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We present Nanopore-DamID, a method to simultaneously detect cytosine methylation and DNA-protein interactions from single molecules, via selective sequencing of adenine-labelled DNA. Assaying LaminB1 and CTCF binding with Nanopore-DamID, we identify escape from LAD-associated repression of hypomethylated promoters amidst generalised hypermethylation of LaminB1-associated regulatory elements. We detect novel CTCF binding sites in highly repetitive regions, and allele-specific CTCF binding to imprinted genes and the active X chromosome. Nanopore-DamID highlights the importance of DNA methylation to transcription factor activity.

Pseudogenes are gene copies presumed to mainly be functionless relics of evolution due to acquired deleterious mutations or transcriptional silencing. When transcribed, pseudogenes may encode proteins or enact RNA-intrinsic regulatory mechanisms. However, the extent, characteristics and functional relevance of the human pseudogene transcriptome are unclear. Short-read sequencing platforms have limited power to resolve and accurately quantify pseudogene transcripts owing to the high sequence similarity of pseudogenes and their parent genes. Using deep full-length PacBio cDNA sequencing of normal human tissues and cancer cell lines, we identify here hundreds of novel transcribed pseudogenes. Pseudogene transcripts are expressed in tissue-specific patterns, exhibit complex splicing patterns and contribute to the coding sequences of known genes. We survey pseudogene transcripts encoding intact open reading frames (ORFs), representing potential unannotated protein-coding genes, and demonstrate their efficient translation in cultured cells. To assess the impact of noncoding pseudogenes on the cellular transcriptome, we delete the nucleus-enriched pseudogene PDCL3P4 transcript from HAP1 cells and observe hundreds of perturbed genes. This study highlights pseudogenes as a complex and dynamic component of the transcriptional landscape underpinning human biology and disease.

The Endoplasmic Reticulum (ER) is responsible for the folding and post-translational modification of secretory proteins, as well as for triaging misfolded proteins. During folding, there is a complex yet only partially understood interplay between disulfide bond formation, which is an enzyme catalyzed event in the oxidizing environment of the ER, along with other post-translational modifications (PTMs) and chaperone-supported protein folding. Here, we used the glycoprotein torsinA as a model substrate to explore the impact of ER redox homeostasis on PTMs and protein biogenesis. TorsinA is a AAA+ ATPase with unusual oligomeric properties and controversial functions. The deletion of a C-terminal glutamic acid residue (∆E) is associated with the development of Early-Onset Torsion Dystonia, a severe movement disorder. TorsinA differs from other AAA+ ATPases since it is an ER resident, and as a result of its entry into the ER torsinA contains two N-linked glycans and at least one disulfide bond. The role of these PTMs on torsinA biogenesis and function and the identity of the enzymes that catalyze them are poorly defined. Using a yeast torsinA expression system, we demonstrate that a specific protein disulfide isomerase, Pdi1, affects the folding and N-linked glycosylation of torsinA and torsinA∆E in a redox-dependent manner, suggesting that the acquisition of early torsinA folding intermediates is sensitive to perturbed interactions between Cys residues and the quality control machinery. We also highlight the role of specific Cys residues during torsinA biogenesis and demonstrate that torsinA∆E is more sensitive than torsinA when these Cys residues are mutated.