Genome-wide DNA hypomethylation and RNA:DNA hybrid accumulation in Aicardi-Goutières syndrome.

Aicardi-Goutières syndrome (AGS) is a severe childhood inflammatory disorder that shows clinical and genetic overlap with systemic lupus erythematosus (SLE). AGS is thought to arise from the accumulation of incompletely metabolized endogenous nucleic acid species owing to mutations in nucleic acid-degrading enzymes TREX1 (AGS1), RNase H2 (AGS2, 3 and 4), and SAMHD1 (AGS5). However, the identity and source of such immunogenic nucleic acid species remain undefined. Using genome-wide approaches, we show that fibroblasts from AGS patients with AGS1-5 mutations are burdened by excessive loads of RNA:DNA hybrids. Using MethylC-seq, we show that AGS fibroblasts display pronounced and global loss of DNA methylation and demonstrate that AGS-specific RNA:DNA hybrids often occur within DNA hypomethylated regions. Altogether, our data suggest that RNA:DNA hybrids may represent a common immunogenic form of nucleic acids in AGS and provide the first evidence of epigenetic perturbations in AGS, furthering the links between AGS and SLE

Prevalent, Dynamic, and Conserved R-Loop Structures Associate with Specific Epigenomic Signatures in Mammals.

R-loops are three-stranded nucleic acid structures formed upon annealing of an RNA strand to one strand of duplex DNA. We profiled R-loops using a high-resolution, strand-specific methodology in human and mouse cell types. R-loops are prevalent, collectively occupying up to 5% of mammalian genomes. R-loop formation occurs over conserved genic hotspots such as promoter and terminator regions of poly(A)-dependent genes. In most cases, R-loops occur co-transcriptionally and undergo dynamic turnover. Detailed epigenomic profiling revealed that R-loops associate with specific chromatin signatures. At promoters, R-loops associate with a hyper-accessible state characteristic of unmethylated CpG island promoters. By contrast, terminal R-loops associate with an enhancer- and insulator-like state and define a broad class of transcription terminators. Together, this suggests that the retention of nascent RNA transcripts at their site of expression represents an abundant, dynamic, and programmed component of the mammalian chromatin that affects chromatin patterning and the control of gene expression

Genome-wide DNA hypomethylation and RNA:DNA hybrid accumulation in Aicardi-Goutières syndrome.

Aicardi-Goutières syndrome (AGS) is a severe childhood inflammatory disorder that shows clinical and genetic overlap with systemic lupus erythematosus (SLE). AGS is thought to arise from the accumulation of incompletely metabolized endogenous nucleic acid species owing to mutations in nucleic acid-degrading enzymes TREX1 (AGS1), RNase H2 (AGS2, 3 and 4), and SAMHD1 (AGS5). However, the identity and source of such immunogenic nucleic acid species remain undefined. Using genome-wide approaches, we show that fibroblasts from AGS patients with AGS1-5 mutations are burdened by excessive loads of RNA:DNA hybrids. Using MethylC-seq, we show that AGS fibroblasts display pronounced and global loss of DNA methylation and demonstrate that AGS-specific RNA:DNA hybrids often occur within DNA hypomethylated regions. Altogether, our data suggest that RNA:DNA hybrids may represent a common immunogenic form of nucleic acids in AGS and provide the first evidence of epigenetic perturbations in AGS, furthering the links between AGS and SLE

Prevalent, Dynamic, and Conserved R-Loop Structures Associate with Specific Epigenomic Signatures in Mammals.

R-loops are three-stranded nucleic acid structures formed upon annealing of an RNA strand to one strand of duplex DNA. We profiled R-loops using a high-resolution, strand-specific methodology in human and mouse cell types. R-loops are prevalent, collectively occupying up to 5% of mammalian genomes. R-loop formation occurs over conserved genic hotspots such as promoter and terminator regions of poly(A)-dependent genes. In most cases, R-loops occur co-transcriptionally and undergo dynamic turnover. Detailed epigenomic profiling revealed that R-loops associate with specific chromatin signatures. At promoters, R-loops associate with a hyper-accessible state characteristic of unmethylated CpG island promoters. By contrast, terminal R-loops associate with an enhancer- and insulator-like state and define a broad class of transcription terminators. Together, this suggests that the retention of nascent RNA transcripts at their site of expression represents an abundant, dynamic, and programmed component of the mammalian chromatin that affects chromatin patterning and the control of gene expression

Preferential Identification of Agonistic OX40 Antibodies by Using Cell Lysate to Pan Natively Paired, Humanized Mouse-Derived Yeast Surface Display Libraries

To discover therapeutically relevant antibody candidates, many groups use mouse immunization followed by hybridoma generation or B cell screening. One modern approach is to screen B cells by generating natively paired single chain variable fragment (scFv) display libraries in yeast. Such methods typically rely on soluble antigens for scFv library screening. However, many therapeutically relevant cell-surface targets are difficult to express in a soluble protein format, complicating discovery. In this study, we developed methods to screen humanized mouse-derived yeast scFv libraries using recombinant OX40 protein in cell lysate. We used deep sequencing to compare screening with cell lysate to screening with soluble OX40 protein, in the context of mouse immunizations using either soluble OX40 or OX40-expressing cells and OX40-encoding DNA vector. We found that all tested methods produce a unique diversity of scFv binders. However, when we reformatted forty-one of these scFv as full-length monoclonal antibodies (mAbs), we observed that mAbs identified using soluble antigen immunization with cell lysate sorting always bound cell surface OX40, whereas other methods had significant false positive rates. Antibodies identified using soluble antigen immunization and cell lysate sorting were also significantly more likely to activate OX40 in a cellular assay. Our data suggest that sorting with OX40 protein in cell lysate is more likely than other methods to retain the epitopes required for antibody-mediated OX40 agonism