UCSF RNA Journal Club

A newsletter announcing the next presenter for RNA Journal Club

Ryan Wagner

Genome-wide mapping of autonomous promoter activity in human cells
van Arensbergen J1, FitzPatrick VD2,3, de Haas M1, Pagie L1, Sluimer J1, Bussemaker HJ2,3, van Steensel B1.
Nat Biotechnol. 2017 Feb;35(2):145-153. doi: 10.1038/nbt.3754. Epub 2016 Dec 26.
February 1, 2017
1Division of Gene Regulation, Netherlands Cancer Institute, Amsterdam, the Netherlands. 2Department of Biological Sciences, Columbia University, New York, New York, USA. 3Department of Systems Biology, Columbia University Medical Center, New York, New York, USA.
Previous methods to systematically characterize sequence-intrinsic activity of promoters have been limited by relatively low throughput and the length of the sequences that could be tested. Here we present 'survey of regulatory elements' (SuRE), a method that assays more than 108 DNA fragments, each 0.2-2 kb in size, for their ability to drive transcription autonomously. In SuRE, a plasmid library of random genomic fragments upstream of a 20-bp barcode is constructed, and decoded by paired-end sequencing. This library is used to transfect cells, and barcodes in transcribed RNA are quantified by high-throughput sequencing. When applied to the human genome, we achieve 55-fold genome coverage, allowing us to map autonomous promoter activity genome-wide in K562 cells. By computational modeling we delineate subregions within promoters that are relevant for their activity. We show that antisense promoter transcription is generally dependent on the sense core promoter sequences, and that most enhancers and several families of repetitive elements act as autonomous transcription initiation sites.
Date: 
March 8, 2017
Where: 
HSW 1057 at noon

Vanille Greiner

Inheritable Silencing of Endogenous Genes by Hit-and-Run Targeted Epigenetic Editing
Amabile A1, Migliara A1, Capasso P2, Biffi M2, Cittaro D3, Naldini L4, Lombardo A5.
Cell. 2016 Sep 22;167(1):219-232.e14. doi: 10.1016/j.cell.2016.09.006.
September 22, 2016
1San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy; Vita-Salute San Raffaele University, Via Olgettina 58, 20132 Milan, Italy. 2San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy. 3Center for Translational Genomics and Bioinformatics, IRCCS San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy. 4San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy; Vita-Salute San Raffaele University, Via Olgettina 58, 20132 Milan, Italy. Electronic address: [email protected] 5San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy; Vita-Salute San Raffaele University, Via Olgettina 58, 20132 Milan, Italy. Electronic address: [email protected] Abstract
Gene silencing is instrumental to interrogate gene function and holds promise for therapeutic applications. Here, we repurpose the endogenous retroviruses' silencing machinery of embryonic stem cells to stably silence three highly expressed genes in somatic cells by epigenetics. This was achieved by transiently expressing combinations of engineered transcriptional repressors that bind to and synergize at the target locus to instruct repressive histone marks and de novo DNA methylation, thus ensuring long-term memory of the repressive epigenetic state. Silencing was highly specific, as shown by genome-wide analyses, sharply confined to the targeted locus without spreading to nearby genes, resistant to activation induced by cytokine stimulation, and relieved only by targeted DNA demethylation. We demonstrate the portability of this technology by multiplex gene silencing, adopting different DNA binding platforms and interrogating thousands of genomic loci in different cell types, including primary T lymphocytes. Targeted epigenome editing might have broad application in research and medicine.
Date: 
March 1, 2017
Where: 
HSW 1057 at noon

Malin Akerblom

Synthetic recording and in situ readout of linage information in single cells
Frieda KL1, Linton JM1, Hormoz S1, Choi J2, Chow KK1, Singer ZS1, Budde MW1, Elowitz MB1,3, Cai L2.
Nature. 2017 Jan 5;541(7635):107-111. doi: 10.1038/nature20777. Epub 2016 Nov 21.
January 5, 2017
1Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, USA. 2Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA. 3Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California 91125, USA.
Reconstructing the lineage relationships and dynamic event histories of individual cells within their native spatial context is a long-standing challenge in biology. Many biological processes of interest occur in optically opaque or physically inaccessible contexts, necessitating approaches other than direct imaging. Here we describe a synthetic system that enables cells to record lineage information and event histories in the genome in a format that can be subsequently read out of single cells in situ. This system, termed memory by engineered mutagenesis with optical in situ readout (MEMOIR), is based on a set of barcoded recording elements termed scratchpads. The state of a given scratchpad can be irreversibly altered by CRISPR/Cas9-based targeted mutagenesis, and later read out in single cells through multiplexed single-molecule RNA fluorescence hybridization (smFISH). Using MEMOIR as a proof of principle, we engineered mouse embryonic stem cells to contain multiple scratchpads and other recording components. In these cells, scratchpads were altered in a progressive and stochastic fashion as the cells proliferated. Analysis of the final states of scratchpads in single cells in situ enabled reconstruction of lineage information from cell colonies. Combining analysis of endogenous gene expression with lineage reconstruction in the same cells further allowed inference of the dynamic rates at which embryonic stem cells switch between two gene expression states. Finally, using simulations, we show how parallel MEMOIR systems operating in the same cell could enable recording and readout of dynamic cellular event histories. MEMOIR thus provides a versatile platform for information recording and in situ, single-cell readout across diverse biological systems.
Date: 
February 15, 2017
Where: 
HSW 1057 at noon

John Gagnon

An Argonaute phosphorylation cycle promotes microRNA-mediated silencing
Golden RJ1,2, Chen B3,4, Li T1, Braun J1, Manjunath H1, Chen X1, Wu J5, Schmid V6, Chang TC1, Kopp F1, Ramirez-Martinez A1, Tagliabracci VS1, Chen ZJ1,7, Xie Y3,4,8, Mendell JT1,7,8,9.
Nature. 2017 Jan 23. doi: 10.1038/nature21025. [Epub ahead of print]
February 23, 2017
1Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA. 2Medical Scientist Training Program, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA. 3Quantitative Biomedical Research Center, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA. 4Department of Clinical Sciences, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA. 5Department of Microbiology and Immunology, University of California San Francisco, San Francisco, California 94143, USA. 6Eugene McDermott Center for Human Growth &Development, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA. 7Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA. 8Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA. 9Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
MicroRNAs (miRNAs) perform critical functions in normal physiology and disease by associating with Argonaute proteins and downregulating partially complementary messenger RNAs (mRNAs). Here we use clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) genome-wide loss-of-function screening coupled with a fluorescent reporter of miRNA activity in human cells to identify new regulators of the miRNA pathway. By using iterative rounds of screening, we reveal a novel mechanism whereby target engagement by Argonaute 2 (AGO2) triggers its hierarchical, multi-site phosphorylation by CSNK1A1 on a set of highly conserved residues (S824-S834), followed by rapid dephosphorylation by the ANKRD52-PPP6C phosphatase complex. Although genetic and biochemical studies demonstrate that AGO2 phosphorylation on these residues inhibits target mRNA binding, inactivation of this phosphorylation cycle globally impairs miRNA-mediated silencing. Analysis of the transcriptome-wide binding profile of non-phosphorylatable AGO2 reveals a pronounced expansion of the target repertoire bound at steady-state, effectively reducing the active pool of AGO2 on a per-target basis. These findings support a model in which an AGO2 phosphorylation cycle stimulated by target engagement regulates miRNA:target interactions to maintain the global efficiency of miRNA-mediated silencing.
Date: 
February 8, 2017
Where: 
HSW 1057 at noon

Maryia Barnett

piggyBac mediates efficient in vivo CRISPR library screening for tumorigenesis in mice
Xu C1, Qi X1, Du X1, Zou H1, Gao F1, Feng T1, Lu H1, Li S2,3, An X1, Zhang L1, Wu Y4, Liu Y2,3,5, Li N1, Capecchi MR6, Wu S7.
Proc Natl Acad Sci U S A. 2017 Jan 6. pii: 201615735. doi: 10.1073/pnas.1615735114. [Epub ahead of print]
January 6, 2017
1State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China. 2Department of Neurosurgery, Medical School, University of Texas Health Science Center at Houston, Houston, TX 77030. 3Center for Stem Cell and Regenerative Medicine, University of Texas Health Science Center at Houston, Houston, TX 77030. 4Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, UT 84112. 5The Senator Lloyd & B. A. Bentsen Center for Stroke Research, the Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston, Houston, TX 77030. 6Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, UT 84112; [email protected] [email protected] 7State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China; [email protected] [email protected]
CRISPR/Cas9 is becoming an increasingly important tool to functionally annotate genomes. However, because genome-wide CRISPR libraries are mostly constructed in lentiviral vectors, in vivo applications are severely limited as a result of difficulties in delivery. Here, we examined the piggyBac (PB) transposon as an alternative vehicle to deliver a guide RNA (gRNA) library for in vivo screening. Although tumor induction has previously been achieved in mice by targeting cancer genes with the CRISPR/Cas9 system, in vivo genome-scale screening has not been reported. With our PB-CRISPR libraries, we conducted an in vivo genome-wide screen in mice and identified genes mediating liver tumorigenesis, including known and unknown tumor suppressor genes (TSGs). Our results demonstrate that PB can be a simple and nonviral choice for efficient in vivo delivery of CRISPR libraries.
Date: 
January 25, 2017
Where: 
HSW 1057 at noon

TBD

TBA
Date: 
January 18, 2016
Where: 
HSW 1057 at noon

Gabriel Eades

Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR–Cas9 library
Shiyou Zhu, Wei Li, Jingze Liu, Chen-Hao Chen, Qi Liao, Ping Xu, Han Xu, Tengfei Xiao, Zhongzheng Cao, Jingyu Peng, Pengfei Yuan, Myles Brown, Xiaole Shirley Liu & Wensheng Wei
Nat Biotechnol. 2016 Dec;34(12):1279-1286. doi: 10.1038/nbt.3715. Epub 2016 Oct 31.
October 31, 2016
1Biodynamic Optical Imaging Center (BIOPIC), Beijing Advanced Innovation Center for Genomics, Peking-Tsinghua Center for Life Sciences, State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China. 2Peking University-Tsinghua University-National Institute of Biological Sciences Joint Graduate Program (PTN), Peking University, China. 3Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA. 4Center for Functional Cancer Epigenetics, Dana-Farber Cancer Institute, Boston, Massachusetts, USA. 5Department of Prevention Medicine, School of Medicine, Ningbo University, Ningbo, Zhejiang, China. 6Broad Institute of MIT and Harvard, Cambridge Center, Cambridge, Massachusetts, USA. 7Division of Molecular and Cellular Oncology, Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA. 8Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China. 9Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA.
CRISPR-Cas9 screens have been widely adopted to analyze coding-gene functions, but high-throughput screening of non-coding elements using this method is more challenging because indels caused by a single cut in non-coding regions are unlikely to produce a functional knockout. A high-throughput method to produce deletions of non-coding DNA is needed. We report a high-throughput genomic deletion strategy to screen for functional long non-coding RNAs (lncRNAs) that is based on a lentiviral paired-guide RNA (pgRNA) library. Applying our screening method, we identified 51 lncRNAs that can positively or negatively regulate human cancer cell growth. We validated 9 of 51 lncRNA hits using CRISPR-Cas9-mediated genomic deletion, functional rescue, CRISPR activation or inhibition and gene-expression profiling. Our high-throughput pgRNA genome deletion method will enable rapid identification of functional mammalian non-coding elements.
Date: 
January 11, 2017
Where: 
HSW 1057 at noon

Not in Session

Winter Break
Date: 
January 4, 2017
Where: 
HSW 1057 at noon

Canceled

N/A
Date: 
July 13, 2016
Where: 
HSW 1057 at noon

Canceled

N/A
Where: 
HSW 1057 at noon