High-sensitive nascent transcript sequencing reveals BRD4-specific control of widespread enhancer and target gene transcription

Gene transcription by RNA polymerase II (Pol II) is under control of promoters and distal regulatory elements known as enhancers. Enhancers are themselves transcribed by Pol II correlating with their activity. How enhancer transcription is regulated and coordinated with transcription at target genes has remained unclear. Here, we developed a high-sensitive native elongating transcript sequencing approach, called HiS-NET-seq, to provide an extended high-resolution view on transcription, especially at lowly transcribed regions such as enhancers. HiS-NET-seq uncovers new transcribed enhancers in human cells. A multi-omics analysis shows that genome-wide enhancer transcription depends on the BET family protein BRD4. Specifically, BRD4 co-localizes to enhancer and promoter-proximal gene regions, and is required for elongation activation at enhancers and their genes. BRD4 keeps a set of enhancers and genes in proximity through long-range contacts. From these studies BRD4 emerges as a general regulator of enhancer transcription that may link transcription at enhancers and genes

An siRNA Screen Identifies the U2 snRNP Spliceosome as a Host Restriction Factor for Recombinant Adeno-associated Viruses

Adeno-associated viruses (AAV) have evolved to exploit the dynamic reorganization of host cell machinery during co-infection by adenoviruses and other helper viruses. In the absence of helper viruses, host factors such as the proteasome and DNA damage response machinery have been shown to effectively inhibit AAV transduction by restricting processes ranging from nuclear entry to second-strand DNA synthesis. To identify host factors that might affect other key steps in AAV infection, we screened an siRNA library that revealed several candidate genes including the PHD finger-like domain protein 5A (PHF5A), a U2 snRNP-associated protein. Disruption of PHF5A expression selectively enhanced transgene expression from AAV by increasing transcript levels and appears to influence a step after second-strand synthesis in a serotype and cell type-independent manner. Genetic disruption of U2 snRNP and associated proteins, such as SF3B1 and U2AF1, also increased expression from AAV vector, suggesting the critical role of U2 snRNP spliceosome complex in this host-mediated restriction. Notably, adenoviral co-infection and U2 snRNP inhibition appeared to target a common pathway in increasing expression from AAV vectors. Moreover, pharmacological inhibition of U2 snRNP by meayamycin B, a potent SF3B1 inhibitor, substantially enhanced AAV vector transduction of clinically relevant cell types. Further analysis suggested that U2 snRNP proteins suppress AAV vector transgene expression through direct recognition of intact AAV capsids. In summary, we identify U2 snRNP and associated splicing factors, which are known to be affected during adenoviral infection, as novel host restriction factors that effectively limit AAV transgene expression. Concurrently, we postulate that pharmacological/genetic manipulation of components of the spliceosomal machinery might enable more effective gene transfer modalities with recombinant AAV vectors

Natural Variation in the Thermotolerance of Neural Function and Behavior due to a cGMP-Dependent Protein Kinase

Although it is acknowledged that genetic variation contributes to individual differences in thermotolerance, the specific genes and pathways involved and how they are modulated by the environment remain poorly understood. We link natural variation in the thermotolerance of neural function and behavior in Drosophila melanogaster to the foraging gene (for, which encodes a cGMP-dependent protein kinase (PKG)) as well as to its downstream target, protein phosphatase 2A (PP2A). Genetic and pharmacological manipulations revealed that reduced PKG (or PP2A) activity caused increased thermotolerance of synaptic transmission at the larval neuromuscular junction. Like synaptic transmission, feeding movements were preserved at higher temperatures in larvae with lower PKG levels. In a comparative assay, pharmacological manipulations altering thermotolerance in a central circuit of Locusta migratoria demonstrated conservation of this neuroprotective pathway. In this circuit, either the inhibition of PKG or PP2A induced robust thermotolerance of neural function. We suggest that PKG and therefore the polymorphism associated with the allelic variation in for may provide populations with natural variation in heat stress tolerance. for's function in behavior is conserved across most organisms, including ants, bees, nematodes, and mammals. PKG's role in thermotolerance may also apply to these and other species. Natural variation in thermotolerance arising from genes involved in the PKG pathway could impact the evolution of thermotolerance in natural populations

A BRD4-mediated elongation control point primes transcribing RNA polymerase II for 3′-processing and termination

Transcription elongation has emerged as a regulatory hub in gene expression of metazoans. A major control point occurs during early elongation before RNA polymerase II (Pol II) is released into productive elongation. Prior research has linked BRD4 with transcription elongation. Here, we use rapid BET protein and BRD4-selective degradation along with quantitative genome-wide approaches to investigate direct functions of BRD4 in Pol II transcription regulation. Notably, as an immediate consequence of acute BRD4 loss, promoter-proximal pause release is impaired, and transcriptionally engaged Pol II past this checkpoint undergoes readthrough transcription. An integrated proteome-wide analysis uncovers elongation and 3′-RNA processing factors as core BRD4 interactors. BRD4 ablation disrupts the recruitment of general 3′-RNA processing factors at the 5′-control region, which correlates with RNA cleavage and termination defects. These studies, performed in human cells, reveal a BRD4-mediated checkpoint and begin to establish a molecular link between 5′-elongation control and 3′-RNA processing

TriPepSVM: De novo prediction of RNA-binding proteins based on short amino acid motifs.

In recent years, hundreds of novel RNA-binding proteins (RBPs) have been identified, leading to the discovery of novel RNA-binding domains. Furthermore, unstructured or disordered low-complexity regions of RBPs have been identified to play an important role in interactions with nucleic acids. However, these advances in understanding RBPs are limited mainly to eukaryotic species and we only have limited tools to faithfully predict RNA-binders in bacteria. Here, we describe a support vector machine-based method, called TriPepSVM, for the prediction of RNA-binding proteins. TriPepSVM applies string kernels to directly handle protein sequences using tri-peptide frequencies. Testing the method in human and bacteria, we find that several RBP-enriched tri-peptides occur more often in structurally disordered regions of RBPs. TriPepSVM outperforms existing applications, which consider classical structural features of RNA-binding or homology, in the task of RBP prediction in both human and bacteria. Finally, we predict 66 novel RBPs in Salmonella Typhimurium and validate the bacterial proteins ClpX, DnaJ and UbiG to associate with RNA in vivo

High-sensitive nascent transcript sequencing reveals BRD4-specific control of widespread enhancer and target gene transcription

Abstract Gene transcription by RNA polymerase II (Pol II) is under control of promoters and distal regulatory elements known as enhancers. Enhancers are themselves transcribed by Pol II correlating with their activity. How enhancer transcription is regulated and coordinated with transcription at target genes has remained unclear. Here, we developed a high-sensitive native elongating transcript sequencing approach, called HiS-NET-seq, to provide an extended high-resolution view on transcription, especially at lowly transcribed regions such as enhancers. HiS-NET-seq uncovers new transcribed enhancers in human cells. A multi-omics analysis shows that genome-wide enhancer transcription depends on the BET family protein BRD4. Specifically, BRD4 co-localizes to enhancer and promoter-proximal gene regions, and is required for elongation activation at enhancers and their genes. BRD4 keeps a set of enhancers and genes in proximity through long-range contacts. From these studies BRD4 emerges as a general regulator of enhancer transcription that may link transcription at enhancers and genes

PHF5A and U2 snRNP component SF3B1 interact with AAV capsid.

<p><b>(A)</b> HeLa or PHF5A-HA-expressing cell lysates were used to pull-down the HA-tagged PHF5A by anti-HA agarose beads. After 15 washes, the HA-tagged PHF5A was detected by anti-HA antibody. <b>(B)</b> Control or PHF5A-HA-expressing HeLa cells were transduced by AAV2 and AAV9 CMV-Luc vectors (MOI 4 x 10⁵) and total AAV genome copies in the HA pulldown were determined by quantitative real-time PCR. <b>(C)</b> AAV2 CMV-Luc vector (3 x 10¹⁰ genome copies) was unheated or preheated for 30 min at 65°C. PHF5A-HA-over-expressing HeLa cell lysates were then incubated with vectors for 1 hour at 4°C, followed by pulldown of PHF5A-HA. AAV vector genome copies in the precipitates were determined by quantitative real-time PCR. <b>(D)</b> HeLa cells were infected with the AAV2 CMV-Luc vector (4 x 10¹⁰ genome copies/well) for 5 min, 4 or 12 hours. Confocal microscopy analysis was performed to detect the subcellular localizations of AAV vector particles (green) and PHF5A (red). Nuclei were counterstained by DAPI (blue). <b>(E)</b> HeLa cells were infected with AAV2 CMV-Luc vectors (4 x 10¹⁰ genome copies/well) or equivalent amounts of empty AAV2 vectors for 4 hours, and cells were analyzed for co-localization of AAV2 capsid and endogenous PHF5A signals. Prominent co-localized signals were indicated by white arrows. <b>(F)</b> HeLa cells were treated with the PHF5A siRNA for 24 hours, followed by transduction with the AAV2 vector as in E for 4 hours. AAV2 vector particles were detected by anti-AAV2 capsid A20 antibody, and the patterns of cytoplasmic and nuclear accumulations of AAV2 vector particles were compared between control and PHF5A-ablated cells. Representative Z-stack images of the middle sections (slices 3 and 4) from control and PHF5A knockdown cells are shown. <b>(G)</b> Schematic representation for the iodixanol cushion method to enrich cellular factors interacting with particulated AAV capsids. <b>(H)</b> HeLa cell lysates were incubated with AAV2 CMV-Luc vectors (5 x 10¹⁰ genome copies) for 1 hour at 4°C. After centrifugation over 25% iodixanol, three layers (the upper phase, lower phase, and pellet) were separately harvested for Western blotting. AAV capsid proteins VP1, 2 and 3, phospho-SF3B1, and endogenous PHF5A were detected by A20, anti-SF3B1, and anti-PHF5A antibodies, respectively. <b>(I)</b> Same as H for AAV capsid proteins, except that empty AAV2 VP3 only capsids were used for SF3B1 co-precipitation. <b>(J)</b> Control or AAV VP1-over-expressing 293T cell lysates were used to pull-down the AAV VP1 protein by A20 antibody. After 15 washes, the pellets were probed for SF3B1 enrichment by anti-SF3B1 antibody.</p

Meayamycin B increases AAV vector transduction of clinically relevant cell types.

<p><b>(A)</b> Primary mouse islets were infected with AAV8 CMV-GFP in the presence or absence of 2 nM meayamycin B, and GFP expression was monitored for three days. <b>(B)</b> Primary human islets were treated with AAV2 or AAV9 CMV-Luc vectors for 7 hours and then treated with 0, 2, 5 or 10 nM meayamycin B. Luciferase expression was analyzed 48 hours p.i. <b>(C)</b> Neonatal rat cardiomyocytes were infected with AAV2 CMV-Luc or scAAV9 CMV-GFP vectors and treated with meayamycin B, 3 hours p.i. Luciferase activity was measured 3 days p.i., while GFP expression was monitored at 5 days p.i. <b>(D)</b> Porcine hepatocytes were infected with AAV2 or AAV9 CMV-Luc vectors for 8 hours, virus was then removed and cells were treated with 0, 2, or 20 nM meayamycin B. Cells were harvested 48 hours p.i. for the luciferase assay. In A-D, an MOI of 10⁴ was used.</p

Screening of the siRNA library for proteasomal pathway genes identifies PHF5A as a factor blocking the transduction by AAV9 vector.

<p><b>(A)</b> Screening of the siRNA library was carried out by reverse transfection of HeLa cells with siRNAs, followed by infection with luciferase-expressing AAV9 vectors (AAV9 CMV-Luc) at a multiplicity of infection (MOI) of 10⁴, and assessment of luciferase expression. Screening of the library identified 12 candidate genes that increased transduction by AAV9 vectors over 10-fold. Further studies were carried out in HeLa cells transfected/transduced with specific siRNAs or shRNA lentivectors for each of the 12 genes to verify the screening candidates. <b>(B)</b> Quantitative real-time RT-PCR was performed to determine the levels of PHF5A transcripts in cells treated with control or PHF5A siRNAs at 48 hours. <b>(C)</b> HeLa cells were transfected with control or PHF5A siRNAs for 24 hours, followed by infection with AAV9 CMV-Luc vectors (MOI 10⁴) for an additional 48 hours. The luciferase assay was performed in order to determine relative luciferase activities in treated cells. <b>(D)</b> Same as C, except that a luciferase-expressing adenoviral vector at an MOI of 3 x 10² or an HIV-1-based lentiviral vector (MOI 0.3) were used to infect siRNA-treated HeLa cells. <b>(E)</b> Lentiviral vector pSIN-PHF5A-Escape with the PHF5A-HA Escape transgene was generated through introduction of three silent mutations in the PHF5A siRNA#1-targeted sequence. Western blotting was performed to verify the expression of the PHF5A-HA-Escape and its resistance to the PHF5A siRNA#1 treatment. Anti-PHF5A antibody was used to detect endogenous and over-expressed PHF5A-HA, while anti-HA antibody detected the HA-tagged PHF5A. <b>(F)</b> HeLa cell lines stably expressing the PHF5A-HA-Escape mutant were generated through lentiviral transduction of the escape mutant, followed by puromycin selection. Upon treatment with the PHF5A siRNA and AAV9 CMV-Luc vector (MOI 10⁴), luciferase expression was determined in control HeLa and PHF5A-HA-Escape-expressing HeLa cells. <b>(B-D, F)</b> Data are shown as averages of three independent experiments with error bars representing standard error of the mean. *p<0.05.</p