Angela N. Brooks, Ph.D. - US grants

Project Summary    A major challenge for the cancer genomics community is determining which somatic mutations are contributing  to tumorigenesis and which are ?passenger? (neutral) mutations. Somatic mutations that cause altered mRNA  splicing have recently been appreciated as oncogenic driver events, such as the case with skipping of exon 14  in the MET proto-­oncogene. Additionally, lung cancer patients with skipping of MET exon 14 respond well to  targeted drug inhibitors. Through computational analysis of both DNA and mRNA sequencing data of 495 lung  cancer donor samples, we have identified 635 cases of exon-­skipping associated with somatic mutation;?  however, we lack experimental evidence to know which of these exons might also represent oncogenic driver  events. This project will test the hypothesis that a subset of exon-­skipping events associated with  somatic mutations are novel oncogenic alterations. To test this hypothesis, our first aim will determine if  previously uncharacterized exon-­skipping events, associated with somatic mutations in RAS pathway genes,  are oncogenic. RAS pathway genes are known to be recurrently mutated in lung cancer and many are  targetable alterations;? therefore, we will focus our initial studies on exon-­skipping events which are most likely  to be oncogenic and most likely to lead to a therapeutic target. Our second aim will develop a novel high-­ throughput CRISPR-­Cas9 screen targeting all 635 exons to test if any of these exons are oncogenic when they  are skipped. Our final aim will use homology-­directed CRISPR-­Cas9 to knock-­in candidate splice mutations  and validate that these mutations cause exon-­skipping, which leads to an oncogenic gene alteration.  Completion of this study will set the framework for more broad studies of somatic mutations that affect gene  function through alternative splicing and to investigate novel targeted therapies.   

Project Summary A major focus of biomedical research that involves sequencing of the genome and transcriptome is to understand how genes are regulated and also dysregulated in disease. Integrated analyses of epigenome and epitranscriptome changes are urgently needed to have a complete molecular profile of cellular changes and to understand the molecular mechanisms of gene regulation. Yet, standard sequencing methods are unable to capture the full complexity of the epigenome (DNA modifications and chromatin accessibility) or the epitranscriptome (RNA modification). Problems inherent to current Illumina-based sequencing include: introduction of PCR bias, short-length of the reads, and the inability to directly sequence RNA molecules. To address these needs, we are developing experimental and computational approaches that allow us to (a) simultaneously detect, in vivo, DNA modifications and DNA accessibility on long stretches of single DNA molecule sequences and correlate these changes with effects on the epitranscriptome by (b) directly profiling full-length alternative RNA isoforms, RNA edits, and RNA modifications from single RNA molecule sequences. Our combined approach will rely on Oxford Nanopore long-read technologies which is capable of distinguishing modified bases in DNA and RNA, and on in vivo methods of marking accessible regions of chromatin. To demonstrate the applicability and relevance of our methods, we will perform these experiments under biological conditions known to impact both chromatin structure and the epitranscriptome. We also plan to (c) profile epigenomic and epitranscriptomic changes in response to knockdown of key chromatin remodeling genes and RNA binding proteins to test if, and how broadly, these regulatory factors affect the epigenome and epitranscriptome. Our combined approach of long-range detection of modified and accessible regions of DNA with detection of isoform-specific RNA processing events will provide a much-needed broadly applicable tool to elucidate the mechanisms governing cell signaling responses involving chromatin and transcriptome alterations.

? DESCRIPTION (provided by applicant): The UCSC Genomic Sciences Graduate Training Program is an innovative graduate training program that combines cutting-edge computational biology training in a diverse biomedical science and engineering environment. The goals of the program are to provide graduate-level inter-disciplinary training in quantitative genome-scale data collection and analysis. The graduate training program is designed to develop critical thinking skills, provide rigorous hands-on training in computer science, statistics and biological sciences, and to develop scientific communication skills of trainees. Program graduates will extend the tradition of UCSC genome scientists in developing tools and technology to solve biomedical research problems. First-year trainees join the inter-disciplinary genomics and biomedical sciences community at UCSC and do three hands-on laboratory rotations with training faculty. Trainees are encouraged to take advantage of the breadth of research programs and do both a computational and an experimental rotation. During this first year, students also begin the coursework, which includes graduate-level instruction in programming, practical genomics, statistics and other areas in biomedical sciences. Second-year students receive training in scientific writing, prepare a formal thesis proposal, and defend this during a public qualifying examination. In the third year and beyond, trainees develop their thesis projects, mentored by their chosen lab PI. In their thesis work, program trainees have the opportunity to contribute to some of the most cutting-edge and ambitious biomedical genomics research projects in the world. Trainees graduate well positioned to lead their own, independent research programs and become the next generation of genome science leaders. The UCSC Genomic Sciences Graduate Training Program is inter-disciplinary and includes 14 program faculties from 7 departments. Program faculty members are united in the tight-knit UCSC genomics community which we hope to expand via this training grant. We are seeking support for ten trainees which will enable UCSC to move closer to providing the number of graduate training opportunities that are in demand from the volume of high-quality applicants to our program.

PROJECT SUMMARY/ABSTRACT The UCSC Diversity Action Plan at the University of California, Santa Cruz originated in 2002 as a sub-award of the UCSC Genome Browser. On the UCSC campus, our DAP is known as the Research Mentoring Internship Program (RMI), a research education program that improves equity and access to careers in genomic science by increasing the participation of underrepresented minority students. The RMI, now managed by the Genomics Institute Office of Diversity, provides mentored research training and financial support for underrepresented minority (URM) students in both undergraduate and graduate (pre-doctoral) educational training, with the goal of preparing and advancing them toward successful careers in genomic science or its ethical, legal, and social implications (ELSI). Students supported by the RMI are assigned to a faculty research mentor with whom they train 10-20 hours per week. Mentor labs may be in any department in the Division of Physical and Biological Sciences or the School of Engineering, provided that the research focuses on genomic sciences (e.g. computational biology, quantitative sciences, bioinformatics and technology development). STEM research environments may be wet labs or computational labs. ELSI projects are usually conducted under the aegis of a faculty member from the Division of Social Sciences, and commonly approach a specific aspect of genomics in one of the following areas: bioethics, policy, health care, social implications. The RMI provides financial support in the form of scholarships for undergraduates and substantial fellowships for graduate students. In addition to research training, the program offers academic and professional development workshops, one-on-one coaching, and career guidance. The program exposes students to the culture and rigors of a research environment under the close supervision of faculty mentoring, thus enhancing preparation for and success in graduate school and beyond. We recruit from regional community colleges and California State Universities that have high percentages of students from low-income and underserved populations. To ensure successful persistence to degree completion, we implement retention strategies based on best practices to create professional support and programming within a cutting-edge research environment that provides our cohort with the knowledge and tools needed to advance to meaningful careers in genomics.