Project description:RNA-seq is widely used for studying gene expression, but commonly used sequencing platforms produce short reads that only span up to two exon-junctions per read. This makes it difficult to accurately determine the composition and phasing of exons within transcripts. Although long-read sequencing improves this issue, it is not amenable to precise quantitation, which limits its utility for differential expression studies. We used long-read isoform sequencing combined with a novel analysis approach to compare alternative splicing of large, repetitive structural genes in muscles. Analysis of muscle structural genes that produce medium (Nrap - 5kb), large (Nebulin - 22 kb) and very-large (Titin - 106 kb) transcripts in cardiac muscle, and fast and slow skeletal muscles identified unannotated exons for each of these ubiquitous muscle genes. This also identified differential exon usage and phasing for these genes between the different muscle types. By mapping the in-phase transcript structures to known annotations, we also identified and quantified previously unannotated transcripts. Results were confirmed by endpoint PCR and Sanger sequencing, which revealed muscle-type specific differential expression of these novel transcripts. The improved transcript identification and quantification demonstrated by our approach removes previous impediments to studies aimed at quantitative differential expression of ultra-long transcripts.
Project description:The Long-read POG dataset comprises a cohort of 189 patient tumours and 41 matched normal samples sequenced using the Oxford Nanopore Technologies PromethION platform. This dataset from the Personalized Oncogenomics (POG) program and the Marathon of Hope Cancer Centres Network includes accompanying DNA and RNA short-read sequence data, analytics, and clinical information. We show the potential of long-read sequencing for resolving complex cancer-related structural variants, viral integrations, and extrachromosomal circular DNA. Long-range phasing of variants facilitates the discovery of allelically differentially methylated regions (aDMRs) and allele-specific expression, including recurrent aDMRs in the cancer genes RET and CDKN2A. Germline promoter methylation in MLH1 can be directly observed in Lynch syndrome. Promoter methylation in BRCA1 and RAD51C is a likely driver behind patterns of homologous recombination deficiency where no driver mutation was found. This dataset demonstrates applications for long-read sequencing in precision medicine, and is available as a resource for developing analytical approaches using this technology.
Project description:Background: Treatment-related neuroendocrine prostate cancer (t-NEPC) is a highly aggressive form of prostate cancer (PCa). Understanding the molecular mechanisms driving t-NEPC may provide valuable therapeutic strategies. Methods: we performed a pan-cancer differential mRNA abundance analysis of neuroendocrine tumors (NET) and non-NETs using integrated datasets to identify potential driver genes in NEPC. Gain- and loss-of-function studies were performed in PCa cell lines and subcutaneous xenografts in mouse to characterize the role of Kinesin-like protein (KIF1A) in neuroendocrine (NE) differentiation. Western blot, qRT-PCR, immunoprecipitation (IP), nuclear and cytoplasmic protein extraction, CO-IP and immunofluorescence were performed to study the regulation of KIF1A to OGT. Results: We identified that KIF1A overexpression is highly correlated to NE differentiation. NE differentiation features in PCa, including NE marker gene expression, stemness and EMT, were impaired by KIF1A knockdown and promoted by KIF1A overexpression. Targeting KIF1A inhibited the growth of NE-differentiated PCa cells in vitro and in vivo. Mechanistically, KIF1A bound with OGT and regulated its protein expression and O-linked N-acetylglucosamine transferase activity. OGT nuclear translocation induced by elevated KIF1A inhibited OGT ubiquitin-proteasome pathway degradation in the cytoplasm and promoted intranuclear O-GlcNAcylation of β-catenin and OCT4. More importantly, our data revealed that OGT is critical for KIF1A induced NE differentiation and aggressive growth. Conclusion: KIF1A promotes NE differentiation through modeling OGT O-GlcNAcylation in PCa. Targeting KIF1A - OGT may impede the development of NEPC for a group of PCa patients with elevated KIF1A expression.
Project description:In recent years, long-read sequencing technologies have detected transcript isoforms with unprecedented accuracy and resolution. However, it remains unclear whether long-read sequencing can effectively disentangle the isoform landscape of complex allele-specific loci that arise from genetic or epigenetic differences between alleles. Here, we combine the PacBio Iso-Seq workflow with the established phasing approach WhatsHap to assign long reads to the corresponding allele in polymorphic F1 mouse hybrids. Upon comparing the long-read sequencing results with matched short reads, we observed general consistency in the allele-specific information and were able to confirm the imprinting status of known imprinted genes. We then explored the complex imprinted Gnas locus known for allele-specific non-coding and coding isoforms and were able to benchmark historical observations. This approach also allowed us to detect isoforms from both the active and inactive X chromosomes of genes that escape X chromosome inactivation. The described workflow offers a promising framework and demonstrates the power of long-read transcriptomic data to provide mechanistic insight into complex allele-specific loci.
Project description:KIF1A, a microtubule-based motor protein responsible for axonal transport, is linked to a group of neurological disorders known as KIF1A-associated neurological disorder (KAND). Current therapeutic options for KAND are limited. Here, we introduced the clinically relevant KIF1A(R11Q) variant into the C. elegans homolog UNC-104, resulting in uncoordinated animal behaviors. Through genetic suppressor screens, we identified intragenic mutations in UNC-104's motor domain that rescued synaptic vesicle localization and coordinated movement. We showed that two suppressor mutations partially recovered motor activity in vitro by counteracting the structural defect caused by R11Q at KIF1A's nucleotide-binding pocket. We found that supplementation with fisetin, a plant flavonol, improved KIF1A(R11Q) worms’ movement and morphology. Notably, our biochemical and single-molecule assays revealed that fisetin directly restored the ATPase activity and processive movement of human KIF1A(R11Q) without affecting wild-type KIF1A. These findings suggest fisetin as a potential intervention for enhancing KIF1A(R11Q) activity and alleviating associated defects in KAND.
Project description:Nucleosomes arrange into extended arrays, much like beads on a string. They are often phased at genomic landmarks and are thought to be evenly spaced. Here we tested to what extent this stereotypic organization describes the nucleosome landscape in Saccharomyces cerevisiae using a long-read nucleosome-sequencing technique called Fiber-Seq. Fiber-Seq maps the nucleosome pattern on individual chromatin fibers. As such, it is ideally suited to measure the density of nucleosomes per read and quantitate the nucleosome occupancy throughout the genome. We document substantial deviations from the stereotypical nucleosome organization, with unexpectedly long linker DNAs between individual nucleosomes, genomic regions lacking entire nucleosomes, heterogeneous phasing of arrays, truly irregular spacing of arrays and read-to-read variation in nucleosome densities. We exploited the technology to test mechanistic models for the biogenesis of nucleosome arrays. We can rule out transcription elongation playing a decisive role in array formation and detect signatures for a clamping activity of remodelers of the ISWI and CHD1 families after acute nucleosome depletion in vivo. Given that nucleosomes are cis-regulatory elements, the cell-to-cell heterogeneity that Fiber-Seq uncovers provides much needed information to understand chromatin structure and function.
Project description:The mechanisms by which motor proteins are loaded on vesicles and how cargos are captured at appropriate sites remain unclear. To better understand how KIF1A-based transport is regulated, we identified the KIF1A interactome and focused on three binding partners, the calcium binding protein Calmodulin (CaM) and two synaptic scaffolding proteins: liprin-a and TANC2.