Publications by authors named "Angela N Brooks"

33 Publications

eVIP2: Expression-based variant impact phenotyping to predict the function of gene variants.

PLoS Comput Biol 2021 07 2;17(7):e1009132. Epub 2021 Jul 2.

Department of Biomolecular Engineering, University of California Santa Cruz, Santa Cruz, California, United States of America.

While advancements in genome sequencing have identified millions of somatic mutations in cancer, their functional impact is poorly understood. We previously developed the expression-based variant impact phenotyping (eVIP) method to use gene expression data to characterize the function of gene variants. The eVIP method uses a decision tree-based algorithm to predict the functional impact of somatic variants by comparing gene expression signatures induced by introduction of wild-type (WT) versus mutant cDNAs in cell lines. The method distinguishes between variants that are gain-of-function, loss-of-function, change-of-function, or neutral. We present eVIP2, software that allows for pathway analysis (eVIP Pathways) and usage with RNA-seq data. To demonstrate the eVIP2 software and approach, we characterized two recurrent frameshift variants in RNF43, a negative regulator of Wnt signaling, frequently mutated in colorectal, gastric, and endometrial cancer. RNF43 WT, RNF43 R117fs, RNF43 G659fs, or GFP control cDNA were overexpressed in HEK293T cells. Analysis with eVIP2 predicted that the frameshift at position 117 was a loss-of-function mutation, as expected. The second frameshift at position 659 has been previously described as a passenger mutation that maintains the RNF43 WT function as a negative regulator of Wnt. Surprisingly, eVIP2 predicted G659fs to be a change-of-function mutation. Additional eVIP Pathways analysis of RNF43 G659fs predicted 10 pathways to be significantly altered, including TNF-α via NFκB signaling, KRAS signaling, and hypoxia, highlighting the benefit of a more comprehensive approach when determining the impact of gene variant function. To validate these predictions, we performed reporter assays and found that each pathway activated by expression of RNF43 G659fs, but not expression of RNF43 WT, was identified as impacted by eVIP2, supporting that RNF43 G659fs is a change-of-function mutation and its effect on the identified pathways. Pathway activation was further validated by Western blot analysis. Lastly, we show primary colon adenocarcinoma patient samples with R117fs and G659fs variants have transcriptional profiles similar to BRAF missense mutations with activated RAS/MAPK signaling, consistent with KRAS signaling pathways being GOF in both variants. The eVIP2 method is an important step towards overcoming the current challenge of variant interpretation in the implementation of precision medicine. eVIP2 is available at https://github.com/BrooksLabUCSC/eVIP2.
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http://dx.doi.org/10.1371/journal.pcbi.1009132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8281988PMC
July 2021

Inflammation drives alternative first exon usage to regulate immune genes including a novel iron-regulated isoform of .

Elife 2021 05 28;10. Epub 2021 May 28.

Department of Molecular, Cell and Developmental Biology, University of California Santa Cruz, Santa Cruz, United States.

Determining the layers of gene regulation within the innate immune response is critical to our understanding of the cellular responses to infection and dysregulation in disease. We identified a conserved mechanism of gene regulation in human and mouse via changes in alternative first exon (AFE) usage following inflammation, resulting in changes to the isoforms produced. Of these AFE events, we identified 95 unannotated transcription start sites in mice using a de novo transcriptome generated by long-read native RNA-sequencing, one of which is in the cytosolic receptor for dsDNA and known inflammatory inducible gene, . We show that this unannotated AFE isoform of is the predominant isoform expressed during inflammation and contains an iron-responsive element in its 5'UTR enabling mRNA translation to be regulated by iron levels. This work highlights the importance of examining alternative isoform changes and translational regulation in the innate immune response and uncovers novel regulatory mechanisms of .
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http://dx.doi.org/10.7554/eLife.69431DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8260223PMC
May 2021

Reintroduction of the archaic variant of in cortical organoids alters neurodevelopment.

Science 2021 02;371(6530)

Department of Pediatrics and Department of Cellular & Molecular Medicine, School of Medicine, Center for Academic Research and Training in Anthropogeny (CARTA), Kavli Institute for Brain and Mind, University of California, San Diego, La Jolla, CA 92037, USA.

The evolutionarily conserved splicing regulator neuro-oncological ventral antigen 1 () plays a key role in neural development and function. also includes a protein-coding difference between the modern human genome and Neanderthal and Denisovan genomes. To investigate the functional importance of an amino acid change in humans, we reintroduced the archaic allele into human induced pluripotent cells using genome editing and then followed their neural development through cortical organoids. This modification promoted slower development and higher surface complexity in cortical organoids with the archaic version of Moreover, levels of synaptic markers and synaptic protein coassociations correlated with altered electrophysiological properties in organoids expressing the archaic variant. Our results suggest that the human-specific substitution in , which is exclusive to modern humans since divergence from Neanderthals, may have had functional consequences for our species' evolution.
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http://dx.doi.org/10.1126/science.aax2537DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8006534PMC
February 2021

Proteasome inhibitor-induced modulation reveals the spliceosome as a specific therapeutic vulnerability in multiple myeloma.

Nat Commun 2020 04 22;11(1):1931. Epub 2020 Apr 22.

Department of Laboratory Medicine, University of California, San Francisco, CA, USA.

Enhancing the efficacy of proteasome inhibitors (PI) is a central goal in myeloma therapy. We proposed that signaling-level responses after PI may reveal new mechanisms of action that can be therapeutically exploited. Unbiased phosphoproteomics after treatment with the PI carfilzomib surprisingly demonstrates the most prominent phosphorylation changes on splicing related proteins. Spliceosome modulation is invisible to RNA or protein abundance alone. Transcriptome analysis after PI demonstrates broad-scale intron retention, suggestive of spliceosome interference, as well as specific alternative splicing of protein homeostasis machinery components. These findings lead us to evaluate direct spliceosome inhibition in myeloma, which synergizes with carfilzomib and shows potent anti-tumor activity. Functional genomics and exome sequencing further support the spliceosome as a specific vulnerability in myeloma. Our results propose splicing interference as an unrecognized modality of PI mechanism, reveal additional modes of spliceosome modulation, and suggest spliceosome targeting as a promising therapeutic strategy in myeloma.
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http://dx.doi.org/10.1038/s41467-020-15521-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7176739PMC
April 2020

Full-length transcript characterization of SF3B1 mutation in chronic lymphocytic leukemia reveals downregulation of retained introns.

Nat Commun 2020 03 18;11(1):1438. Epub 2020 Mar 18.

Department of Biomolecular Engineering, University of California, Santa Cruz, CA, 95062, USA.

While splicing changes caused by somatic mutations in SF3B1 are known, identifying full-length isoform changes may better elucidate the functional consequences of these mutations. We report nanopore sequencing of full-length cDNA from CLL samples with and without SF3B1 mutation, as well as normal B cell samples, giving a total of 149 million pass reads. We present FLAIR (Full-Length Alternative Isoform analysis of RNA), a computational workflow to identify high-confidence transcripts, perform differential splicing event analysis, and differential isoform analysis. Using nanopore reads, we demonstrate differential 3' splice site changes associated with SF3B1 mutation, agreeing with previous studies. We also observe a strong downregulation of intron retention events associated with SF3B1 mutation. Full-length transcript analysis links multiple alternative splicing events together and allows for better estimates of the abundance of productive versus unproductive isoforms. Our work demonstrates the potential utility of nanopore sequencing for cancer and splicing research.
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http://dx.doi.org/10.1038/s41467-020-15171-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7080807PMC
March 2020

Genomic basis for RNA alterations in cancer.

Nature 2020 02 5;578(7793):129-136. Epub 2020 Feb 5.

BGI-Shenzhen, Shenzhen, China.

Transcript alterations often result from somatic changes in cancer genomes. Various forms of RNA alterations have been described in cancer, including overexpression, altered splicing and gene fusions; however, it is difficult to attribute these to underlying genomic changes owing to heterogeneity among patients and tumour types, and the relatively small cohorts of patients for whom samples have been analysed by both transcriptome and whole-genome sequencing. Here we present, to our knowledge, the most comprehensive catalogue of cancer-associated gene alterations to date, obtained by characterizing tumour transcriptomes from 1,188 donors of the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). Using matched whole-genome sequencing data, we associated several categories of RNA alterations with germline and somatic DNA alterations, and identified probable genetic mechanisms. Somatic copy-number alterations were the major drivers of variations in total gene and allele-specific expression. We identified 649 associations of somatic single-nucleotide variants with gene expression in cis, of which 68.4% involved associations with flanking non-coding regions of the gene. We found 1,900 splicing alterations associated with somatic mutations, including the formation of exons within introns in proximity to Alu elements. In addition, 82% of gene fusions were associated with structural variants, including 75 of a new class, termed 'bridged' fusions, in which a third genomic location bridges two genes. We observed transcriptomic alteration signatures that differ between cancer types and have associations with variations in DNA mutational signatures. This compendium of RNA alterations in the genomic context provides a rich resource for identifying genes and mechanisms that are functionally implicated in cancer.
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http://dx.doi.org/10.1038/s41586-020-1970-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054216PMC
February 2020

Nanopore native RNA sequencing of a human poly(A) transcriptome.

Nat Methods 2019 12 18;16(12):1297-1305. Epub 2019 Nov 18.

Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA.

High-throughput complementary DNA sequencing technologies have advanced our understanding of transcriptome complexity and regulation. However, these methods lose information contained in biological RNA because the copied reads are often short and modifications are not retained. We address these limitations using a native poly(A) RNA sequencing strategy developed by Oxford Nanopore Technologies. Our study generated 9.9 million aligned sequence reads for the human cell line GM12878, using thirty MinION flow cells at six institutions. These native RNA reads had a median length of 771 bases, and a maximum aligned length of over 21,000 bases. Mitochondrial poly(A) reads provided an internal measure of read-length quality. We combined these long nanopore reads with higher accuracy short-reads and annotated GM12878 promoter regions to identify 33,984 plausible RNA isoforms. We describe strategies for assessing 3' poly(A) tail length, base modifications and transcript haplotypes.
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http://dx.doi.org/10.1038/s41592-019-0617-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7768885PMC
December 2019

A Pan-cancer Transcriptome Analysis Reveals Pervasive Regulation through Alternative Promoters.

Cell 2019 09;178(6):1465-1477.e17

Computational and Systems Biology, Genome Institute of Singapore, Singapore 138672, Singapore; Cellular and Molecular Research, National Cancer Centre, Singapore 169610, Singapore. Electronic address:

Most human protein-coding genes are regulated by multiple, distinct promoters, suggesting that the choice of promoter is as important as its level of transcriptional activity. However, while a global change in transcription is recognized as a defining feature of cancer, the contribution of alternative promoters still remains largely unexplored. Here, we infer active promoters using RNA-seq data from 18,468 cancer and normal samples, demonstrating that alternative promoters are a major contributor to context-specific regulation of transcription. We find that promoters are deregulated across tissues, cancer types, and patients, affecting known cancer genes and novel candidates. For genes with independently regulated promoters, we demonstrate that promoter activity provides a more accurate predictor of patient survival than gene expression. Our study suggests that a dynamic landscape of active promoters shapes the cancer transcriptome, opening new diagnostic avenues and opportunities to further explore the interplay of regulatory mechanisms with transcriptional aberrations in cancer.
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http://dx.doi.org/10.1016/j.cell.2019.08.018DOI Listing
September 2019

A Murine Model of Chronic Lymphocytic Leukemia Based on B Cell-Restricted Expression of Sf3b1 Mutation and Atm Deletion.

Cancer Cell 2019 02 31;35(2):283-296.e5. Epub 2019 Jan 31.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA; Department of Systems Biology, Beckman Research Institute, City of Hope, Monrovia, CA, USA. Electronic address:

SF3B1 is recurrently mutated in chronic lymphocytic leukemia (CLL), but its role in the pathogenesis of CLL remains elusive. Here, we show that conditional expression of Sf3b1-K700E mutation in mouse B cells disrupts pre-mRNA splicing, alters cell development, and induces a state of cellular senescence. Combination with Atm deletion leads to the overcoming of cellular senescence and the development of CLL-like disease in elderly mice. These CLL-like cells show genome instability and dysregulation of multiple CLL-associated cellular processes, including deregulated B cell receptor signaling, which we also identified in human CLL cases. Notably, human CLLs harboring SF3B1 mutations exhibit altered response to BTK inhibition. Our murine model of CLL thus provides insights into human CLL disease mechanisms and treatment.
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http://dx.doi.org/10.1016/j.ccell.2018.12.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6372356PMC
February 2019

Splicing modulation sensitizes chronic lymphocytic leukemia cells to venetoclax by remodeling mitochondrial apoptotic dependencies.

JCI Insight 2018 10 4;3(19). Epub 2018 Oct 4.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.

The identification of targetable vulnerabilities in the context of therapeutic resistance is a key challenge in cancer treatment. We detected pervasive aberrant splicing as a characteristic feature of chronic lymphocytic leukemia (CLL), irrespective of splicing factor mutation status, which was associated with sensitivity to the spliceosome modulator, E7107. Splicing modulation affected CLL survival pathways, including members of the B cell lymphoma-2 (BCL2) family of proteins, remodeling antiapoptotic dependencies of human and murine CLL cells. E7107 treatment decreased myeloid cell leukemia-1 (MCL1) dependence and increased BCL2 dependence, sensitizing primary human CLL cells and venetoclax-resistant CLL-like cells from an Eμ-TCL1-based adoptive transfer murine model to treatment with the BCL2 inhibitor venetoclax. Our data provide preclinical rationale to support the combination of venetoclax with splicing modulators to reprogram apoptotic dependencies in CLL for treating venetoclax-resistant CLL cases.
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http://dx.doi.org/10.1172/jci.insight.121438DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237462PMC
October 2018

An alternative splicing switch in FLNB promotes the mesenchymal cell state in human breast cancer.

Elife 2018 07 30;7. Epub 2018 Jul 30.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, United States.

Alternative splicing of mRNA precursors represents a key gene expression regulatory step and permits the generation of distinct protein products with diverse functions. In a genome-scale expression screen for inducers of the epithelial-to-mesenchymal transition (EMT), we found a striking enrichment of RNA-binding proteins. We validated that QKI and RBFOX1 were necessary and sufficient to induce an intermediate mesenchymal cell state and increased tumorigenicity. Using RNA-seq and eCLIP analysis, we found that QKI and RBFOX1 coordinately regulated the splicing and function of the actin-binding protein FLNB, which plays a causal role in the regulation of EMT. Specifically, the skipping of FLNB exon 30 induced EMT by releasing the FOXC1 transcription factor. Moreover, skipping of FLNB exon 30 is strongly associated with EMT gene signatures in basal-like breast cancer patient samples. These observations identify a specific dysregulation of splicing, which regulates tumor cell plasticity and is frequently observed in human cancer.
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http://dx.doi.org/10.7554/eLife.37184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6103745PMC
July 2018

A Next Generation Connectivity Map: L1000 Platform and the First 1,000,000 Profiles.

Cell 2017 Nov;171(6):1437-1452.e17

Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Harvard Medical School, Boston, MA 02115, USA.

We previously piloted the concept of a Connectivity Map (CMap), whereby genes, drugs, and disease states are connected by virtue of common gene-expression signatures. Here, we report more than a 1,000-fold scale-up of the CMap as part of the NIH LINCS Consortium, made possible by a new, low-cost, high-throughput reduced representation expression profiling method that we term L1000. We show that L1000 is highly reproducible, comparable to RNA sequencing, and suitable for computational inference of the expression levels of 81% of non-measured transcripts. We further show that the expanded CMap can be used to discover mechanism of action of small molecules, functionally annotate genetic variants of disease genes, and inform clinical trials. The 1.3 million L1000 profiles described here, as well as tools for their analysis, are available at https://clue.io.
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http://dx.doi.org/10.1016/j.cell.2017.10.049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5990023PMC
November 2017

RBM25 is a global splicing factor promoting inclusion of alternatively spliced exons and is itself regulated by lysine mono-methylation.

J Biol Chem 2017 08 27;292(32):13381-13390. Epub 2017 Jun 27.

From the Department of Biology, Stanford University, Stanford, California 94305,

In eukaryotes, precursor mRNA (pre-mRNA) splicing removes non-coding intron sequences to produce mature mRNA. This removal is controlled in part by RNA-binding proteins that regulate alternative splicing decisions through interactions with the splicing machinery. RNA binding motif protein 25 (RBM25) is a putative splicing factor strongly conserved across eukaryotic lineages. However, the role of RBM25 in global splicing regulation and its cellular functions are unknown. Here we show that RBM25 is required for the viability of multiple human cell lines, suggesting that it could play a key role in pre-mRNA splicing. Indeed, transcriptome-wide analysis of splicing events demonstrated that RBM25 regulates a large fraction of alternatively spliced exons throughout the human genome. Moreover, proteomic analysis indicated that RBM25 interacts with components of the early spliceosome and regulators of alternative splicing. Previously, we identified an RBM25 species that is mono-methylated at lysine 77 (RBM25K77me1), and here we used quantitative mass spectrometry to show that RBM25K77me1 is abundant in multiple human cell lines. We also identified a region of RBM25 spanning Lys-77 that binds with high affinity to serine- and arginine-rich splicing factor 2 (SRSF2), a crucial protein in exon definition, but only when Lys-77 is unmethylated. Together, our findings uncover a pivotal role for RBM25 as an essential regulator of alternative splicing and reveal a new potential mechanism for regulation of pre-mRNA splicing by lysine methylation of a splicing factor.
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http://dx.doi.org/10.1074/jbc.M117.784371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5555197PMC
August 2017

Transcriptomic Characterization of SF3B1 Mutation Reveals Its Pleiotropic Effects in Chronic Lymphocytic Leukemia.

Cancer Cell 2016 Nov 3;30(5):750-763. Epub 2016 Nov 3.

Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, MA 02115, USA.

Mutations in SF3B1, which encodes a spliceosome component, are associated with poor outcome in chronic lymphocytic leukemia (CLL), but how these contribute to CLL progression remains poorly understood. We undertook a transcriptomic characterization of primary human CLL cells to identify transcripts and pathways affected by SF3B1 mutation. Splicing alterations, identified in the analysis of bulk cells, were confirmed in single SF3B1-mutated CLL cells and also found in cell lines ectopically expressing mutant SF3B1. SF3B1 mutation was found to dysregulate multiple cellular functions including DNA damage response, telomere maintenance, and Notch signaling (mediated through KLF8 upregulation, increased TERC and TERT expression, or altered splicing of DVL2 transcript, respectively). SF3B1 mutation leads to diverse changes in CLL-related pathways.
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http://dx.doi.org/10.1016/j.ccell.2016.10.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5127278PMC
November 2016

High-throughput Phenotyping of Lung Cancer Somatic Mutations.

Cancer Cell 2016 08 28;30(2):214-228. Epub 2016 Jul 28.

Dana-Farber Cancer Institute, Boston, MA.

Recent genome sequencing efforts have identified millions of somatic mutations in cancer. However, the functional impact of most variants is poorly understood. Here we characterize 194 somatic mutations identified in primary lung adenocarcinomas. We present an expression-based variant-impact phenotyping (eVIP) method that uses gene expression changes to distinguish impactful from neutral somatic mutations. eVIP identified 69% of mutations analyzed as impactful and 31% as functionally neutral. A subset of the impactful mutations induces xenograft tumor formation in mice and/or confers resistance to cellular EGFR inhibition. Among these impactful variants are rare somatic, clinically actionable variants including EGFR S645C, ARAF S214C and S214F, ERBB2 S418T, and multiple BRAF variants, demonstrating that rare mutations can be functionally important in cancer.
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http://dx.doi.org/10.1016/j.ccell.2016.06.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5003022PMC
August 2016

Distinct patterns of somatic genome alterations in lung adenocarcinomas and squamous cell carcinomas.

Nat Genet 2016 06 9;48(6):607-16. Epub 2016 May 9.

Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.

To compare lung adenocarcinoma (ADC) and lung squamous cell carcinoma (SqCC) and to identify new drivers of lung carcinogenesis, we examined the exome sequences and copy number profiles of 660 lung ADC and 484 lung SqCC tumor-normal pairs. Recurrent alterations in lung SqCCs were more similar to those of other squamous carcinomas than to alterations in lung ADCs. New significantly mutated genes included PPP3CA, DOT1L, and FTSJD1 in lung ADC, RASA1 in lung SqCC, and KLF5, EP300, and CREBBP in both tumor types. New amplification peaks encompassed MIR21 in lung ADC, MIR205 in lung SqCC, and MAPK1 in both. Lung ADCs lacking receptor tyrosine kinase-Ras-Raf pathway alterations had mutations in SOS1, VAV1, RASA1, and ARHGAP35. Regarding neoantigens, 47% of the lung ADC and 53% of the lung SqCC tumors had at least five predicted neoepitopes. Although targeted therapies for lung ADC and SqCC are largely distinct, immunotherapies may aid in treatment for both subtypes.
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http://dx.doi.org/10.1038/ng.3564DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4884143PMC
June 2016

Comprehensive Molecular Characterization of Papillary Renal-Cell Carcinoma.

N Engl J Med 2016 Jan 4;374(2):135-45. Epub 2015 Nov 4.

Background: Papillary renal-cell carcinoma, which accounts for 15 to 20% of renal-cell carcinomas, is a heterogeneous disease that consists of various types of renal cancer, including tumors with indolent, multifocal presentation and solitary tumors with an aggressive, highly lethal phenotype. Little is known about the genetic basis of sporadic papillary renal-cell carcinoma, and no effective forms of therapy for advanced disease exist.

Methods: We performed comprehensive molecular characterization of 161 primary papillary renal-cell carcinomas, using whole-exome sequencing, copy-number analysis, messenger RNA and microRNA sequencing, DNA-methylation analysis, and proteomic analysis.

Results: Type 1 and type 2 papillary renal-cell carcinomas were shown to be different types of renal cancer characterized by specific genetic alterations, with type 2 further classified into three individual subgroups on the basis of molecular differences associated with patient survival. Type 1 tumors were associated with MET alterations, whereas type 2 tumors were characterized by CDKN2A silencing, SETD2 mutations, TFE3 fusions, and increased expression of the NRF2-antioxidant response element (ARE) pathway. A CpG island methylator phenotype (CIMP) was observed in a distinct subgroup of type 2 papillary renal-cell carcinomas that was characterized by poor survival and mutation of the gene encoding fumarate hydratase (FH).

Conclusions: Type 1 and type 2 papillary renal-cell carcinomas were shown to be clinically and biologically distinct. Alterations in the MET pathway were associated with type 1, and activation of the NRF2-ARE pathway was associated with type 2; CDKN2A loss and CIMP in type 2 conveyed a poor prognosis. Furthermore, type 2 papillary renal-cell carcinoma consisted of at least three subtypes based on molecular and phenotypic features. (Funded by the National Institutes of Health.).
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http://dx.doi.org/10.1056/NEJMoa1505917DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4775252PMC
January 2016

Regulation of alternative splicing in Drosophila by 56 RNA binding proteins.

Genome Res 2015 Nov 20;25(11):1771-80. Epub 2015 Aug 20.

Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA; Department of Plant and Microbial Biology, University of California, Berkeley, California 94720, USA.

Alternative splicing is regulated by RNA binding proteins (RBPs) that recognize pre-mRNA sequence elements and activate or repress adjacent exons. Here, we used RNA interference and RNA-seq to identify splicing events regulated by 56 Drosophila proteins, some previously unknown to regulate splicing. Nearly all proteins affected alternative first exons, suggesting that RBPs play important roles in first exon choice. Half of the splicing events were regulated by multiple proteins, demonstrating extensive combinatorial regulation. We observed that SR and hnRNP proteins tend to act coordinately with each other, not antagonistically. We also identified a cross-regulatory network where splicing regulators affected the splicing of pre-mRNAs encoding other splicing regulators. This large-scale study substantially enhances our understanding of recent models of splicing regulation and provides a resource of thousands of exons that are regulated by 56 diverse RBPs.
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http://dx.doi.org/10.1101/gr.192518.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4617972PMC
November 2015

Breaking down RET breakpoints in lung adenocarcinoma.

J Thorac Oncol 2014 May;9(5):590-2

*Department of Medical Oncology; †Center for Cancer Genome Discovery, Dana-Farber Cancer Institute, Boston; ‡Cancer Program, Broad Institute of Harvard and M.I.T., Cambridge; and §Department of Pathology, Harvard Medical School and Brigham and Women's Hospital, Boston, MA.

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http://dx.doi.org/10.1097/JTO.0000000000000168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4036692PMC
May 2014

SOX2 and p63 colocalize at genetic loci in squamous cell carcinomas.

J Clin Invest 2014 Apr 3;124(4):1636-45. Epub 2014 Mar 3.

The transcription factor SOX2 is an essential regulator of pluripotent stem cells and promotes development and maintenance of squamous epithelia. We previously reported that SOX2 is an oncogene and subject to highly recurrent genomic amplification in squamous cell carcinomas (SCCs). Here, we have further characterized the function of SOX2 in SCC. Using ChIP-seq analysis, we compared SOX2-regulated gene profiles in multiple SCC cell lines to ES cell profiles and determined that SOX2 binds to distinct genomic loci in SCCs. In SCCs, SOX2 preferentially interacts with the transcription factor p63, as opposed to the transcription factor OCT4, which is the preferred SOX2 binding partner in ES cells. SOX2 and p63 exhibited overlapping genomic occupancy at a large number of loci in SCCs; however, coordinate binding of SOX2 and p63 was absent in ES cells. We further demonstrated that SOX2 and p63 jointly regulate gene expression, including the oncogene ETV4, which was essential for SOX2-amplified SCC cell survival. Together, these findings demonstrate that the action of SOX2 in SCC differs substantially from its role in pluripotency. The identification of the SCC-associated interaction between SOX2 and p63 will enable deeper characterization the downstream targets of this interaction in SCC and normal squamous epithelial physiology.
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http://dx.doi.org/10.1172/JCI71545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3973117PMC
April 2014

A pan-cancer analysis of transcriptome changes associated with somatic mutations in U2AF1 reveals commonly altered splicing events.

PLoS One 2014 31;9(1):e87361. Epub 2014 Jan 31.

Cancer Program, Broad Institute of Harvard and Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America ; Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, United States of America ; Department of Pathology, Harvard Medical School, Boston, Massachusetts, United States of America.

Although recurrent somatic mutations in the splicing factor U2AF1 (also known as U2AF35) have been identified in multiple cancer types, the effects of these mutations on the cancer transcriptome have yet to be fully elucidated. Here, we identified splicing alterations associated with U2AF1 mutations across distinct cancers using DNA and RNA sequencing data from The Cancer Genome Atlas (TCGA). Using RNA-Seq data from 182 lung adenocarcinomas and 167 acute myeloid leukemias (AML), in which U2AF1 is somatically mutated in 3-4% of cases, we identified 131 and 369 splicing alterations, respectively, that were significantly associated with U2AF1 mutation. Of these, 30 splicing alterations were statistically significant in both lung adenocarcinoma and AML, including three genes in the Cancer Gene Census, CTNNB1, CHCHD7, and PICALM. Cell line experiments expressing U2AF1 S34F in HeLa cells and in 293T cells provide further support that these altered splicing events are caused by U2AF1 mutation. Consistent with the function of U2AF1 in 3' splice site recognition, we found that S34F/Y mutations cause preferences for CAG over UAG 3' splice site sequences. This report demonstrates consistent effects of U2AF1 mutation on splicing in distinct cancer cell types.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0087361PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3909098PMC
October 2014

Identification and experimental validation of splicing regulatory elements in Drosophila melanogaster reveals functionally conserved splicing enhancers in metazoans.

RNA 2011 Oct 24;17(10):1884-94. Epub 2011 Aug 24.

Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.

RNA sequence elements involved in the regulation of pre-mRNA splicing have previously been identified in vertebrate genomes by computational methods. Here, we apply such approaches to predict splicing regulatory elements in Drosophila melanogaster and compare them with elements previously found in the human, mouse, and pufferfish genomes. We identified 99 putative exonic splicing enhancers (ESEs) and 231 putative intronic splicing enhancers (ISEs) enriched near weak 5' and 3' splice sites of constitutively spliced introns, distinguishing between those found near short and long introns. We found that a significant proportion (58%) of fly enhancer sequences were previously reported in at least one of the vertebrates. Furthermore, 20% of putative fly ESEs were previously identified as ESEs in human, mouse, and pufferfish; while only two fly ISEs, CTCTCT and TTATAA, were identified as ISEs in all three vertebrate species. Several putative enhancer sequences are similar to characterized binding-site motifs for Drosophila and mammalian splicing regulators. To provide additional evidence for the function of putative ISEs, we separately identified 298 intronic hexamers significantly enriched within sequences phylogenetically conserved among 15 insect species. We found that 73 putative ISEs were among those enriched in conserved regions of the D. melanogaster genome. The functions of nine enhancer sequences were verified in a heterologous splicing reporter, demonstrating that these sequences are sufficient to enhance splicing in vivo. Taken together, these data identify a set of predicted positive-acting splicing regulatory motifs in the Drosophila genome and reveal regulatory sequences that are present in distant metazoan genomes.
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http://dx.doi.org/10.1261/rna.2696311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185920PMC
October 2011

The developmental transcriptome of Drosophila melanogaster.

Nature 2011 Mar 22;471(7339):473-9. Epub 2010 Dec 22.

Department of Genetics and Developmental Biology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, Connecticut 06030-6403, USA.

Drosophila melanogaster is one of the most well studied genetic model organisms; nonetheless, its genome still contains unannotated coding and non-coding genes, transcripts, exons and RNA editing sites. Full discovery and annotation are pre-requisites for understanding how the regulation of transcription, splicing and RNA editing directs the development of this complex organism. Here we used RNA-Seq, tiling microarrays and cDNA sequencing to explore the transcriptome in 30 distinct developmental stages. We identified 111,195 new elements, including thousands of genes, coding and non-coding transcripts, exons, splicing and editing events, and inferred protein isoforms that previously eluded discovery using established experimental, prediction and conservation-based approaches. These data substantially expand the number of known transcribed elements in the Drosophila genome and provide a high-resolution view of transcriptome dynamics throughout development.
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http://dx.doi.org/10.1038/nature09715DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3075879PMC
March 2011

Identification of functional elements and regulatory circuits by Drosophila modENCODE.

Science 2010 Dec 22;330(6012):1787-97. Epub 2010 Dec 22.

Computer Science and Artificial Intelligence Laboratory, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA.

To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- and tissue-specific regulators, and enables gene-expression prediction. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation.
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http://dx.doi.org/10.1126/science.1198374DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3192495PMC
December 2010

Conservation of an RNA regulatory map between Drosophila and mammals.

Genome Res 2011 Feb 4;21(2):193-202. Epub 2010 Oct 4.

Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.

Alternative splicing is generally controlled by proteins that bind directly to regulatory sequence elements and either activate or repress splicing of adjacent splice sites in a target pre-mRNA. Here, we have combined RNAi and mRNA-seq to identify exons that are regulated by Pasilla (PS), the Drosophila melanogaster ortholog of mammalian NOVA1 and NOVA2. We identified 405 splicing events in 323 genes that are significantly affected upon depletion of ps, many of which were annotated as being constitutively spliced. The sequence regions upstream and within PS-repressed exons and downstream from PS-activated exons are enriched for YCAY repeats, and these are consistent with the location of these motifs near NOVA-regulated exons in mammals. Thus, the RNA regulatory map of PS and NOVA1/2 is highly conserved between insects and mammals despite the fact that the target gene orthologs regulated by PS and NOVA1/2 are almost entirely nonoverlapping. This observation suggests that the regulatory codes of individual RNA binding proteins may be nearly immutable, yet the regulatory modules controlled by these proteins are highly evolvable.
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http://dx.doi.org/10.1101/gr.108662.110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3032923PMC
February 2011

Genome-wide analysis of alternative pre-mRNA splicing and RNA-binding specificities of the Drosophila hnRNP A/B family members.

Mol Cell 2009 Feb;33(4):438-49

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) have been traditionally seen as proteins packaging RNA nonspecifically into ribonucleoprotein particles (RNPs), but evidence suggests specific cellular functions on discrete target pre-mRNAs. Here we report genome-wide analysis of alternative splicing patterns regulated by four Drosophila homologs of the mammalian hnRNP A/B family (hrp36, hrp38, hrp40, and hrp48). Analysis of the global RNA-binding distributions of each protein revealed both small and extensively bound regions on target transcripts. A significant subset of RNAs were bound and regulated by more than one hnRNP protein, revealing a combinatorial network of interactions. In vitro RNA-binding site selection experiments (SELEX) identified distinct binding motif specificities for each protein, which were overrepresented in their respective regulated and bound transcripts. These results indicate that individual heterogeneous ribonucleoproteins have specific affinities for overlapping, but distinct, populations of target pre-mRNAs controlling their patterns of RNA processing.
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http://dx.doi.org/10.1016/j.molcel.2009.01.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2674966PMC
February 2009

The coupling of alternative splicing and nonsense-mediated mRNA decay.

Adv Exp Med Biol 2007 ;623:190-211

Department of Molecular and Cell Biology, University of California, Berkeley, USA.

Most human genes exhibit alternative splicing, but not all alternatively spliced transcripts produce functional proteins. Computational and experimental results indicate that a substantial fraction of alternative splicing events in humans result in mRNA isoforms that harbor a premature termination codon (PTC). These transcripts are predicted to be degraded by the nonsense-mediated mRNA decay (NMD) pathway. One explanation for the abundance of PTC-containing isoforms is that they represent splicing errors that are identified and degraded by the NMD pathway. Another potential explanation for this startling observation is that cells may link alternative splicing and NMD to regulate the abundance of mRNA transcripts. This mechanism, which we call "Regulated Unproductive Splicing and Translation" (RUST), has been experimentally shown to regulate expression of a wide variety of genes in many organisms from yeast to human. It is frequently employed for autoregulation of proteins that affect the splicing process itself. Thus, alternative splicing and NMD act together to play an important role in regulating gene expression.
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http://dx.doi.org/10.1007/978-0-387-77374-2_12DOI Listing
April 2008
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