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.

A systematic review and meta-analysis of the effect of intravitreal VEGF inhibitors on cardiorenal outcomes.

BACKGROUND: Vascular endothelial growth factor inhibitors (VEGFis) have transformed the treatment of many retinal diseases, including diabetic maculopathy. Increasing evidence supports systemic absorption of intravitreal VEGFi and development of significant cardiorenal side effects. METHODS: We conducted a systematic review and meta-analysis (PROSPERO: CRD42020189037) of randomised controlled trials of intravitreal VEGFi treatments (bevacizumab, ranibizumab and aflibercept) for any eye disease. Outcomes of interest were cardiorenal side effects (hypertension, proteinuria, kidney function decline and heart failure). Fixed effects meta-analyses were conducted where possible. RESULTS: There were 78 trials (81 comparisons; 13 175 participants) that met the criteria for inclusion: 47% were trials in diabetic eye disease. Hypertension (29 trials; 8570 participants) was equally common in VEGFi and control groups {7.3 versus 5.4%; relative risk [RR] 1.08 [95% confidence interval (CI) 0.91-1.28]}. New or worsening heart failure (10 trials; 3384 participants) had a similar incidence in VEGFi and control groups [RR 1.03 (95% CI 0.70-1.51)]. Proteinuria (5 trials; 1902 participants) was detectable in some VEGFi-treated participants (0.2%) but not controls [0.0%; RR 4.43 (95% CI 0.49-40.0)]. Kidney function decline (9 trials; 3471 participants) was similar in VEGFi and control groups. In participants with diabetic eye disease, the risk of all-cause mortality was higher in VEGFi-treated participants [RR 1.62 (95% CI 1.04-2.46)]. CONCLUSION: In trials of intravitreal VEGFi, we did not identify an increased risk of cardiorenal outcomes, although these outcomes were reported in only a minority of cases. There was an increased risk of death in VEGFi-treated participants with diabetic eye disease. Additional scrutiny of post-licensing observational data may improve the recognition of safety concerns in VEGFi-treated patients.

An Unusual Case of Unilateral Maculopathy.

A 44-year-old male presented with unilateral sudden onset reduced visual acuity. The optical coherence tomography (OCT) scan demonstrated submacular fluid with thickening and hyper-reflectivity of the outer retinal layers, together with subfoveal retinal pigment epithelial hyper-reflectivity corresponding to a small area of foveal interdigitation zone/ellipsoid zone (IZ/EZ) loss in the detached retina. An OCT 4 months later showed resolution of the submacular fluid, but the IZ/EZ loss persisted with thinning of the outer nuclear layer, resulting in a poor visual outcome. The clinical findings most likely represent a case of unilateral acute idiopathic maculopathy; however, the OCT features and poor visual outcome are not typical. Differential diagnoses include acute solar maculopathy, central serous chorioretinopathy, poppers maculopathy, whiplash maculopathy, and acute retinal pigment epitheliitis.

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.

Anatomical and functional outcomes following switching from aflibercept to ranibizumab in neovascular age-related macular degeneration in Europe: SAFARI study.

BACKGROUND/AIMS: Prospective data on switching anti-vascular endothelial growth factors in patients with neovascular age-related macular degeneration (nAMD) who have previously shown no/partial response are limited. This prospective study assessed the effect of switching from aflibercept to ranibizumab on anatomical and functional outcomes in patients with persistent/recurrent disease activity. METHODS: SAFARI (NCT02161575) was a 6-month, prospective, single-arm study conducted in the UK and Germany. Patients, meeting strict eligibility criteria for one of two subgroups (primary treatment failure or suboptimal treatment response), received 3 monthly intravitreal ranibizumab injections (0.5 mg). Thereafter, ranibizumab was administered pro re nata at monthly visits. The primary endpoint was change from baseline (CfB) to day 90 in central subfield retinal thickness (CSRT). Best-corrected visual acuity (BCVA) and retinal morphology parameters were assessed. RESULTS: One hundred patients were enrolled (primary treatment failure, 1; suboptimal treatment response, 99). In the overall population, there was a significant CfB in median CSRT of -30.75 µm (95% CI -59.50,-20.50; p<0.0001) to day 90. Improvements were also observed in other quantitative and qualitative optical coherence tomography parameters. In Early Treatment Diabetic Retinopathy Study letters assessed by category, 55% and 59% of patients gained 0-≥15 letters versus baseline at day 90 and day 180, respectively. However, mean improvements in BCVA (CfB) to each time point were small (≤2 letters). No new safety signals were identified. CONCLUSION: Switching from aflibercept to ranibizumab led to a significant improvement in CSRT, with ~60% experiencing stabilised/improved BCVA. Therefore, patients with nAMD who have shown a suboptimal response to aflibercept may benefit from switching to ranibizumab.

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.

Incidence of submacular haemorrhage (SMH) in Scotland: a Scottish Ophthalmic Surveillance Unit (SOSU) study.

PURPOSE: Submacular haemorrhage (SMH) is a cause of severe visual loss in neovascular age-related macular degeneration (nAMD). The incidence is uncertain and furthermore there is no widely used classification system nor agreed best practice. The aim of this national surveillance study was to identify the incidence, presenting features and clinical course of new fovea-involving submacular haemorrhage associated with nAMD. METHODS: A questionnaire was sent monthly to every ophthalmic specialist in Scotland over a 12-month period asking them to report all newly presenting patients with acute SMH secondary to nAMD of at least two disc diameters (DDs) in greatest linear diameter. A follow-up questionnaire was sent 6 months after initial presentation. Cases related to other causes were excluded. RESULTS: Twenty-nine cases were reported giving an incidence of 5.4 per million per annum (range 2-15). The mean age was 83 years (range 66-96) and females accounted for 17/29 (59%). Fifteen of the 29 cases (52%) had a past history of AMD, of which 7 had nAMD. Nineteen of the 29 cases (66%) presented within 7 days of onset and the majority had SMH of < 11 DD (20/29, 69%). Treatment options comprised the following: observation (n = 6, 21%), anti-VEGF alone (n = 6, 21%) or vitrectomy with co-application of tissue plasminogen activator (TPA), anti-VEGF and gas (n = 17, 58%). The vitrectomy group experienced the greatest change in vision from logMAR 1.89-1.50 (p = 0.374). Four of 20 (20%) cases with 6 months follow-up suffered a re-bleed at a mean time of 96 days. CONCLUSIONS: The incidence, clinical features and course of a consecutive national cohort of patients with SMH secondary to nAMD are presented.

Photodynamic Therapy for Circumscribed Choroidal Haemangioma in a Scottish Cohort.

PURPOSE: The aim of this study was to evaluate the safety and efficacy of photodynamic therapy (PDT) with verteporfin as a treatment for circumscribed choroidal haemangioma (CCH). PROCEDURES: This is a retrospective cohort study of all treatment-naïve patients undergoing PDT with verteporfin for CCH in a single centre between April 1, 2007, and September 30, 2016. Best corrected visual acuity (BCVA; using ETDRS letter score), optical coherence tomography (OCT) measurements and a subjective measurement of visual function were recorded before treatment, at 3-month follow-up and at each annual follow-up. RESULTS: Seventeen Caucasian patients with CCH received PDT, with a re-treatment rate of 23.5% (n = 4). Mean (±SD) follow-up was 36.5 (±32.6) months (range 2-106). Mean (±SD) pre-PDT BCVA was 58.5 (±15.5) letters, with a mean improvement from baseline of 8.2 letters at 3 months, of 13.8 letters at 1 year, of 21.1 letters at 2 years and of 19.5 letters at 3 years of follow-up. Subjective visual improvement was noted in 67% at 3 months, in 93% at 1 year, in 86% at 2 years and in 100% at 3 years of follow-up. OCT demonstrated no intraretinal/subretinal fluid in 63% at 3 months, in 77% at 1 year, in 86% at 2 years and in 100% at 3 years of follow-up. No complications of PDT were noted during the study. CONCLUSIONS: PDT is a safe and effective treatment for CCH which results in both structural and functional improvements, and these findings are particularly applicable to patients of Caucasian ethnicity. OCT provides a useful and readily available option to monitor CCH disease activity and its response to PDT.

Pre-treatment clinical features in central retinal vein occlusion that predict visual outcome following intravitreal ranibizumab.

BACKGROUND: Predicting how patients with central retinal vein occlusion (CRVO) will respond to intravitreal anti-VEGF is challenging. The purpose of this study was to identify pre-treatment clinical features in CRVO that predict visual acuity (VA) following intravitreal ranibizumab. METHODS: Medical records, fundus images and optical coherence tomography (OCT) scans of treatment naïve patients with CRVO receiving PRN intravitreal ranibizumab were retrospectively reviewed. Early Treatment Diabetic Retinopathy Study (ETDRS) VA and central retinal thickness (CRT) were recorded at baseline, 3 and 12 months after starting therapy. Regression analysis was used to determine independent predictors of VA at 3 and 12 months follow-up. Possible predictors included baseline VA, age, presence of cotton wool spots (CWS), haemorrhages (few scattered or multiple deep), foveal detachment, CRT, time from presentation to treatment, number of injections given, presence of RAPD, and cause of CRVO. RESULTS: Data from 52 eyes of 50 patients receiving intravitreal ranibizumab treatment for CRVO were analyzed. The mean pre-treatment VA was 43.3 (SD 22.5) letters, which improved to 52.0 (SD 24.3) letters at 3 months, then dropped to 42.0 (SD 30.26) at 12 months. Baseline CRT reduced from 616.7 µm (SD 272.4) to 346.0 µm (SD 205.2) at 3 months and 304.0 µm (SD 168.3) at 12 months. The following features were predictive of poorer VA after starting intravitreal ranibizumab: Poorer pretreatment VA (3-months, P = 0.010; 12-months, P = 0.006), increasing age (3-months, P = < 0.001; 12-months, P = 0.006), and presence of CWS (3-months, P < 0.001; 12-months, P = 0.045). CONCLUSION: Pre-treatment VA, older age, and presence of CWS are easily identifiable clinical features in the hospital setting which help predict visual outcome in patients with CRVO receiving intravitreal ranibizumab.

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.