Temporal genomic analysis of melanoma rejection identifies regulators of tumor immune evasion.

Decreased intra-tumor heterogeneity (ITH) correlates with increased patient survival and immunotherapy response. However, even highly homogenous tumors may display variability in their aggressiveness, and how immunologic-factors impinge on their aggressiveness remains understudied. Here we studied the mechanisms responsible for the immune-escape of murine tumors with low ITH. We compared the temporal growth of homogeneous, genetically-similar single-cell clones that are rejected vs. those that are not-rejected after transplantation in-vivo using single-cell RNA sequencing and immunophenotyping. Non-rejected clones showed high infiltration of tumor-associated-macrophages (TAMs), lower T-cell infiltration, and increased T-cell exhaustion compared to rejected clones. Comparative analysis of rejection-associated gene expression programs, combined with in-vivo CRISPR knockout screens of candidate mediators, identified Mif (macrophage migration inhibitory factor) as a regulator of immune rejection. Mif knockout led to smaller tumors and reversed non-rejection-associated immune composition, particularly, leading to the reduction of immunosuppressive macrophage infiltration. Finally, we validated these results in melanoma patient data.

Alternative polyadenylation factor CPSF6 regulates temperature compensation of the mammalian circadian clock.

A defining property of circadian clocks is temperature compensation, characterized by the resilience of their near 24-hour free-running periods against changes in environmental temperature within the physiological range. While temperature compensation is evolutionary conserved across different taxa of life and has been studied within many model organisms, its molecular underpinnings remain elusive. Posttranscriptional regulations such as temperature-sensitive alternative splicing or phosphorylation have been described as underlying reactions. Here, we show that knockdown of cleavage and polyadenylation specificity factor subunit 6 (CPSF6), a key regulator of 3'-end cleavage and polyadenylation, significantly alters circadian temperature compensation in human U-2 OS cells. We apply a combination of 3'-end-RNA-seq and mass spectrometry-based proteomics to globally quantify changes in 3' UTR length as well as gene and protein expression between wild-type and CPSF6 knockdown cells and their dependency on temperature. Since changes in temperature compensation behavior should be reflected in alterations of temperature responses within one or all of the 3 regulatory layers, we statistically assess differential responses upon changes in ambient temperature between wild-type and CPSF6 knockdown cells. By this means, we reveal candidate genes underlying circadian temperature compensation, including eukaryotic translation initiation factor 2 subunit 1 (EIF2S1).

Large-Scale Immunopeptidome Analysis Reveals Recurrent Posttranslational Splicing of Cancer- and Immune-Associated Genes.

Posttranslational spliced peptides (PTSPs) are a unique class of peptides that have been found to be presented by HLA class-I molecules in cancer. Thus far, no consensus has been reached on the proportion of PTSPs in the immunopeptidome, with estimates ranging from 2% to as high as 45% and stirring significant debate. Furthermore, the role of the HLA class-II pathway in PTSP presentation has been studied only in diabetes. Here, we exploit our large-scale cancer peptidomics database and our newly devised pipeline for filtering spliced peptide predictions to identify recurring spliced peptides, both for HLA class-I and class-II complexes. Our results indicate that HLA class-I-spliced peptides account for a low percentage of the immunopeptidome (less than 3.1%) yet are larger in number relative to other types of identified aberrant peptides. Therefore, spliced peptides significantly contribute to the repertoire of presented peptides in cancer cells. In addition, we identified HLA class-II-bound spliced peptides, but to a lower extent (less than 0.5%). The identified spliced peptides include cancer- and immune-associated genes, such as the MITF oncogene, DAPK1 tumor suppressor, and HLA-E, which were validated using synthetic peptides. The potential immunogenicity of the DAPK1- and HLA-E-derived PTSPs was also confirmed. In addition, a reanalysis of our published mouse single-cell clone immunopeptidome dataset showed that most of the spliced peptides were found repeatedly in a large number of the single-cell clones. Establishing a novel search-scheme for the discovery and evaluation of recurring PTSPs among cancer patients may assist in identifying potential novel targets for immunotherapy.

circMbl functions in cis and in trans to regulate gene expression and physiology in a tissue-specific fashion.

Muscleblind (mbl) is an essential muscle and neuronal splicing regulator. Mbl hosts multiple circular RNAs (circRNAs), including circMbl, which is conserved from flies to humans. Here, we show that mbl-derived circRNAs are key regulators of MBL by cis- and trans-acting mechanisms. By generating fly lines to specifically modulate the levels of all mbl RNA isoforms, including circMbl, we demonstrate that the two major mbl protein isoforms, MBL-O/P and MBL-C, buffer their own levels by producing different types of circRNA isoforms in the eye and fly brain, respectively. Moreover, we show that circMbl has unique functions in trans, as knockdown of circMbl results in specific morphological and physiological phenotypes. In addition, depletion of MBL-C or circMbl results in opposite behavioral phenotypes, showing that they also regulate each other in trans. Together, our results illuminate key aspects of mbl regulation and uncover cis and trans functions of circMbl in vivo.

Anti-tumour immunity induces aberrant peptide presentation in melanoma.

Extensive tumour inflammation, which is reflected by high levels of infiltrating T cells and interferon-γ (IFNγ) signalling, improves the response of patients with melanoma to checkpoint immunotherapy1,2. Many tumours, however, escape by activating cellular pathways that lead to immunosuppression. One such mechanism is the production of tryptophan metabolites along the kynurenine pathway by the enzyme indoleamine 2,3-dioxygenase 1 (IDO1), which is induced by IFNγ3-5. However, clinical trials using inhibition of IDO1 in combination with blockade of the PD1 pathway in patients with melanoma did not improve the efficacy of treatment compared to PD1 pathway blockade alone6,7, pointing to an incomplete understanding of the role of IDO1 and the consequent degradation of tryptophan in mRNA translation and cancer progression. Here we used ribosome profiling in melanoma cells to investigate the effects of prolonged IFNγ treatment on mRNA translation. Notably, we observed accumulations of ribosomes downstream of tryptophan codons, along with their expected stalling at the tryptophan codon. This suggested that ribosomes bypass tryptophan codons in the absence of tryptophan. A detailed examination of these tryptophan-associated accumulations of ribosomes-which we term 'W-bumps'-showed that they were characterized by ribosomal frameshifting events. Consistently, reporter assays combined with proteomic and immunopeptidomic analyses demonstrated the induction of ribosomal frameshifting, and the generation and presentation of aberrant trans-frame peptides at the cell surface after treatment with IFNγ. Priming of naive T cells from healthy donors with aberrant peptides induced peptide-specific T cells. Together, our results suggest that IDO1-mediated depletion of tryptophan, which is induced by IFNγ, has a role in the immune recognition of melanoma cells by contributing to diversification of the peptidome landscape.

An in vivo strategy for knockdown of circular RNAs.

Exonic circular RNAs (circRNAs) are highly abundant RNAs generated mostly from exons of protein-coding genes. Assaying the functions of circRNAs is not straightforward as common approaches for circRNA depletion tend to also alter the levels of mRNAs generated from the hosting gene. Here we describe a methodology for specific knockdown of circRNAs in vivo with tissue and cell resolution. We also describe an experimental and computational platform for determining the potential off-target effects as well as for verifying the obtained phenotypes. Briefly, we utilize shRNAs targeted to the circRNA-specific back-splice junction to specifically downregulate the circRNA. We utilized this methodology to downregulate five circRNAs that are highly expressed in Drosophila. There were no effects on the levels of their linear counterparts or any RNA with complementarity to the expressed shRNA. Interestingly, downregulation of circCtrip resulted in developmental lethality that was recapitulated with a second shRNA. Moreover, downregulation of individual circRNAs caused specific changes in the fly head transcriptome, suggesting roles for these circRNAs in the fly nervous system. Together, our results provide a methodological approach that enables the comprehensive study of circRNAs at the organismal and cellular levels and generated for the first time flies in which specific circRNAs are downregulated.

PhISCS-BnB: a fast branch and bound algorithm for the perfect tumor phylogeny reconstruction problem.

MOTIVATION: Recent advances in single-cell sequencing (SCS) offer an unprecedented insight into tumor emergence and evolution. Principled approaches to tumor phylogeny reconstruction via SCS data are typically based on general computational methods for solving an integer linear program, or a constraint satisfaction program, which, although guaranteeing convergence to the most likely solution, are very slow. Others based on Monte Carlo Markov Chain or alternative heuristics not only offer no such guarantee, but also are not faster in practice. As a result, novel methods that can scale up to handle the size and noise characteristics of emerging SCS data are highly desirable to fully utilize this technology. RESULTS: We introduce PhISCS-BnB (phylogeny inference using SCS via branch and bound), a branch and bound algorithm to compute the most likely perfect phylogeny on an input genotype matrix extracted from an SCS dataset. PhISCS-BnB not only offers an optimality guarantee, but is also 10-100 times faster than the best available methods on simulated tumor SCS data. We also applied PhISCS-BnB on a recently published large melanoma dataset derived from the sublineages of a cell line involving 20 clones with 2367 mutations, which returned the optimal tumor phylogeny in <4 h. The resulting phylogeny agrees with and extends the published results by providing a more detailed picture on the clonal evolution of the tumor. AVAILABILITY AND IMPLEMENTATION: https://github.com/algo-cancer/PhISCS-BnB. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Thermosensitive alternative splicing senses and mediates temperature adaptation in Drosophila.

Circadian rhythms are generated by the cyclic transcription, translation, and degradation of clock gene products, including timeless (tim), but how the circadian clock senses and adapts to temperature changes is not completely understood. Here, we show that temperature dramatically changes the splicing pattern of tim in Drosophila. We found that at 18°C, TIM levels are low because of the induction of two cold-specific isoforms: tim-cold and tim-short and cold. At 29°C, another isoform, tim-medium, is upregulated. Isoform switching regulates the levels and activity of TIM as each isoform has a specific function. We found that tim-short and cold encodes a protein that rescues the behavioral defects of tim01 mutants, and that flies in which tim-short and cold is abrogated have abnormal locomotor activity. In addition, miRNA-mediated control limits the expression of some of these isoforms. Finally, data that we obtained using minigenes suggest that tim alternative splicing might act as a thermometer for the circadian clock.

UVB-Induced Tumor Heterogeneity Diminishes Immune Response in Melanoma.

Although clonal neo-antigen burden is associated with improved response to immune therapy, the functional basis for this remains unclear. Here we study this question in a novel controlled mouse melanoma model that enables us to explore the effects of intra-tumor heterogeneity (ITH) on tumor aggressiveness and immunity independent of tumor mutational burden. Induction of UVB-derived mutations yields highly aggressive tumors with decreased anti-tumor activity. However, single-cell-derived tumors with reduced ITH are swiftly rejected. Their rejection is accompanied by increased T cell reactivity and a less suppressive microenvironment. Using phylogenetic analyses and mixing experiments of single-cell clones, we dissect two characteristics of ITH: the number of clones forming the tumor and their clonal diversity. Our analysis of melanoma patient tumor data recapitulates our results in terms of overall survival and response to immune checkpoint therapy. These findings highlight the importance of clonal mutations in robust immune surveillance and the need to quantify patient ITH to determine the response to checkpoint blockade.

High-accuracy determination of internal circadian time from a single blood sample.

BACKGROUND: The circadian clock is a fundamental and pervasive biological program that coordinates 24-hour rhythms in physiology, metabolism, and behavior, and it is essential to health. Whereas therapy adapted to time of day is increasingly reported to be highly successful, it needs to be personalized, since internal circadian time is different for each individual. In addition, internal time is not a stable trait, but is influenced by many factors, including genetic predisposition, age, sex, environmental light levels, and season. An easy and convenient diagnostic tool is currently missing. METHODS: To establish a validated test, we followed a 3-stage biomarker development strategy: (a) using circadian transcriptomics of blood monocytes from 12 individuals in a constant routine protocol combined with machine learning approaches, we identified biomarkers for internal time; and these biomarkers (b) were migrated to a clinically relevant gene expression profiling platform (NanoString) and (c) were externally validated using an independent study with 28 early or late chronotypes. RESULTS: We developed a highly accurate and simple assay (BodyTime) to estimate the internal circadian time in humans from a single blood sample. Our assay needs only a small set of blood-based transcript biomarkers and is as accurate as the current gold standard method, dim-light melatonin onset, at smaller monetary, time, and sample-number cost. CONCLUSION: The BodyTime assay provides a new diagnostic tool for personalization of health care according to the patient's circadian clock. FUNDING: This study was supported by the Bundesministerium für Bildung und Forschung, Germany (FKZ: 13N13160 and 13N13162) and Intellux GmbH, Germany.

Dynamic hyper-editing underlies temperature adaptation in Drosophila.

In Drosophila, A-to-I editing is prevalent in the brain, and mutations in the editing enzyme ADAR correlate with specific behavioral defects. Here we demonstrate a role for ADAR in behavioral temperature adaptation in Drosophila. Although there is a higher level of editing at lower temperatures, at 29°C more sites are edited. These sites are less evolutionarily conserved, more disperse, less likely to be involved in secondary structures, and more likely to be located in exons. Interestingly, hypomorph mutants for ADAR display a weaker transcriptional response to temperature changes than wild-type flies and a highly abnormal behavioral response upon temperature increase. In sum, our data shows that ADAR is essential for proper temperature adaptation, a key behavior trait that is essential for survival of flies in the wild. Moreover, our results suggest a more general role of ADAR in regulating RNA secondary structures in vivo.

Clonally stable Vκ allelic choice instructs Igκ repertoire.

Although much has been done to understand how rearrangement of the Igκ locus is regulated during B-cell development, little is known about the way the variable (V) segments themselves are selected. Here we show, using B6/Cast hybrid pre-B-cell clones, that a limited number of V segments on each allele is stochastically activated as characterized by the appearance of non-coding RNA and histone modifications. The activation states are clonally distinct, stable across cell division and developmentally important in directing the Ig repertoire upon differentiation. Using a new approach of allelic ATAC-seq, we demonstrate that the Igκ V alleles have differential chromatin accessibility, which may serve as the underlying basis of clonal maintenance at this locus, as well as other instances of monoallelic expression throughout the genome. These findings highlight a new level of immune system regulation that optimizes gene diversity.

Defining the 5΄ and 3΄ landscape of the Drosophila transcriptome with Exo-seq and RNaseH-seq.

Cells regulate biological responses in part through changes in transcription start sites (TSS) or cleavage and polyadenylation sites (PAS). To fully understand gene regulatory networks, it is therefore critical to accurately annotate cell type-specific TSS and PAS. Here we present a simple and straightforward approach for genome-wide annotation of 5΄- and 3΄-RNA ends. Our approach reliably discerns bona fide PAS from false PAS that arise due to internal poly(A) tracts, a common problem with current PAS annotation methods. We applied our methodology to study the impact of temperature on the Drosophila melanogaster head transcriptome. We found hundreds of previously unidentified TSS and PAS which revealed two interesting phenomena: first, genes with multiple PASs tend to harbor a motif near the most proximal PAS, which likely represents a new cleavage and polyadenylation signal. Second, motif analysis of promoters of genes affected by temperature suggested that boundary element association factor of 32 kDa (BEAF-32) and DREF mediates a transcriptional program at warm temperatures, a result we validated in a fly line where beaf-32 is downregulated. These results demonstrate the utility of a high-throughput platform for complete experimental and computational analysis of mRNA-ends to improve gene annotation.

Translation of CircRNAs.

Circular RNAs (circRNAs) are abundant and evolutionarily conserved RNAs of largely unknown function. Here, we show that a subset of circRNAs is translated in vivo. By performing ribosome footprinting from fly heads, we demonstrate that a group of circRNAs is associated with translating ribosomes. Many of these ribo-circRNAs use the start codon of the hosting mRNA, are bound by membrane-associated ribosomes, and have evolutionarily conserved termination codons. In addition, we found that a circRNA generated from the muscleblind locus encodes a protein, which we detected in fly head extracts by mass spectrometry. Next, by performing in vivo and in vitro translation assays, we show that UTRs of ribo-circRNAs (cUTRs) allow cap-independent translation. Moreover, we found that starvation and FOXO likely regulate the translation of a circMbl isoform. Altogether, our study provides strong evidence for translation of circRNAs, revealing the existence of an unexplored layer of gene activity.

Marked Differences in C9orf72 Methylation Status and Isoform Expression between C9/ALS Human Embryonic and Induced Pluripotent Stem Cells.

We established two human embryonic stem cell (hESC) lines with a GGGGCC expansion in the C9orf72 gene (C9), and compared them with haploidentical and unrelated C9 induced pluripotent stem cells (iPSCs). We found a marked difference in C9 methylation between the cells. hESCs and parental fibroblasts are entirely unmethylated while the iPSCs are hypermethylated. In addition, we show that the expansion alters promoter usage and interferes with the proper splicing of intron 1, eventually leading to the accumulation of repeat-containing mRNA following neural differentiation. These changes are attenuated in C9 iPSCs, presumably owing to hypermethylation. Altogether, this study highlights the importance of neural differentiation in the pathogenesis of disease and points to the potential role of hypermethylation as a neuroprotective mechanism against pathogenic mRNAs, envisaging a milder phenotype in C9 iPSCs.

A pseudouridylation switch in rRNA is implicated in ribosome function during the life cycle of Trypanosoma brucei.

The protozoan parasite Trypanosoma brucei, which causes devastating diseases in humans and animals in sub-Saharan Africa, undergoes a complex life cycle between the mammalian host and the blood-feeding tsetse fly vector. However, little is known about how the parasite performs most molecular functions in such different environments. Here, we provide evidence for the intriguing possibility that pseudouridylation of rRNA plays an important role in the capacity of the parasite to transit between the insect midgut and the mammalian bloodstream. Briefly, we mapped pseudouridines (Ψ) on rRNA by Ψ-seq in procyclic form (PCF) and bloodstream form (BSF) trypanosomes. We detected 68 Ψs on rRNA, which are guided by H/ACA small nucleolar RNAs (snoRNA). The small RNome of both life cycle stages was determined by HiSeq and 83 H/ACAs were identified. We observed an elevation of 21 Ψs modifications in BSF as a result of increased levels of the guiding snoRNAs. Overexpression of snoRNAs guiding modification on H69 provided a slight growth advantage to PCF parasites at 30 °C. Interestingly, these modifications are predicted to significantly alter the secondary structure of the large subunit (LSU) rRNA suggesting that hypermodified positions may contribute to the adaption of ribosome function during cycling between the two hosts.

Clk post-transcriptional control denoises circadian transcription both temporally and spatially.

The transcription factor CLOCK (CLK) is essential for the development and maintenance of circadian rhythms in Drosophila. However, little is known about how CLK levels are controlled. Here we show that Clk mRNA is strongly regulated post-transcriptionally through its 3' UTR. Flies expressing Clk transgenes without normal 3' UTR exhibit variable CLK-driven transcription and circadian behaviour as well as ectopic expression of CLK-target genes in the brain. In these flies, the number of the key circadian neurons differs stochastically between individuals and within the two hemispheres of the same brain. Moreover, flies carrying Clk transgenes with deletions in the binding sites for the miRNA bantam have stochastic number of pacemaker neurons, suggesting that this miRNA mediates the deterministic expression of CLK. Overall our results demonstrate a key role of Clk post-transcriptional control in stabilizing circadian transcription, which is essential for proper development and maintenance of circadian rhythms in Drosophila.

The transcription factor Cabut coordinates energy metabolism and the circadian clock in response to sugar sensing.

Nutrient sensing pathways adjust metabolism and physiological functions in response to food intake. For example, sugar feeding promotes lipogenesis by activating glycolytic and lipogenic genes through the Mondo/ChREBP-Mlx transcription factor complex. Concomitantly, other metabolic routes are inhibited, but the mechanisms of transcriptional repression upon sugar sensing have remained elusive. Here, we characterize cabut (cbt), a transcription factor responsible for the repressive branch of the sugar sensing transcriptional network in Drosophila. We demonstrate that cbt is rapidly induced upon sugar feeding through direct regulation by Mondo-Mlx. We found that CBT represses several metabolic targets in response to sugar feeding, including both isoforms of phosphoenolpyruvate carboxykinase (pepck). Deregulation of pepck1 (CG17725) in mlx mutants underlies imbalance of glycerol and glucose metabolism as well as developmental lethality. Furthermore, we demonstrate that cbt provides a regulatory link between nutrient sensing and the circadian clock. Specifically, we show that a subset of genes regulated by the circadian clock are also targets of CBT. Moreover, perturbation of CBT levels leads to deregulation of the circadian transcriptome and circadian behavioral patterns.

Circular RNAs in the Mammalian Brain Are Highly Abundant, Conserved, and Dynamically Expressed.

Circular RNAs (circRNAs) are an endogenous class of animal RNAs. Despite their abundance, their function and expression in the nervous system are unknown. Therefore, we sequenced RNA from different brain regions, primary neurons, isolated synapses, as well as during neuronal differentiation. Using these and other available data, we discovered and analyzed thousands of neuronal human and mouse circRNAs. circRNAs were extraordinarily enriched in the mammalian brain, well conserved in sequence, often expressed as circRNAs in both human and mouse, and sometimes even detected in Drosophila brains. circRNAs were overall upregulated during neuronal differentiation, highly enriched in synapses, and often differentially expressed compared to their mRNA isoforms. circRNA expression correlated negatively with expression of the RNA-editing enzyme ADAR1. Knockdown of ADAR1 induced elevated circRNA expression. Together, we provide a circRNA brain expression atlas and evidence for important circRNA functions and values as biomarkers.

Differentiation of human parthenogenetic pluripotent stem cells reveals multiple tissue- and isoform-specific imprinted transcripts.

Parental imprinting results in monoallelic parent-of-origin-dependent gene expression. However, many imprinted genes identified by differential methylation do not exhibit complete monoallelic expression. Previous studies demonstrated complex tissue-dependent expression patterns for some imprinted genes. Still, the complete magnitude of this phenomenon remains largely unknown. By differentiating human parthenogenetic induced pluripotent stem cells into different cell types and combining DNA methylation with a 5' RNA sequencing methodology, we were able to identify tissue- and isoform-dependent imprinted genes in a genome-wide manner. We demonstrate that nearly half of all imprinted genes express both biallelic and monoallelic isoforms that are controlled by tissue-specific alternative promoters. This study provides a global analysis of tissue-specific imprinting in humans and suggests that alternative promoters are central in the regulation of imprinted genes.