UV radiation (UVR) drives high mutational burdens, yet precursor melanocytes accumulate these mutations without triggering immune clearance. Here, we investigated whether melanocyte-intrinsic transcriptional program(s) underlie immune tolerance to mutations resulting from UVR exposure. In primary human melanocytes, expression of PD-L1 (CD274) was dependent on microphthalmia-associated transcription factor (MITF), a crucial regulator of melanocyte development and an intermediate in the UV-tanning pathway. MITF directly activated PD-L1 transcription by binding a conserved upstream enhancer containing functional E-box elements. MITF determined both baseline melanocytic PD-L1 expression in healthy skin and its induction following UVR, independent of interferon signaling. Melanocyte-restricted Pd-l1 deletion in mice triggered CD8+ T cell infiltration and depigmentation after long-term UVB exposure, recapitulating features of human vitiligo. PD-L1-deficient human induced pluripotent stem cell (iPSC)-derived melanocytes underwent increased apoptosis and were more susceptible than PD-L1-intact melanocytes to gp100-specific CD8+ T cell killing. Thus, a melanocyte-intrinsic MITF-PD-L1 tolerance program protects melanocytes from autoimmune destruction, potentially facilitating early immune evasion during melanoma development and conversely underlying the responsiveness of melanoma to PD-1/PD-L1 blockade.

Background: Chromosome 3p (chr3p) is frequently deleted in multiple cancers, indicating the presence of shared tumor suppressors. In aggressive uveal melanomas (UVM), this deletion often co-occurs with chr8q amplification (8q+), suggesting strong selection pressure during UVM evolution. Methods: To understand the pattern of genomic alterations mediated by chr3p deletion, we have developed an algorithm for detecting isochromosomes in 10,632 TCGA cancer patients. We further perform integrative genomics analysis to investigate how chr3p deletion could affect subsequent cancer genome evolution and synthetic lethality in UVM. Results: Analysis of genomic alterations in 33 different cancer types implicates the deletion or deleterious mutations of SET-domain-containing 2 (SETD2) at chr3p21 in significantly facilitating the formation of isochromosomes, thereby promoting genomic instability conducive to rapid cancer genome evolution. Fracturing of dicentric isochromosomes during cell division is pervasive and follows the dynamic fragmentation pattern of solids under impulse. In the most aggressive UVM subtype, chr3 deletion includes MITF, a master regulator of melanocyte survival and differentiation, and co-occurs with 8q+. We demonstrate that MITF is a master transcriptional regulator of GNAQ/GNA11 and associated synthetic-lethal genes in UVM. MITF maintains MAPK and calcium homeostasis in UVM, and its hemizygous deletion is thus accidental, likely creating an early crisis during oncogenesis. We further show that MITF, MYC, and GNAQ/GNA11 form coupled regulatory feedback loops in the melanocyte lineage, and MITF deletion in UVM creates acute dependency on MYC-mediated rescue via 8q+. The discovered feedback loops predict both overall and relapse-free patient survival within the most aggressive UVM subtype, explain sensitivity to therapeutic gene perturbations, and inform effective combinatorial therapies. Conclusions: SETD2 deletion potentiates isochromosome formation across diverse cancers. Combinatorial targeting of MITF together with a previously identified synthetic lethal gene may benefit UVM patients harboring both chr3 deletion and 8q+.

Activating mutations in the telomerase reverse transcriptase (TERT) promoter are prevalent in cancer and enable limitless cell division characteristic of immortal cells. Solving the immortality mechanism represents a major step toward selective reversal in cancer cells. TERT promoter (TERTp) mutations create a de novo E26 transformation-specific (ETS) transcription factor binding motif. Here, we analyzed 53 cell lines representing 16 cancer types and 6 recurrent TERTp mutations and found that the GA-binding protein (GABP) tetramer is responsible for promoter activation in all cases. Surprisingly, TERT expression is maintained after tetramer depletion. Further investigation revealed an underlying network of auto-suppression among the GABP subunits. Release from it drives TERT maintenance via upregulated GABP dimers or a paralogous tetramer. The GABPB1L tetramer is therefore a pan-cancer, pan-mutation activator of the mutant TERT promoter, but it is replaceable. Domains shared by the three GABP complexes, rather than solely the B1L tetramer, are mutation-specific vulnerabilities.

Prime editor (PE) is a highly versatile CRISPR-Cas9 genome editing technique. The current constructs, however, have variable efficiency and may require laborious experimental optimization. This study presents statistical models for learning the salient epigenomic and sequence features of target sites modulating the editing efficiency and provides guidelines for designing optimal PEs. We found that both regional constitutive heterochromatin and local nucleosome occlusion of target sites impede editing, while position-specific G/C nucleotides in the primer binding site (PBS) and reverse transcription (RT) template regions of PE guide-RNA (pegRNA) yield high editing efficiency, especially for short PBS designs. The presence of G/C nucleotides was most critical immediately 5' to the protospacer adjacent motif (PAM) site for all designs. The effects of different last templated nucleotides were quantified and seen to depend on both PBS and RT template lengths. Our models found AGG to be the preferred PAM and detected a guanine nucleotide four bases downstream of PAM to facilitate editing, suggesting a hitherto-unrecognized interaction with Cas9. A neural network interpretation method based on nonextensive statistical mechanics further revealed multi-nucleotide preferences, indicating dependency among several bases across pegRNA. Our work clarifies previous conflicting observations and uncovers context-dependent features important for optimizing PE designs.

Supplementary Table 1. CB cis-sQTL. Supplementary Table 2. SCG cis-sQTL. Supplementary Table 3. trans-sQTL. Supplementary Table 4. Genes differentially expressed on Chr10 in 95% CI. Supplementary Table 5. Genes differentially expressed on ChrX in 95% CI. Supplementary Table 6. Cis-sQTL Co-localizing with SCNVs. Supplementary Table 7. Genotyping markers used in this study. Supplementary Table 8. Oligonucleotides used in this study.

Supplementary Figure S1. DDX26B and RBMX2 Influence Alternative Splicing. Supplementary Figure S2. Astn2 possesses a cis-sQTL. Supplementary Figure S3. SCNVs Identified by WGS. Supplementary Figure S4. Fubp1 RT-PCR From Parental Mouse Strains. Supplementary Figure S5. FUBP1 Expression Correlates with Survival in Two Neuroblastoma Datasets. Supplementary Figure S6. Total Set of Identified Unique Splicing Motifs. Supplementary Figure S7. Motifs with Matches to an RNA Binding database. Supplementary Figure S8. Recurrent Somatic Mutations Occur in Intronic Splicing Motifs in GBM.

Understanding the impact of regulatory variants on complex phenotypes is a significant challenge because the genes and pathways that are targeted by such variants and the cell type context in which regulatory variants operate are typically unknown. Cell-type-specific long-range regulatory interactions that occur between a distal regulatory sequence and a gene offer a powerful framework for examining the impact of regulatory variants on complex phenotypes. However, high-resolution maps of such long-range interactions are available only for a handful of cell types. Furthermore, identifying specific gene subnetworks or pathways that are targeted by a set of variants is a significant challenge. We have developed L-HiC-Reg, a Random Forests regression method to predict high-resolution contact counts in new cell types, and a network-based framework to identify candidate cell-type-specific gene networks targeted by a set of variants from a genome-wide association study (GWAS). We applied our approach to predict interactions in 55 Roadmap Epigenomics Mapping Consortium cell types, which we used to interpret regulatory single nucleotide polymorphisms (SNPs) in the NHGRI-EBI GWAS catalogue. Using our approach, we performed an in-depth characterization of fifteen different phenotypes including schizophrenia, coronary artery disease (CAD) and Crohn's disease. We found differentially wired subnetworks consisting of known as well as novel gene targets of regulatory SNPs. Taken together, our compendium of interactions and the associated network-based analysis pipeline leverages long-range regulatory interactions to examine the context-specific impact of regulatory variation in complex phenotypes.

Supplementary Tables 1-2, Figures 1-10 from CCCTC-Binding Factor Confines the Distal Action of Estrogen Receptor

Supplementary Figure Legends, Table Legends

Schwannomas are common peripheral nerve sheath tumors that can cause severe morbidity due to their stereotypic intracranial and paraspinal locations. However, the molecular drivers responsible for a substantial subset remain unknown. Through genomic profiling of 96 schwannomas, we identified novel in-frame insertion/deletion mutations in the SOX10 gene in 29% of sporadic tumors which uniformly lacked alterations in known nerve sheath tumor genes including NF1, NF2, LZTR1, and SMARCB1. These indel mutations clustered at the C-terminal end of the HMG-box domain, the DNA binding motif of the SOX10 transcription factor critical for Schwann cell differentiation. Schwannomas arising from non-vestibular cranial nerves (e.g. facial, trigeminal, vagus) were highly enriched for SOX10 mutations, whereas the vast majority of vestibular schwannomas had NF2 inactivation. DNA methylation profiling revealed that SOX10 mutant schwannomas clustered together with schwannomas harboring NF2 mutation or SH3PXD2A-HTRA1 fusion, indicating that SOX10 indel mutations define a novel schwannoma molecular subtype and not a distinct nerve sheath tumor entity. To investigate the mechanism by which SOX10 indel mutations promote schwannoma development, we stably transduced human fetal glial cells with either wildtype SOX10 or two different tumor-derived insertion mutants. RNA sequencing revealed that both SOX10 indel mutants, in contrast to wildtype SOX10, failed to activate expression of myelination and glial differentiation gene programs, including PMP2 which encodes the major peripheral myelin protein in mature Schwann cells. Electrophoretic mobility shift assay revealed that the indel mutants retained their DNA binding capacity to the SOX10 binding site in the PMP2 promoter, while a luciferase reporter assay revealed transactivation at the PMP2 promoter by wildtype SOX10 but not by the mutants. In summary, we identified a new recurrent genetic event in 29% of sporadic schwannomas that we speculate drives schwannoma development through impaired transactivation of myelination gene expression programs causing blocked differentiation of immature Schwann cells.

Abstract Activating mutations in the Telomerase Reverse Transcriptase ( TERT ) promoter are the single most common non-coding mutation in cancer and enable limitless cell division characteristic of immortal cells 1–12 . Solving the immortality mechanism represents a major step towards selectively reversing it in cancer cells. TERT promoter mutations create a de novo E26 transformation specific (ETS) transcription factor binding motif, however most of the 28 ETS factors and many other transcriptional regulators have been implicated 13–22 . Cancer type and mutation specific mechanisms have also been proposed. Here, we uniformly and robustly analyzed fifty-three cell lines representing sixteen cancer types and six recurrent mutations and found that a tetramer of the GA-binding protein (GABP) is specifically responsible for mutant TERT promoter activation in all cases, with no such role in TERT promoter wild type cells. Strikingly, TERT expression is maintained in tetramer depleted tumor cells. We show how and why the tetramer is serially replaced, not by other transcription factors, but by GABP dimers and then weakly by a paralogous tetramer complex. Elimination of the tetramer and dimer reinstates epigenetic repression of TERT , activates checkpoint programs and prevents cancer cell division. We conclude that unique features of the GABP tetramer must therefore determine positive selection of nearly all TERT promoter mutations in human cancer. Furthermore, domains shared among the three GABP complexes present pan-cancer vulnerabilities.

Differential chromatin interaction analysis between MCF-7 and T-47D based on Hi-C data.

Background Schwannomas are common peripheral nerve sheath tumors that can cause severe morbidity given their stereotypic intracranial and paraspinal locations. Similar to many solid tumors, schwannomas and other nerve sheath tumors are primarily thought to arise due to aberrant hyperactivation of the RAS growth factor signaling pathway. Here, we sought to further define the molecular pathogenesis of schwannomas. Methods We performed comprehensive genomic profiling on a cohort of 96 human schwannomas, as well as DNA methylation profiling on a subset. Functional studies including RNA sequencing, chromatin immunoprecipitation-DNA sequencing, electrophoretic mobility shift assay, and luciferase reporter assays were performed in a fetal glial cell model following transduction with wildtype and tumor-derived mutant isoforms of SOX10. Results We identified that nearly one-third of sporadic schwannomas lack alterations in known nerve sheath tumor genes and instead harbor novel recurrent in-frame insertion/deletion mutations in SOX10, which encodes a transcription factor responsible for controlling Schwann cell differentiation and myelination. SOX10 indel mutations were highly enriched in schwannomas arising from nonvestibular cranial nerves (eg facial, trigeminal, vagus) and were absent from vestibular nerve schwannomas driven by NF2 mutation. Functional studies revealed these SOX10 indel mutations have retained DNA binding capacity but impaired transactivation of glial differentiation and myelination gene programs. Conclusions We thus speculate that SOX10 indel mutations drive a unique subtype of schwannomas by impeding proper differentiation of immature Schwann cells.

Supplementary Figures 1-5 from Nkx3-1 and LEF-1 Function as Transcriptional Inhibitors of Estrogen Receptor Activity

Abstract Telomerase Reverse Transcriptase promoter mutations enable tumor cell immortality in millions of cancer patients annually. While prior therapies targeting telomerase lacked tumor selectivity and were poorly tolerated, TERT promoter mutations and their regulation present a unique opportunity for tumor specific reversal of cellular immortality. The two hotspot mutations, G228A and G250A, generate identical de novo E26 transformation specific (ETS) transcription factor binding sites. However, the 28 ETS factors share a similar binding site preference, raising the question of whether all ETS factors can reactivate TERT. In glioblastoma and a few other cancers, we have shown the de novo ETS site along with a nearby native ETS site (ETS-195/200) recruit one ETS factor, the GA-binding protein (GABP) tetramer, to reactivate TERT. This GABP-mediated mechanism may be restricted to specific cancer types and the two hotspot mutations or could be widely relevant to any cancer with a de novo ETS TERT promoter mutation. We have found that the GABP tetramer activates 6 distinct mutant TERT promoters across 16 different cancer types. However, TERT expression is maintained in most tetramer depleted tumor cells as the tetramer is serially replaced by GABP dimers and weakly by a paralogous tetramer complex that increase following disengagement of GABP tetramer-mediated negative feedback loops. Elimination of both the tetramer and dimer reinstates epigenetic repression of TERT, shortens telomeres, and prevents cancer cell division. However, some knockout cancer cells avoid this fate through weak activation of TERT by the paralogous GABP tetramer complex. The compensatory maintenance of TERT expression can be overcome with a dominant negative GABPB1, leading to TERT silencing, evidenced by gene expression and non-invasive metabolic imaging correlates, and telomere shortening in TERT promoter mutant glioblastoma. We are currently investigating the delivery of this dominant negative to tumor cells via clinically approved retroviral replicating vectors.

Some of inherited human genetic variation can contribute to important phenotypic diversity, such as the varying degrees of individual susceptibility to developing certain health conditions and individual response to therapeutic interventions. To date, over 490,000 genotypephenotype associations have been discovered through large-scale genome-wide association studies (GWAS) [1]; however, molecular functions of most of these discovered GWAS variants remain unknown. There are several technical challenges hindering our understanding: (1) the effect size of a typical genetic variant, as measured in terms of the odds ratio of genotype occurrence in case versus control populations, is very small, suggesting that macroscopic systems-level phenotypic differences modulated by each variant may also be small and difficult to detect; (2) most reported variants reside in non-proteincoding regions of the human genome, indicating that they are likely affecting the regulation of some unknown target genes’ expression; and, (3) the discovered variants may not be functional themselves, but be merely in genetic linkage disequilibrium with other functional variants. A promising approach to address these challenges is to integrate genomic, epigenomic, transcriptomic and machine learning methods to identify functional genetic variants and characterize their mode of action in regulating target genes. One particular mode of regulatory function amenable to this integrative analysis is altering the binding affinity of transcription factors (TF) to DNA recognition sequences [2]. That is, assuming that a causative variant perturbs the binding activity of a TF, one can focus on the variants that are genetically linked to a given GWAS variant and located in transcriptionally active open chromatin regions annotated via epigenomic profiling – e.g., DNase-seq, ATAC-seq, and histone modification signatures of enhancers and promoters, often available in public databases such as the Encyclopedia of DNA Elements (ENCODE), Roadmap Epigenomics Mapping Consortium (REMC) and Gene Expression Omnibus (GEO) [3–5]. The ability of these epigenomically filtered candidate variants to perturb the binding activity of a specific TF can then be assessed computationally by training machine learning algorithms on TF ChIP-seq and HT-SELEX-seq data to learn the salient features of preferred DNA recognition sequences and to predict how the variants in the context of surrounding nucleotides alter the strength of TF-DNA interaction [2, 6–12]. Allelespecific binding preferences of predicted TFs can be verified by searching for skewed allele frequencies of the candidate variants in raw ChIP-seq reads, appropriately taking into account potential mapping biases. Target genes that are differentially expressed between case and control populations as a result of the predicted perturbation of TF binding activity may then be identified via expression quantitative trait loci and allele-specific expression analyses using processed and raw RNA-seq data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) projects [13–15]; further support can

Single-cell sequencing technologies that simultaneously generate multimodal cellular profiles present opportunities for improved understanding of cell heterogeneity in tissues. How the multimodal information can be integrated to obtain a common cell type identification, however, poses a computational challenge. Multilayer graphs provide a natural representation of multi-omic single-cell sequencing datasets, and finding cell clusters may be understood as a multilayer graph partition problem. We introduce two spectral algorithms on multilayer graphs, spectral clustering on multilayer graphs (SCML) and the weighted locally linear (WLL) method, to cluster cells in multi-omic single-cell sequencing datasets. We connect these algorithms through a unifying mathematical framework that represents each layer using a Hamiltonian operator and a mixture of its eigenstates to integrate the multiple graph layers, demonstrating in the process that the WLL method is a rigorous multilayer spectral graph theoretic reformulation of the popular Seurat weighted nearest neighbor (WNN) algorithm. Implementing our algorithms and applying them to a CITE-seq dataset of cord blood mononuclear cells yields results similar to the Seurat WNN analysis. Our work thus extends spectral methods to multimodal single-cell data analysis. The code used in this study can be found at https://github.com/jssong-lab/sc-spectrum

Loss-of-function mutations of JAK1/2 impair cancer cell responsiveness to IFNγ and immunogenicity. Therefore, an understanding of compensatory pathways to activate IFNγ signaling in cancer cells is clinically important for the success of immunotherapy. Here we demonstrate that the transcription factor SOX10 hinders immunogenicity of melanoma cells through the IRF4-IRF1 axis. Genetic and pharmacologic approaches revealed that SOX10 repressed IRF1 transcription via direct induction of a negative regulator, IRF4. The SOX10-IRF4-IRF1 axis regulated PD-L1 expression independently of JAK-STAT pathway activity, and suppression of SOX10 increased the efficacy of combination therapy with an anti-PD-1 antibody and histone deacetylase inhibitor against a clinically relevant melanoma model. Thus, the SOX10-IRF4-IRF1 axis serves as a potential target that can bypass JAK-STAT signaling to immunologically warm up melanoma with a "cold" tumor immune microenvironment. SIGNIFICANCE: This study identifies a novel SOX10/IRF4 pathway that regulates noncanonical induction of IRF1 independent of the JAK-STAT pathway and can be targeted to improve the efficacy of anti-PD-1 therapy in melanoma.

Background. Large-scale genome-wide association studies (GWAS) have implicated thousands of germline genetic variants in modulating individuals' risk to various diseases, including cancer. At least 25 risk loci have been identified for low-grade gliomas (LGGs), but their molecular functions remain largely unknown. Methods. We hypothesized that GWAS loci contain causal single nucleotide polymorphisms (SNPs) that reside in accessible open chromatin regions and modulate the expression of target genes by perturbing the binding affinity of transcription factors (TFs). We performed an integrative analysis of genomic and epigenomic data from The Cancer Genome Atlas and other public repositories to identify candidate causal SNPs within linkage disequilibrium blocks of LGG GWAS loci. We assessed their potential regulatory role via in silico TF binding sequence perturbations, convolutional neural network trained onTF binding data, and simulated annealing-based interpretation methods. Results. We built an interactive website (http://education.knoweng.org/alg3/) summarizing the functional footprinting of 280 variants in 25 LGG GWAS regions, providing rich information for further computational and experimental scrutiny. We identified as case studies PHLDB1 and SLC25A26 as candidate target genes of rs12803321 and rs11706832, respectively, and predicted the GWAS variant rs648044 to be the causal SNP modulating ZBTB16, a known tumor suppressor in multiple cancers. We showed that rs648044 likely perturbed the binding affinity of the TF MAFF, as supported by RNA interference and in vitro MAFF binding experiments. Conclusions. The identified candidate (causal SNP, target gene, TF) triplets and the accompanying resource will help accelerate our understanding of the molecular mechanisms underlying genetic risk factors for gliomas.