Func%onal Genomic Screens in the RNAi Pla7orm

Transcription

1 Func%onal Genomic Screens in the RNAi Pla7orm Broad Ins*tute of MIT and Harvard Broad RNAi Platform

2 Func%onal Genomic Screens in the RNAi Pla7orm Func*onal genomics à Gene func*on in context. in cells What to expect if you go down this road I. Overview II. Perturba*ons III. Planning a screen the idea & all the key ingredients IV. Op*miza*on/Execu*on V. Data analysis VI. Gene func*on, purng it all together: Figures 3-7

3 What s the gene that causes 5q- syndrome?

4 What is 5q- syndrome? - myelodisplasia 5q- syndrome pa%ents: Severe anemia Reduced erythroid development Normal numbers of megakaryocyte Hypolobated micromegakaryoctes Normal/elevated platelets Normal/low neutrophils Occasional progression to AML van Den Berghe, Nature, 251, (1974) Dele%on (1.5Mb) 40 genes, which one? (Haploinsufficiency) A 40- year hunt among 40 genes.but no luck Ben Ebert, Todd Golub Nature 2008

5 Focused screen of common deleted region A key to screen design: a model for disease Hemopoetic progenitor differentiation - increased megakaryocyte/erythrocyte ratio Ratio Megakaryocyte/Erythroid TNIP1 CSF1R GLRA1 Differentiate hemopoetic progenitor cells while suppressing a candidate gene SH3TC2 SYNPO PCYOX1L AFAPIL1 CDX1 MYOZ3 FLJ41603 ANXA6 ZNF300 CD74 SPARC PPARGC1B PDGFRB PDE6A IL17B DCTN4 CSNK1A1 ATOX1 SLC36A2 G3BP DKFZP434C171 SLC36A3 Hairpin targets RPS14 Ribosomal subunit protein GPX3 ABLIM3 TIGD6 SLC26A2 GRPEL2 NDST1 NID67 CAMK2A SLC36A1 SLC6A7 FAT2 TCOF1 GM2A RBM22 RPS14 Ben Ebert, Todd Golub

6 RPS14 overexpression reverts 5q- patient cells 5q - patients Non-5q - MDS patients Erythroid / megakaryocyte Empty Vector 8.62% 0.36% 3.2% RPS14 0.2% GlyA/CD GlyA/CD CD41 CD41 0 Empty RPS14 Empty RPS14 Empty 0 RSP14 Empty RSP14 Empty RSP14 RPS14 Empty RSP14 RPS % 39.5% 29.2% 67.4% Patient 1 Patient 2 Normal 3 Normal 4 GlyA GlyA 2.5 Erythroid / meyeloid 2.5 Empty Vector 54.6% 0.61% RPS % 1.36% GlyA/CD11b Empty RPS14 Empty RPS14 Empty RSP14 Empty RSP14 GlyA/CD11b Empty RSP14 Empty RSP14 Empty RPS14 Empty RPS14 CD11b 37.1% 7.71% CD11b 32.4% 24.1% Patient 1 Patient 2 Normal 3 Normal 4 GlyA GlyA Ben Ebert, Todd Golub

7 Which host cell genes restrict flu infec%on?

8 Knock down genes and monitor flu infection levels in U2OS cells IFIT3 was noted as an interferon-induced gene. Follow-up shows IFITM family are all flu restriction factors. Brass, Farzan, Elledge Cell 2009

9 Systematic gene function discovery: - Perturb each gene and look for change in cell behavior - Knockdown, overexpression, re-engineer, etc. X X X X X X

10 Func*onal genomics à Probe func*on by perturbing gene ac*vity in cells It can be complicated - Mul*ple func*ons per gene - Mul*ple isoforms, protein modifica*ons - Context- dependence func*ons: cell type, cell state, environment

11 Cell-based loss-of-function screens Human and mouse What happens to the cells? Need: Reagents to perturb every gene Good tissue culture model Good readouts of cell state

12 Define project Screen projects Define ques*on: Find genes that do XYZ Define biological model system Define assays to read out phenotypes of interest Primary screen feasibility and execu%on Op*mize model system, assay(s); posi*ve and nega*ve controls Select gene set to interrogate Execute pilot and primary screen select hits What happens to the cells? Need: Follow up on interes%ng genes/pathways Reagents to perturb every gene Good tissue culture model Good readouts of cell state Confirm assay result Confirm target gene specificity mul*ple RNAi reagents, target KD Elaborate the biological effects, e.g. mechanism generality/context, biomedical sig? The screen is just one early step of a project

13 Gene perturba5ons Genome- scale in mammalian cells RNA interference last 10 years Overexpression possible for decades, rare in prac*ce Inser%onal mutagenesis in haploid cells a few years Other strategies: (scale varies) Genome engineering: Zn fingers, TALENS, CRIPR mirna sponges, an%sense

14 Disease models Cancer Barbie, D. A., P. Tamayo, et al. (2009). "Systema*c RNA interference reveals that oncogenic KRAS- driven cancers require TBK1." Nature 462: Boehm, J. S., J. J. Zhao, et al. (2007). "Integra*ve genomic approaches iden*fy IKBKE as a breast cancer oncogene." Cell 129: Anemia Ebert, B. L., J. Pretz, et al. (2008). "Iden*fica*on of RPS14 as a 5q- syndrome gene by RNA interference screen." Nature 451: Osteoporosis Jones, D. C., M. N. Wein, et al. (2006). "Regula*on of adult bone mass by the zinc finger adapter protein Schnurri- 3." Science 312: Mitochondrial Dysfunc*on Pagliarini, D. J., S. E. Calvo, et al. (2008). "A mitochondrial protein compendium elucidates complex I disease biology." Cell 134: Immunology and systems immunology Amit, I., M. Garber, et al. (2009). "Unbiased reconstruc*on of a mammalian transcrip*onal network media*ng pathogen responses." Science 326(5950): Oberdoerffer, S., L. F. Moita, et al. (2008). "Regula*on of CD45 alterna*ve splicing by heterogeneous ribonucleoprotein, hnrnpll." Science 321:

15 Challenges to the systema5c func5onal annota5on of the genome Cell- based screening: Tissue culture model, phenotypic assay Gene5c perturba5ons issues, for example RNAi: Delivery efficiency, effects Gene knockdown effec5veness Specificity: Off- target, non- specific effects Follow- up bolleneck Cost Partly an art experience helps a lot!

16 Publica%ons using the TRC library in direct collabora%on or consulta%on with the Broad RNAi Pla7orm Amit, I. et al. (2009) "Unbiased reconstruc*on of a mammalian transcrip*onal network media*ng pathogen responses." Science 326: Barbie, D. A. et al. (2009) "Systema*c RNA interference reveals that oncogenic KRAS- driven cancers require TBK1." Nature 462: Barre*na, J. et al. (2010) Subtype- specific genomic altera*ons define new targets for sot- *ssue sarcoma therapy. Nature Gene%cs 42: Bilodeau, S. et al. (2009) SetDB1 contributes to repression of genes encoding developmental regulators and maintenance of ES cell state. Genes & Dev 23: Boehm, J. S. et al. (2007) "Integra*ve genomic approaches iden*fy IKBKE as a breast cancer oncogene." Cell 129: Ebert, B. L. et al. (2008) "Iden*fica*on of RPS14 as a 5q- syndrome gene by RNA interference screen." Nature 451: Eguchi, J. et al. (2008) "Interferon regulatory factors are transcrip*onal regulators of adipogenesis." Cell Metab 7: Firestein, R. et al. (2008) "CDK8 is a colorectal cancer oncogene that regulates beta- catenin ac*vity." Nature 455: Frohling, S. et al. (2007) "Iden*fica*on of driver and passenger muta*ons of FLT3 by high- throughput DNA sequence analysis and func*onal assessment of candidate alleles." Cancer Cell 12: Gohil, V. M. et al. (2010) "Mitochondrial and nuclear genomic responses to loss of LRPPRC expression." J Biol Chem 285: Johannessen, C.M. et al. (2010) COT drives resistance to RAF inhibi*on through MAP kinase pathway reac*va*on. Nature 468: Jones, D. C. et al. (2006) "Regula*on of adult bone mass by the zinc finger adapter protein Schnurri- 3." Science 312: Jones,T.R.et al.(2009)"scoring diverse cellular morphologies in image- based screens with itera*ve feedback and machine learning. PNAS106: Kagey, M.H. et al. (2010) Mediator and cohesin connect gene expression and chroma*n architecture. Nature 467: Kim, S. Y. (2010) "CK1epsilon is required for breast cancers dependent on beta- catenin ac*vity." PLoS One 5: e8979. Moffat, J. et al. (2006) "A len*viral RNAi library for human and mouse genes applied to an arrayed viral high- content screen." Cell 1246: Oberdoerffer, S. et al. (2008) "Regula*on of CD45 alterna*ve splicing by heterogeneous ribonucleoprotein, hnrnpll." Science 321: Pagliarini, D. J. et al. (2008) "A mitochondrial protein compendium elucidates complex I disease biology." Cell 134: Pavri, R. et al. (2010) Ac*va*on- Induced Cy*dine Deaminase Targets DNA at Sites of RNA Pol. II Stalling by Interac*on with Spt5. Cell143: Perocchi F. et al. (2010) MICU1 encodes a mitochondrial EF hand protein required for Ca2+ uptake. Nature, 467: Root, D. E. et al. (2006) "Genome- scale loss- of- func*on screening with a len*viral RNAi library." Nat Methods 3: Sancak, Y. et al. (2007) "PRAS40 is an insulin- regulated inhibitor of the mtorc1 protein kinase." Mol Cell 25: Savina, A. et al. (2009) "The small GTPase Rac2 controls phagosomal alkaliniza*on and an*gen crosspresenta*on selec*vely in CD8(+) dendri*c cells." Immunity 30: Scholl, C. et al. (2007) "The homeobox gene CDX2 is aberrantly expressed in most cases of acute myeloid leukemia and promotes leukemogenesis." J Clin Invest 117: Scholl,C.etal.(2009)"Synthe*c lethal interac*on between oncogenic KRAS dependency and STK33 suppression in human cancer cells. Cell137: Smolen, G.A. et al. (2010) A genome- wide RNAi screen iden*fies mul*ple RSK- dependent regulators of cell migra*on Genes & Dev. 24: Sugiana, C.et al.(2008) Muta*on of C20orf7 disrupts complex I assembly and causes lethal neonatal mitochondrial disease."amjhumgenet 83: Vasudevan, K. M. et al. (2009) "AKT- independent signaling downstream of oncogenic PIK3CA muta*ons in human cancer." Cancer Cell 16: Vicent, S. et al.(2010) Wilms tumor 1 regulates KRAS- driven oncogenesis and senescence in mouse and human models. J Clin Invest120: Yang, W. S. et. al. (2008) "Inhibi*on of casein kinase 1- epsilon induces cancer- cell- selec*ve PERIOD2- dependent growth arrest." Genome Biol 9: R92. Zhang, Q. et al. (2009) "Control of cyclin D1 and breast tumorigenesis by the EglN2 prolyl hydroxylase." Cancer Cell 16(5):

17 RNAi Pla7orm what do we do? Genomic screening projects fct. genomics exper5se R & D in func5onal genomics tools, strategies Infrastructure for maintaining, distribu5ng, employing gene perturba5on reagents Libraries, reagents crea5on

18 The Broad RNAi Platform ~35 People Production of shrna + ORF libraries; others Screening collaborations Informatics and computational biology Research on functional genomics methods; design of new reagents Researchers in Broad community The research T world

19 Platform Organization Production: Virus, DNA, Process Development Screening: Guide and support collaborations Director: David Root Achilles: Set up, execute proliferation screens Admin: Emmi Snyder Informatics: Software engineering and support R & D: Vector, tools, computational biology

20 Platform Organization Production: Virus, DNA, Process Development William Harrington Director : David Root Screening: Guide and support collaborations Achilles: Set up, execute proliferation screens Rakela Lubonja Admin: Emmi Snyder Informatics: Software engineering and support Nancy Tran R & D: Vector, tools, computational biology Xiaoping Yang

21 Platform Organization Production: Virus, DNA, Process Development Mukta Bagul Screening: Guide and support collaborations Director: David Root John Doench Achilles: Set up, execute proliferation screens Admin: Emmi Snyder Informatics: Daniel Lam Software engineering and support R & D: Vector, tools, computational biology Thomas Nieland

22 Platform Organization Production: Virus, DNA, Process Development Levi Ali Simon Aoyama Screening: Guide and support collaborations Director: David Root Achilles: Set up, execute proliferation screens Glenn Cowley Bill Gerath Admin: Emmi Snyder Informatics: Software engineering and support R & D: Vector, tools, computational biology Jessica Hsiao Guozhi Jiang

23 Platform Organization Production: Virus, DNA, Process Development Douglas Alan Director: David Root Admin: Emmi Snyder Screening: Guide and support collaborations Achilles: Set up, execute proliferation screens Informatics: Software engineering and support Thomas Green Adam Brown R & D: Vector, tools, computational biology Matthew Greene

24 Platform Organization Production: Virus, DNA, Process Development Ella Hartenian Screening: Guide and support collaborations Director: David Root Admin: Emmi Snyder Achilles: Set up, execute proliferation screens Informatics: Software engineering and support Peyton Greenside Jen Grenier Mudra Hegde R & D: Vector, tools, computational biology Cong Zhu

25 RNAi Pla7orm Get information at our Broad site: Come see us about: Screen project ideas gene discovery How to use gene perturba*on tools for non- screen based projects Projects to inves*gate new strategies, technologies for fct. genomics