For quality, interpretation, reproducibility and sharing value, microscopy images should be accompanied by detailed descriptions of the conditions that were used to produce them. Micro-Meta App is an intuitive, highly interoperable, open-source software tool that was developed in the context of the 4D Nucleome (4DN) consortium and is designed to facilitate the extraction and collection of relevant microscopy metadata as specified by the recent 4DN-BINA-OME tiered-system of Microscopy Metadata specifications. In addition to substantially lowering the burden of quality assurance, the visual nature of Micro-Meta App makes it particularly suited for training purposes.
BACKGROUND: Retrotransposons have been implicated as causes of Mendelian disease, but their role in autism spectrum disorder (ASD) has not been systematically defined, because they are only called with adequate sensitivity from whole genome sequencing (WGS) data and a large enough cohort for this analysis has only recently become available. RESULTS: We analyzed WGS data from a cohort of 2288 ASD families from the Simons Simplex Collection by establishing a scalable computational pipeline for retrotransposon insertion detection. We report 86,154 polymorphic retrotransposon insertions-including > 60% not previously reported-and 158 de novo retrotransposition events. The overall burden of de novo events was similar between ASD individuals and unaffected siblings, with 1 de novo insertion per 29, 117, and 206 births for Alu, L1, and SVA respectively, and 1 de novo insertion per 21 births total. However, ASD cases showed more de novo L1 insertions than expected in ASD genes. Additionally, we observed exonic insertions in loss-of-function intolerant genes, including a likely pathogenic exonic insertion in CSDE1, only in ASD individuals. CONCLUSIONS: These findings suggest a modest, but important, impact of intronic and exonic retrotransposon insertions in ASD, show the importance of WGS for their analysis, and highlight the utility of specific bioinformatic tools for high-throughput detection of retrotransposon insertions.
The vertebrate retina is generated by retinal progenitor cells (RPCs), which produce >100 cell types. Although some RPCs produce many cell types, other RPCs produce restricted types of daughter cells, such as a cone photoreceptor and a horizontal cell (HC). We used genome-wide assays of chromatin structure to compare the profiles of a restricted cone/HC RPC and those of other RPCs in chicks. These data nominated regions of regulatory activity, which were tested in tissue, leading to the identification of many cis-regulatory modules (CRMs) active in cone/HC RPCs and developing cones. Two transcription factors, Otx2 and Oc1, were found to bind to many of these CRMs, including those near genes important for cone development and function, and their binding sites were required for activity. We also found that Otx2 has a predicted autoregulatory CRM. These results suggest that Otx2, Oc1 and possibly other Onecut proteins have a broad role in coordinating cone development and function. The many newly discovered CRMs for cones are potentially useful reagents for gene therapy of cone diseases.
Histone chaperones are critical for controlling chromatin integrity during transcription, DNA replication, and DNA repair. Three conserved and essential chaperones, Spt6, Spn1/Iws1, and FACT, associate with elongating RNA polymerase II and interact with each other physically and/or functionally; however, there is little understanding of their individual functions or their relationships with each other. In this study, we selected for suppressors of a temperature-sensitive spt6 mutation that disrupts the Spt6-Spn1 physical interaction and that also causes both transcription and chromatin defects. This selection identified novel mutations in FACT. Surprisingly, suppression by FACT did not restore the Spt6-Spn1 interaction, based on coimmunoprecipitation, ChIP, and mass spectrometry experiments. Furthermore, suppression by FACT bypassed the complete loss of Spn1. Interestingly, the FACT suppressor mutations cluster along the FACT-nucleosome interface, suggesting that they alter FACT-nucleosome interactions. In agreement with this observation, we showed that the spt6 mutation that disrupts the Spt6-Spn1 interaction caused an elevated level of FACT association with chromatin, while the FACT suppressors reduced the level of FACT-chromatin association, thereby restoring a normal Spt6-FACT balance on chromatin. Taken together, these studies reveal previously unknown regulation between histone chaperones that is critical for their essential in vivo functions.
Negative elongation factor (NELF) is a critical transcriptional regulator that stabilizes paused RNA polymerase to permit rapid gene expression changes in response to environmental cues. Although NELF is essential for embryonic development, its role in adult stem cells remains unclear. In this study, through a muscle-stem-cell-specific deletion, we showed that NELF is required for efficient muscle regeneration and stem cell pool replenishment. In mechanistic studies using PRO-seq, single-cell trajectory analyses and myofiber cultures revealed that NELF works at a specific stage of regeneration whereby it modulates p53 signaling to permit massive expansion of muscle progenitors. Strikingly, transplantation experiments indicated that these progenitors are also necessary for stem cell pool repopulation, implying that they are able to return to quiescence. Thus, we identified a critical role for NELF in the expansion of muscle progenitors in response to injury and revealed that progenitors returning to quiescence are major contributors to the stem cell pool repopulation.
Idiopathic normal pressure hydrocephalus (iNPH) is a neurological disorder that occurs in about 1% of individuals over age 60 and is characterized by enlarged cerebral ventricles, gait difficulty, incontinence, and cognitive decline. The cause and pathophysiology of iNPH are largely unknown. We performed whole exome sequencing of DNA obtained from 53 unrelated iNPH patients. Two recurrent heterozygous loss of function deletions in CWH43 were observed in 15% of iNPH patients and were significantly enriched 6.6-fold and 2.7-fold, respectively, when compared to the general population. Cwh43 modifies the lipid anchor of glycosylphosphatidylinositol-anchored proteins. Mice heterozygous for CWH43 deletion appeared grossly normal but displayed hydrocephalus, gait and balance abnormalities, decreased numbers of ependymal cilia, and decreased localization of glycosylphosphatidylinositol-anchored proteins to the apical surfaces of choroid plexus and ependymal cells. Our findings provide novel mechanistic insights into the origins of iNPH and demonstrate that it represents a distinct disease entity.
Homologous recombination (HR)-deficient cancers are sensitive to poly-ADP ribose polymerase inhibitors (PARPi), which have shown clinical efficacy in the treatment of high-grade serous cancers (HGSC). However, the majority of patients will relapse, and acquired PARPi resistance is emerging as a pressing clinical problem. Here we generated seven single-cell clones with acquired PARPi resistance derived from a PARPi-sensitive TP53 -/- and BRCA1 -/- epithelial cell line generated using CRISPR/Cas9. These clones showed diverse resistance mechanisms, and some clones presented with multiple mechanisms of resistance at the same time. Genomic analysis of the clones revealed unique transcriptional and mutational profiles and increased genomic instability in comparison with a PARPi-sensitive cell line. Clonal evolutionary analyses suggested that acquired PARPi resistance arose via clonal selection from an intrinsically unstable and heterogenous cell population in the sensitive cell line, which contained preexisting drug-tolerant cells. Similarly, clonal and spatial heterogeneity in tumor biopsies from a clinical patient with BRCA1-mutant HGSC with acquired PARPi resistance was observed. In an imaging-based drug screening, the clones showed heterogenous responses to targeted therapeutic agents, indicating that not all PARPi-resistant clones can be targeted with just one therapy. Furthermore, PARPi-resistant clones showed mechanism-dependent vulnerabilities to the selected agents, demonstrating that a deeper understanding on the mechanisms of resistance could lead to improved targeting and biomarkers for HGSC with acquired PARPi resistance. SIGNIFICANCE: This study shows that BRCA1-deficient cells can give rise to multiple genomically and functionally heterogenous PARPi-resistant clones, which are associated with various vulnerabilities that can be targeted in a mechanism-specific manner.
Although germline de novo copy number variants (CNVs) are known causes of autism spectrum disorder (ASD), the contribution of mosaic (early-developmental) copy number variants (mCNVs) has not been explored. In this study, we assessed the contribution of mCNVs to ASD by ascertaining mCNVs in genotype array intensity data from 12,077 probands with ASD and 5,500 unaffected siblings. We detected 46 mCNVs in probands and 19 mCNVs in siblings, affecting 2.8-73.8% of cells. Probands carried a significant burden of large (>4-Mb) mCNVs, which were detected in 25 probands but only one sibling (odds ratio = 11.4, 95% confidence interval = 1.5-84.2, P = 7.4 × 10). Event size positively correlated with severity of ASD symptoms (P = 0.016). Surprisingly, we did not observe mosaic analogues of the short de novo CNVs recurrently observed in ASD (eg, 16p11.2). We further experimentally validated two mCNVs in postmortem brain tissue from 59 additional probands. These results indicate that mCNVs contribute a previously unexplained component of ASD risk.
Spn1/Iws1 is a conserved protein involved in transcription and chromatin dynamics, yet its general in vivo requirement for these functions is unknown. Using a Spn1 depletion system in Saccharomyces cerevisiae, we demonstrate that Spn1 broadly influences several aspects of gene expression on a genome-wide scale. We show that Spn1 is globally required for normal mRNA levels and for normal splicing of ribosomal protein transcripts. Furthermore, Spn1 maintains the localization of H3K36 and H3K4 methylation across the genome and is required for normal histone levels at highly expressed genes. Finally, we show that the association of Spn1 with the transcription machinery is strongly dependent on its binding partner, Spt6, while the association of Spt6 and Set2 with transcribed regions is partially dependent on Spn1. Taken together, our results show that Spn1 affects multiple aspects of gene expression and provide additional evidence that it functions as a histone chaperone in vivo.
In the context of human disease, the mechanisms whereby transcription factors reprogram gene expression in reparative responses to injury are not well understood. We have studied the mechanisms of transcriptional reprogramming in disease using murine kidney podocytes as a model for tissue injury. Podocytes are a crucial component of glomeruli, the filtration units of each nephron. Podocyte injury is the initial event in many processes that lead to end-stage kidney disease. Wilms tumor-1 (WT1) is a master regulator of gene expression in podocytes, binding nearly all genes known to be crucial for maintenance of the glomerular filtration barrier. Using murine models and human kidney organoids, we investigated WT1-mediated transcriptional reprogramming during the course of podocyte injury. Reprogramming the transcriptome involved highly dynamic changes in the binding of WT1 to target genes during a reparative injury response, affecting chromatin state and expression levels of target genes.
The Genomics of Malignant Peripheral Nerve Sheath Tumor (GeM) Consortium is an international collaboration focusing on multi-omic analysis of malignant peripheral nerve sheath tumors (MPNSTs), the most aggressive tumor associated with neurofibromatosis type 1 (NF1). Here we present a summary of current knowledge gaps, a description of our consortium and the cohort we have assembled, and an overview of our plans for multi-omic analysis of these tumors. We propose that our analysis will lead to a better understanding of the order and timing of genetic events related to MPNST initiation and progression. Our ten institutions have assembled 96 fresh frozen NF1-related (63%) and sporadic MPNST specimens from 86 subjects with corresponding clinical and pathological data. Clinical data have been collected as part of the International MPNST Registry. We will characterize these tumors with bulk whole genome sequencing, RNAseq, and DNA methylation profiling. In addition, we will perform multiregional analysis and temporal sampling, with the same methodologies, on a subset of nine subjects with NF1-related MPNSTs to assess tumor heterogeneity and cancer evolution. Subsequent multi-omic analyses of additional archival specimens will include deep exome sequencing (500×) and high density copy number arrays for both validation of results based on fresh frozen tumors, and to assess further tumor heterogeneity and evolution. Digital pathology images are being collected in a cloud-based platform for consensus review. The result of these efforts will be the largest MPNST multi-omic dataset with correlated clinical and pathological information ever assembled.
Touat M, Li YY, Boynton AN, Spurr LF, Iorgulescu BJ, Bohrson CL, Cortes-Ciriano I, Birzu C, Geduldig JE, Pelton K, Lim-Fat MJ, Pal S, Ferrer-Luna R, Ramkissoon SH, Dubois F, Bellamy C, Currimjee N, Bonardi J, Qian K, Ho P, Malinowski S, Taquet L, Jones RE, Shetty A, Chow K-H, Sharaf R, Pavlick D, Albacker LA, Younan N, Baldini C, Verreault M, Giry M, Guillerm E, Ammari S, Beuvon F, Mokhtari K, Alentorn A, Dehais C, Houillier C, Laigle-Donadey F, Psimaras D, Lee EQ, Nayak L, McFaline-Figueroa RJ, Carpentier A, Cornu P, Capelle L, Mathon B, Barnholtz-Sloan JS, Chakravarti A, Bi WL, Chiocca AE, Fehnel KP, Alexandrescu S, Chi SN, Haas-Kogan D, Batchelor TT, Frampton GM, Alexander BM, Huang RY, Ligon AH, Coulet F, Delattre J-Y, Hoang-Xuan K, Meredith DM, Santagata S, Duval A, Sanson M, Cherniack AD, Wen PY, Reardon DA, Marabelle A, Park PJ, Idbaih A, Beroukhim R, Bandopadhayay P, Bielle F, Ligon KL. Mechanisms and therapeutic implications of hypermutation in gliomas. Nature 2020;580(7804):517-523.Abstract
A high tumour mutational burden (hypermutation) is observed in some gliomas; however, the mechanisms by which hypermutation develops and whether it predicts the response to immunotherapy are poorly understood. Here we comprehensively analyse the molecular determinants of mutational burden and signatures in 10,294 gliomas. We delineate two main pathways to hypermutation: a de novo pathway associated with constitutional defects in DNA polymerase and mismatch repair (MMR) genes, and a more common post-treatment pathway, associated with acquired resistance driven by MMR defects in chemotherapy-sensitive gliomas that recur after treatment with the chemotherapy drug temozolomide. Experimentally, the mutational signature of post-treatment hypermutated gliomas was recapitulated by temozolomide-induced damage in cells with MMR deficiency. MMR-deficient gliomas were characterized by a lack of prominent T cell infiltrates, extensive intratumoral heterogeneity, poor patient survival and a low rate of response to PD-1 blockade. Moreover, although bulk analyses did not detect microsatellite instability in MMR-deficient gliomas, single-cell whole-genome sequencing analysis of post-treatment hypermutated glioma cells identified microsatellite mutations. These results show that chemotherapy can drive the acquisition of hypermutated populations without promoting a response to PD-1 blockade and supports the diagnostic use of mutational burden and signatures in cancer.
Elucidating the lineage relationships among different cell types is key to understanding human brain development. Here we developed parallel RNA and DNA analysis after deep sequencing (PRDD-seq), which combines RNA analysis of neuronal cell types with analysis of nested spontaneous DNA somatic mutations as cell lineage markers, identified from joint analysis of single-cell and bulk DNA sequencing by single-cell MosaicHunter (scMH). PRDD-seq enables simultaneous reconstruction of neuronal cell type, cell lineage, and sequential neuronal formation ("birthdate") in postmortem human cerebral cortex. Analysis of two human brains showed remarkable quantitative details that relate mutation mosaic frequency to clonal patterns, confirming an early divergence of precursors for excitatory and inhibitory neurons, and an "inside-out" layer formation of excitatory neurons as seen in other species. In addition our analysis allows an estimate of excitatory neuron-restricted precursors (about 10) that generate the excitatory neurons within a cortical column. Inhibitory neurons showed complex, subtype-specific patterns of neurogenesis, including some patterns of development conserved relative to mouse, but also some aspects of primate cortical interneuron development not seen in mouse. PRDD-seq can be broadly applied to characterize cell identity and lineage from diverse archival samples with single-cell resolution and in potentially any developmental or disease condition.
Combined PARP and immune checkpoint inhibition has yielded encouraging results in ovarian cancer, but predictive biomarkers are lacking. We performed immunogenomic profiling and highly multiplexed single-cell imaging on tumor samples from patients enrolled in a Phase I/II trial of niraparib and pembrolizumab in ovarian cancer (NCT02657889). We identify two determinants of response; mutational signature 3 reflecting defective homologous recombination DNA repair, and positive immune score as a surrogate of interferon-primed exhausted CD8 + T-cells in the tumor microenvironment. Presence of one or both features associates with an improved outcome while concurrent absence yields no responses. Single-cell spatial analysis reveals prominent interactions of exhausted CD8 + T-cells and PD-L1 + macrophages and PD-L1 + tumor cells as mechanistic determinants of response. Furthermore, spatial analysis of two extreme responders shows differential clustering of exhausted CD8 + T-cells with PD-L1 + macrophages in the first, and exhausted CD8 + T-cells with cancer cells harboring genomic PD-L1 and PD-L2 amplification in the second.
A key mutational process in cancer is structural variation, in which rearrangements delete, amplify or reorder genomic segments that range in size from kilobases to whole chromosomes1-7. Here we develop methods to group, classify and describe somatic structural variants, using data from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA), which aggregated whole-genome sequencing data from 2,658 cancers across 38 tumour types8. Sixteen signatures of structural variation emerged. Deletions have a multimodal size distribution, assort unevenly across tumour types and patients, are enriched in late-replicating regions and correlate with inversions. Tandem duplications also have a multimodal size distribution, but are enriched in early-replicating regions-as are unbalanced translocations. Replication-based mechanisms of rearrangement generate varied chromosomal structures with low-level copy-number gains and frequent inverted rearrangements. One prominent structure consists of 2-7 templates copied from distinct regions of the genome strung together within one locus. Such cycles of templated insertions correlate with tandem duplications, and-in liver cancer-frequently activate the telomerase gene TERT. A wide variety of rearrangement processes are active in cancer, which generate complex configurations of the genome upon which selection can act.
Rodriguez-Martin B, Alvarez EG, Baez-Ortega A, Zamora J, Supek F, Demeulemeester J, Santamarina M, Ju YS, Temes J, Garcia-Souto D, Detering H, Li Y, Rodriguez-Castro J, Dueso-Barroso A, Bruzos AL, Dentro SC, Blanco MG, Contino G, Ardeljan D, Tojo M, Roberts ND, Zumalave S, Edwards PAW, Weischenfeldt J, Puiggròs M, Chong Z, Chen K, Lee EA, Wala JA, Raine K, Butler A, Waszak SM, Navarro FCP, Schumacher SE, Monlong J, Maura F, Bolli N, Bourque G, Gerstein M, Park PJ, Wedge DC, Beroukhim R, Torrents D, Korbel JO, Martincorena I, Fitzgerald RC, Van Loo P, Kazazian HH, Burns KH, Group PCAWGSVW, Campbell PJ, Tubio JMC, Consortium PCAWG. Pan-cancer analysis of whole genome identifies driver rearrangements promoted by LINE-1 retrotransposition [Internet]. Nature Genetics 2020;52(3):306-319. Publisher's VersionAbstract
About half of all cancers have somatic integrations of retrotransposons. Here, to characterize their role in oncogenesis, we analyzed the patterns and mechanisms of somatic retrotransposition in 2,954 cancer genomes from 38 histological cancer subtypes within the framework of the Pan-Cancer Analysis of Whole Genomes (PCAWG) project. We identified 19,166 somatically acquired retrotransposition events, which affected 35% of samples and spanned a range of event types. Long interspersed nuclear element (LINE-1; L1 hereafter) insertions emerged as the first most frequent type of somatic structural variation in esophageal adenocarcinoma, and the second most frequent in head-and-neck and colorectal cancers. Aberrant L1 integrations can delete megabase-scale regions of a chromosome, which sometimes leads to the removal of tumor-suppressor genes, and can induce complex translocations and large-scale duplications. Somatic retrotranspositions can also initiate breakage-fusion-bridge cycles, leading to high-level amplification of oncogenes. These observations illuminate a relevant role of L1 retrotransposition in remodeling the cancer genome, with potential implications for the development of human tumors.
Cancers require telomere maintenance mechanisms for unlimited replicative potential. They achieve this through TERT activation or alternative telomere lengthening associated with ATRX or DAXX loss. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, we dissect whole-genome sequencing data of over 2500 matched tumor-control samples from 36 different tumor types aggregated within the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium to characterize the genomic footprints of these mechanisms. While the telomere content of tumors with ATRX or DAXX mutations (ATRX/DAXXtrunc) is increased, tumors with TERT modifications show a moderate decrease of telomere content. One quarter of all tumor samples contain somatic integrations of telomeric sequences into non-telomeric DNA. This fraction is increased to 80% prevalence in ATRX/DAXXtrunc tumors, which carry an aberrant telomere variant repeat (TVR) distribution as another genomic marker. The latter feature includes enrichment or depletion of the previously undescribed singleton TVRs TTCGGG and TTTGGG, respectively. Our systematic analysis provides new insight into the recurrent genomic alterations associated with telomere maintenance mechanisms in cancer.
Cancer is often seen as a disease of mutations and chromosomal abnormalities. However, some cancers, including pediatric rhabdoid tumors (RTs), lack recurrent alterations targetable by current drugs and need alternative, informed therapeutic options. To nominate potential targets, we performed a high-throughput small-molecule screen complemented by a genome-scale CRISPR-Cas9 gene-knockout screen in a large number of RT and control cell lines. These approaches converged to reveal several receptor tyrosine kinases (RTKs) as therapeutic targets, with RTK inhibition effective in suppressing RT cell growth in vitro and against a xenograft model in vivo. RT cell lines highly express and activate (phosphorylate) different RTKs, creating dependency without mutation or amplification. Downstream of RTK signaling, we identified PTPN11, encoding the pro-growth signaling protein SHP2, as a shared dependency across all RT cell lines. This study demonstrates that large-scale perturbational screening can uncover vulnerabilities in cancers with "quiet" genomes.
Howard TP, Arnoff TE, Song MR, Giacomelli AO, Wang X, Hong AL, Dharia NV, Wang S, Vazquez F, Pham M-T, Morgan AM, Wachter F, Bird GH, Kugener G, Oberlick EM, Rees MG, Tiv HL, Hwang JH, Walsh KH, Cook A, Krill-Burger JM, Tsherniak A, Gokhale PC, Park PJ, Stegmaier K, Walensky LD, Hahn WC, Roberts CWM. MDM2 and MDM4 Are Therapeutic Vulnerabilities in Malignant Rhabdoid Tumors. Cancer Research 2019;79(9)Abstract
Malignant rhabdoid tumors (MRT) are highly aggressive pediatric cancers that respond poorly to current therapies. In this study, we screened several MRT cell lines with large-scale RNAi, CRISPR-Cas9, and small-molecule libraries to identify potential drug targets specific for these cancers. We discovered MDM2 and MDM4, the canonical negative regulators of p53, as significant vulnerabilities. Using two compounds currently in clinical development, idasanutlin (MDM2-specific) and ATSP-7041 (MDM2/4-dual), we show that MRT cells were more sensitive than other p53 wild-type cancer cell lines to inhibition of MDM2 alone as well as dual inhibition of MDM2/4. These compounds caused significant upregulation of the p53 pathway in MRT cells, and sensitivity was ablated by CRISPR-Cas9–mediated inactivation of TP53. We show that loss of SMARCB1, a subunit of the SWI/SNF (BAF) complex mutated in nearly all MRTs, sensitized cells to MDM2 and MDM2/4 inhibition by enhancing p53-mediated apoptosis. Both MDM2 and MDM2/4 inhibition slowed MRT xenograft growth in vivo, with a 5-day idasanutlin pulse causing marked regression of all xenografts, including durable complete responses in 50% of mice. Together, these studies identify a genetic connection between mutations in the SWI/SNF chromatin-remodeling complex and the tumor suppressor gene TP53 and provide preclinical evidence to support the targeting of MDM2 and MDM4 in this often-fatal pediatric cancer.