is an essential binding scaffold for transcription-associated factors. Metazoan CTDs have well-conserved lengths and sequence compositions arising from the evolution of divergent motifs, features thought to be essential for development. On the contrary, we show that a truncated CTD composed solely of YSPTSPS repeats supports Drosophila viability but that a CTD with enough YSPTSPS repeats to match the length of the wild-type Drosophila CTD is defective. Furthermore, a fluorescently tagged CTD lacking the rest of Pol II dynamically enters transcription compartments, indicating that the CTD functions as a signal sequence. However, CTDs with too many YSPTSPS repeats are more prone to localize to static nuclear foci separate from the chromosomes. We propose that the sequence complexity of the CTD offsets aberrant behavior caused by excessive repetitive sequences without compromising its targeting function.

Genetic analysis of the RNA polymerase II CTD in Drosophila.

The Carboxy-terminal Domain (CTD) of RNA polymerase II (Pol II) plays essential roles in regulating gene expression in eukaryotes. Here, we describe multiple genetic approaches for studying the CTD in Drosophila that complement pre-existing molecular analyses of the Pol II CTD in other experimental models. These approaches will allow one to assess the effects of any CTD mutations in a developmentally complex organism. The approaches discussed in this work can in principle, be applied to analyze other transcription components in eukaryotes.

GFZF, a Glutathione S-Transferase Protein Implicated in Cell Cycle Regulation and Hybrid Inviability, Is a Transcriptional Coactivator.

The core promoters of protein-encoding genes play a central role in regulating transcription. M1BP is a transcriptional activator that associates with a core promoter element known as Motif 1 that resides at thousands of genes in Drosophila To gain insight into how M1BP functions, we identified an interacting protein called GFZF. GFZF had been previously identified in genetic screens for factors involved in maintenance of hybrid inviability, the G2-M DNA damage checkpoint, and RAS/mitogen-activated protein kinase (MAPK) signaling, but its contribution to these processes was unknown. Here, we show that GFZF resides in the nucleus and functions as a transcriptional coactivator. In addition, we show that GFZF is a glutathione S-transferase (GST). Thus, GFZF is the first transcriptional coactivator with intrinsic GST activity, and its identification as a transcriptional coactivator provides an explanation for its role in numerous biological processes.

A sequence-specific core promoter-binding transcription factor recruits TRF2 to coordinately transcribe ribosomal protein genes.

Ribosomal protein (RP) genes must be coordinately expressed for proper assembly of the ribosome yet the mechanisms that control expression of RP genes in metazoans are poorly understood. Recently, TATA-binding protein-related factor 2 (TRF2) rather than the TATA-binding protein (TBP) was found to function in transcription of RP genes in Drosophila. Unlike TBP, TRF2 lacks sequence-specific DNA binding activity, so the mechanism by which TRF2 is recruited to promoters is unclear. We show that the transcription factor M1BP, which associates with the core promoter region, activates transcription of RP genes. Moreover, M1BP directly interacts with TRF2 to recruit it to the RP gene promoter. High resolution ChIP-exo was used to analyze in vivo the association of M1BP, TRF2 and TFIID subunit, TAF1. Despite recent work suggesting that TFIID does not associate with RP genes in Drosophila, we find that TAF1 is present at RP gene promoters and that its interaction might also be directed by M1BP. Although M1BP associates with thousands of genes, its colocalization with TRF2 is largely restricted to RP genes, suggesting that this combination is key to coordinately regulating transcription of the majority of RP genes in Drosophila.

Structural heterogeneity in the intrinsically disordered RNA polymerase II C-terminal domain.

RNA polymerase II contains a repetitive, intrinsically disordered, C-terminal domain (CTD) composed of heptads of the consensus sequence YSPTSPS. The CTD is heavily phosphorylated and serves as a scaffold, interacting with factors involved in transcription initiation, elongation and termination, RNA processing and chromatin modification. Despite being a nexus of eukaryotic gene regulation, the structure of the CTD and the structural implications of phosphorylation are poorly understood. Here we present a biophysical and biochemical interrogation of the structure of the full length CTD of Drosophila melanogaster, which we conclude is a compact random coil. Surprisingly, we find that the repetitive CTD is structurally heterogeneous. Phosphorylation causes increases in radius, protein accessibility and stiffness, without disrupting local structural heterogeneity. Additionally, we show the human CTD is also structurally heterogeneous and able to substitute for the D. melanogaster CTD in supporting fly development to adulthood. This finding implicates conserved structural organization, not a precise array of heptad motifs, as important to CTD function.

Phosphorylation induces sequence-specific conformational switches in the RNA polymerase II C-terminal domain.

The carboxy-terminal domain (CTD) of the RNA polymerase II (Pol II) large subunit cycles through phosphorylation states that correlate with progression through the transcription cycle and regulate nascent mRNA processing. Structural analyses of yeast and mammalian CTD are hampered by their repetitive sequences. Here we identify a region of the Drosophila melanogaster CTD that is essential for Pol II function in vivo and capitalize on natural sequence variations within it to facilitate structural analysis. Mass spectrometry and NMR spectroscopy reveal that hyper-Ser5 phosphorylation transforms the local structure of this region via proline isomerization. The sequence context of this switch tunes the activity of the phosphatase Ssu72, leading to the preferential de-phosphorylation of specific heptads. Together, context-dependent conformational switches and biased dephosphorylation suggest a mechanism for the selective recruitment of cis-proline-specific regulatory factors and region-specific modulation of the CTD code that may augment gene regulation in developmentally complex organisms.

Identification of Regions in the Spt5 Subunit of DRB Sensitivity-inducing Factor (DSIF) That Are Involved in Promoter-proximal Pausing.

DRB sensitivity-inducing factor (DSIF or Spt4/5) is a conserved transcription elongation factor that both inhibits and stimulates transcription elongation in metazoans. In Drosophila and vertebrates, DSIF together with negative elongation factor (NELF) associates with RNA polymerase II during early elongation and causes RNA polymerase II to pause in the promoter-proximal region of genes. The mechanism of how DSIF establishes pausing is not known. We constructed Spt5 mutant forms of DSIF and tested their capacity to restore promoter-proximal pausing to DSIF-depleted Drosophila nuclear extracts. The C-terminal repeat region of Spt5, which has been implicated in both inhibition and stimulation of elongation, is dispensable for promoter-proximal pausing. A region encompassing KOW4 and KOW5 of Spt5 is essential for pausing, and mutations in KOW5 specifically shift the location of the pause. RNA cross-linking analysis reveals that KOW5 directly contacts the nascent transcript, and deletion of KOW5 disrupts this interaction. Our results suggest that KOW5 is involved in promoter-proximal pausing through contact with the nascent RNA.

Mapping the Phosphorylation Pattern of Drosophila melanogaster RNA Polymerase II Carboxyl-Terminal Domain Using Ultraviolet Photodissociation Mass Spectrometry.

Phosphorylation of the C-terminal domain of RNA polymerase II (CTD) plays an essential role in eukaryotic transcription by recruiting transcriptional regulatory factors to the active polymerase. However, the scarcity of basic residues and repetitive nature of the CTD sequence impose a huge challenge for site-specific characterization of phosphorylation, hindering our understanding of this crucial biological process. Herein, we apply LC-UVPD-MS methods to analyze post-translational modification along native sequence CTDs. Application of our method to the Drosophila melanogaster CTD reveals the phosphorylation pattern of this model organism for the first time. The divergent nature of fly CTD allows us to derive rules defining how flanking residues affect phosphorylation choice by CTD kinases. Our data support the use of LC-UVPD-MS to decipher the CTD code and determine rules that program its function.

Reconstitution of factor-dependent, promoter proximal pausing in Drosophila nuclear extracts.

Genomic analyses reveal that RNA polymerase II initiates transcription but pauses shortly downstream on thousands of promoters in Drosophila and mammalian cells. Here, we describe the reconstitution of this promoter proximal pausing in nuclear extracts from Drosophila embryos. This approach is useful for dissecting the role(s) of transcription factors in promoter proximal pausing. Most of our studies employ the hsp70 heat shock gene promoter; however, this technique has successfully reconstituted RNA polymerase II pausing downstream of several other Drosophila promoters. A pulse/chase method is employed to restrict incorporation of radiolabel to the 5' portion of the RNA such that the specific activity of most transcripts are nearly identical and the intensity of radioactive RNA bands detected on gels reflects the molar ratios and quantities of each RNA product, regardless of length. The radiolabeled RNAs are isolated by hybridization to a biotinylated oligonucleotide and captured on magnetic beads. We also describe the use of antibodies to investigate mechanistic aspects of promoter proximal pausing.

RNAi screen in Drosophila larvae identifies histone deacetylase 3 as a positive regulator of the hsp70 heat shock gene expression during heat shock.

The transcription regulation of the Drosophila hsp70 gene is a complex process that involves the regulation of multiple steps, including the establishment of paused Pol II and release of Pol II into elongation upon heat shock activation. While the major players involved in the regulation of gene expression have been studied in detail, additional factors involved in this process continue to be discovered. To identify factors involved in hsp70 expression, we developed a screen that capitalizes on a visual assessment of heat shock activation using a hsp70-beta galactosidase reporter and publicly available RNAi fly lines to deplete candidate proteins. We validated the screen by showing that the depletion of HSF, CycT, Cdk9, Nurf 301, or ELL prevented the full induction of hsp70 by heat shock. Our screen also identified the histone deacetylase HDAC3 and its associated protein SMRTER as positive regulators of hsp70 activation. Additionally, we show that HDAC3 and SMRTER contribute to hsp70 gene expression at a step subsequent to HSF-mediated activation and release of the paused Pol II that resides at the promoter prior to heat shock induction.

Negative elongation factor (NELF) coordinates RNA polymerase II pausing, premature termination, and chromatin remodeling to regulate HIV transcription.

A barrier to eradicating HIV infection is targeting and eliminating latently infected cells. Events that contribute to HIV transcriptional latency include repressive chromatin structure, transcriptional interference, the inability of Tat to recruit positive transcription factor b, and poor processivity of RNA polymerase II (RNAP II). In this study, we investigated mechanisms by which negative elongation factor (NELF) establishes and maintains HIV latency. Negative elongation factor (NELF) induces RNAP II promoter proximal pausing and limits provirus expression in HIV-infected primary CD4(+) T cells. Decreasing NELF expression overcomes RNAP II pausing to enhance HIV transcription elongation in infected primary T cells, demonstrating the importance of pausing in repressing HIV transcription. We also show that RNAP II pausing is coupled to premature transcription termination and chromatin remodeling. NELF interacts with Pcf11, a transcription termination factor, and diminishing Pcf11 in primary CD4(+) T cells induces HIV transcription elongation. In addition, we identify NCoR1-GPS2-HDAC3 as a NELF-interacting corepressor complex that is associated with repressed HIV long terminal repeats. We propose a model in which NELF recruits Pcf11 and NCoR1-GPS2-HDAC3 to paused RNAP II, reinforcing repression of HIV transcription and establishing a critical checkpoint for HIV transcription and latency.

Kinetic competition between elongation rate and binding of NELF controls promoter-proximal pausing.

Pausing of RNA polymerase II (Pol II) 20-60 bp downstream of transcription start sites is a major checkpoint during transcription in animal cells. Mechanisms that control pausing are largely unknown. We developed permanganate-ChIP-seq to evaluate the state of Pol II at promoters throughout the Drosophila genome, and a biochemical system that reconstitutes promoter-proximal pausing to define pausing mechanisms. Stable open complexes of Pol II are largely absent from the transcription start sites of most mRNA genes but are present at snRNA genes and the highly transcribed heat shock genes following their induction. The location of the pause is influenced by the timing between when NELF loads onto Pol II and how fast Pol II escapes the promoter region. Our biochemical analysis reveals that the sequence-specific transcription factor, GAF, orchestrates efficient pausing by recruiting NELF to promoters before transcription initiation and by assisting in loading NELF onto Pol II after initiation.

Distinct mechanisms of transcriptional pausing orchestrated by GAGA factor and M1BP, a novel transcription factor.

Thousands of genes in Drosophila have Pol II paused in the promoter proximal region. Almost half of these genes are associated with either GAGA factor (GAF) or a newly discovered factor we call M1BP. Although both factors dictate the association of Pol II at their target promoters, they are nearly mutually exclusive on the genome and mediate different mechanisms of regulation. High-resolution mapping of Pol II using permanganate-ChIP-seq indicates that pausing on M1BP genes is transient and could involve the +1 nucleosome. In contrast, pausing on GAF genes is much stronger and largely independent of nucleosomes. Distinct regulatory mechanisms are reflected by transcriptional plasticity: M1BP genes are constitutively expressed throughout development while GAF genes exhibit much greater developmental specificity. M1BP binds a core promoter element called Motif 1. Motif 1 potentially directs a distinct transcriptional mechanism from the canonical TATA box, which does not correlate with paused Pol II on the genomic scale. In contrast to M1BP and GAF genes, a significant portion of TATA box genes appear to be controlled at preinitiation complex formation.

Paused Pol II coordinates tissue morphogenesis in the Drosophila embryo.

Paused RNA polymerase (Pol II) is a pervasive feature of Drosophila embryos and mammalian stem cells, but its role in development is uncertain. Here, we demonstrate that a spectrum of paused Pol II determines the "time to synchrony"-the time required to achieve coordinated gene expression across the cells of a tissue. To determine whether synchronous patterns of gene activation are significant in development, we manipulated the timing of snail expression, which controls the coordinated invagination of ∼1,000 mesoderm cells during gastrulation. Replacement of the strongly paused snail promoter with moderately paused or nonpaused promoters causes stochastic activation of snail expression and increased variability of mesoderm invagination. Computational modeling of the dorsal-ventral patterning network recapitulates these variable and bistable gastrulation profiles and emphasizes the importance of timing of gene activation in development. We conclude that paused Pol II and transcriptional synchrony are essential for coordinating cell behavior during morphogenesis.

Chromatin-associated RNA interference components contribute to transcriptional regulation in Drosophila.

RNA interference (RNAi) pathways have evolved as important modulators of gene expression that operate in the cytoplasm by degrading RNA target molecules through the activity of short (21-30 nucleotide) RNAs. RNAi components have been reported to have a role in the nucleus, as they are involved in epigenetic regulation and heterochromatin formation. However, although RNAi-mediated post-transcriptional gene silencing is well documented, the mechanisms of RNAi-mediated transcriptional gene silencing and, in particular, the role of RNAi components in chromatin dynamics, especially in animal multicellular organisms, are elusive. Here we show that the key RNAi components Dicer 2 (DCR2) and Argonaute 2 (AGO2) associate with chromatin (with a strong preference for euchromatic, transcriptionally active, loci) and interact with the core transcription machinery. Notably, loss of function of DCR2 or AGO2 showed that transcriptional defects are accompanied by the perturbation of RNA polymerase II positioning on promoters. Furthermore, after heat shock, both Dcr2 and Ago2 null mutations, as well as missense mutations that compromise the RNAi activity, impaired the global dynamics of RNA polymerase II. Finally, the deep sequencing of the AGO2-associated small RNAs (AGO2 RIP-seq) revealed that AGO2 is strongly enriched in small RNAs that encompass the promoter regions and other regions of heat-shock and other genetic loci on both the sense and antisense DNA strands, but with a strong bias for the antisense strand, particularly after heat shock. Taken together, our results show that DCR2 and AGO2 are globally associated with transcriptionally active loci and may have a pivotal role in shaping the transcriptome by controlling the processivity of RNA polymerase II.

Cohesin selectively binds and regulates genes with paused RNA polymerase.

BACKGROUND: The cohesin complex mediates sister chromatid cohesion and regulates gene transcription. Prior studies show that cohesin preferentially binds and regulates genes that control growth and differentiation and that even mild disruption of cohesin function alters development. Here we investigate how cohesin specifically recognizes and regulates genes that control development in Drosophila. RESULTS: Genome-wide analyses show that cohesin selectively binds genes in which RNA polymerase II (Pol II) pauses just downstream of the transcription start site. These genes often have GAGA factor (GAF) binding sites 100 base pairs (bp) upstream of the start site, and GT dinucleotide repeats 50 to 800 bp downstream in the plus strand. They have low levels of histone H3 lysine 36 trimethylation (H3K36me3) associated with transcriptional elongation, even when highly transcribed. Cohesin depletion does not reduce polymerase pausing, in contrast to depletion of the NELF (negative elongation factor) pausing complex. Cohesin, NELF, and Spt5 pausing and elongation factor knockdown experiments indicate that cohesin does not inhibit binding of polymerase to promoters or physically block transcriptional elongation, but at genes that it strongly represses, it hinders transition of paused polymerase to elongation at a step distinct from those controlled by Spt5 and NELF. CONCLUSIONS: Our findings argue that cohesin and pausing factors are recruited independently to the same genes, perhaps by GAF and the GT repeats, and that their combined action determines the level of actively elongating RNA polymerase.