Func%onal Genomic Screens in the RNAi Pla7orm
Broad Ins*tute of MIT and Harvard
Broad RNAi Platform
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”
What’s the gene that causes 5q-‐ syndrome?
van Den Berghe, Nature, 251, 437-438 (1974)
Ben Ebert, Todd Golub Nature 2008
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
Dele%on (1.5Mb)
40 genes, which one? (Haploinsufficiency)
A 40-‐year hunt among 40 genes ….but no luck
-5
5
15
25
35
45
55
TNIP1
CSF1R
GLRA1
SH3TC2
SYNPO
PCYOX1L
AFAPIL1
CDX1
MYOZ3
FLJ41603
ANXA6
ZNF300
CD74
SPARC
PPARGC1B
PDGFRB
PDE6A
IL17B
DCTN4
CSNK1A1
ATOX1
SLC36A2
G3BP
DKFZP434C171
SLC36A3
GPX3
ABLIM3
TIGD6
SLC26A2
GRPEL2
NDST1
NID67
CAMK2A
SLC36A1
SLC6A7
FAT2
TCOF1
GM2A
RBM22
RPS14
Focused screen of common deleted region
Hairpin targets
Rat
io M
egak
aryo
cyte
/Ery
thro
id
RPS14 Ribosomal subunit protein
A key to screen design: a model for disease Hemopoetic progenitor differentiation - increased megakaryocyte/erythrocyte ratio
Differentiate hemopoetic progenitor cells while suppressing a candidate gene
Ben Ebert, Todd Golub
51.5%
Patient 1
0
0.5
1
1.5
2
2.5
Empty RPS14 Empty RPS14
0
0.5
1
1.5
2
2.5
3
Empty
RPS14 Empty
RPS14
Empty Vector RPS14
RPS14
Patient 1 Patient 2
Patient 2
Gly
A/C
D41
Gly
A/C
D41
Gly
A/C
D11
b
Normal 3 Normal 4
CD
41
8.62%
29.2%
32.4%
0.36%
39.5% 67.4%
0.2% 3.2%
42.3% 1.36%
24.1%
GlyA
7.71% 37.1%
GlyA
CD
11b
Empty Vector 54.6% 0.61%
Empty
Empty
Empty Empty Empty
Empty
RSP14 RSP14 RSP14 RSP14
RSP14 RSP14
GlyA
CD
41
CD
11b
CD
11b
GlyA
5q- patients Non-5q- MDS patients
0
0
0.5
1
1.5
2
2.5
0
0.5
1
1.5
2
2.5
3
Empty RPS14 Empty RPS140
0.5
1
1.5
2
2.5
3 3
Gly
A/C
D11
b
Normal 3 Normal 4
Empty RSP14 Empty RSP14 0
0.5
1
1.5
2
2.5
Empty RPS14 Empty RPS140
0.5
1
1.5
2
2.5
0.5
1
1.5
2
2.5
RPS14 overexpression ‘reverts’ 5q- patient cells
Erythroid / megakaryocyte
Erythroid / meyeloid
Ben Ebert, Todd Golub
Which host cell genes restrict flu infec%on?
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
X X X X X X
Systematic gene function discovery: - Perturb each gene and look for change in cell behavior - Knockdown, overexpression, re-engineer, etc.
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
Cell-based loss-of-function screens Human and mouse
What happens to the cells?
Need:
Good tissue culture model
Reagents to perturb every gene
Good readouts of cell state
• Define ques*on: “Find genes that do XYZ” • Define biological model system • Define assays to read out phenotypes of interest
Screen projects
• Op*mize model system, assay(s); posi*ve and nega*ve controls • Select gene set to interrogate • Execute pilot and primary screen – select hits
Define project
Primary screen – feasibility and execu%on
• Confirm assay result • Confirm target gene specificity – mul*ple RNAi reagents, target KD • Elaborate the biological effects,
e.g. mechanism generality/context, biomedical sig?
Follow up on interes%ng genes/pathways
What happens to the cells?
Need:
Good tissue culture model
Reagents to perturb every gene
Good readouts of cell state
The screen is just one early step of a project
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
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: 108-‐12.
– Boehm, J. S., J. J. Zhao, et al. (2007). "Integra*ve genomic approaches iden*fy IKBKE as a breast cancer oncogene." Cell 129: 1065-‐79.
• 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: 335-‐9. • 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: 1223-‐7.
• Mitochondrial Dysfunc*on – Pagliarini, D. J., S. E. Calvo, et al. (2008). "A mitochondrial protein compendium
elucidates complex I disease biology." Cell 134: 112-‐23. • 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): 257-‐63.
– Oberdoerffer, S., L. F. Moita, et al. (2008). "Regula*on of CD45 alterna*ve splicing by heterogeneous ribonucleoprotein, hnRNPLL." Science 321: 686-‐91.
Challenges to the systema5c func5onal annota5on of the genome
Gene5c perturba5ons issues, for example RNAi: • Delivery – efficiency, effects
• Gene knockdown effec5veness • Specificity: Off-‐target, non-‐specific effects • Follow-‐up boLleneck • Cost
Cell-‐based screening: • Tissue culture model, phenotypic assay
Partly an ‘art’ – experience helps a lot!
Publica%ons using the TRC library in direct collabora%on or consulta%on with the Broad RNAi Pla7orm 2006-‐2010
Amit, I. et al. (2009) "Unbiased reconstruc*on of a mammalian transcrip*onal network media*ng pathogen responses." Science 326: 257-‐63. Barbie, D. A. et al. (2009) "Systema*c RNA interference reveals that oncogenic KRAS-‐driven cancers require TBK1." Nature 462: 108-‐12. Barre*na, J. et al. (2010) “Subtype-‐specific genomic altera*ons define new targets for sot-‐*ssue sarcoma therapy.” Nature Gene%cs 42: 715–721. Bilodeau, S. et al. (2009) “SetDB1 contributes to repression of genes encoding developmental regulators and maintenance of ES cell state.” Genes & Dev 23: 2484-‐2489. Boehm, J. S. et al. (2007) "Integra*ve genomic approaches iden*fy IKBKE as a breast cancer oncogene." Cell 129: 1065-‐79. Ebert, B. L. et al. (2008) "Iden*fica*on of RPS14 as a 5q-‐ syndrome gene by RNA interference screen." Nature 451: 335-‐9. Eguchi, J. et al. (2008) "Interferon regulatory factors are transcrip*onal regulators of adipogenesis." Cell Metab 7: 86-‐94. Firestein, R. et al. (2008) "CDK8 is a colorectal cancer oncogene that regulates beta-‐catenin ac*vity." Nature 455: 547-‐51. 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: 501-‐13. Gohil, V. M. et al. (2010) "Mitochondrial and nuclear genomic responses to loss of LRPPRC expression." J Biol Chem 285: 13742-‐7. Johannessen, C.M. et al. (2010) “COT drives resistance to RAF inhibi*on through MAP kinase pathway reac*va*on.” Nature 468:968–972 Jones, D. C. et al. (2006) "Regula*on of adult bone mass by the zinc finger adapter protein Schnurri-‐3." Science 312: 1223-‐7. Jones,T.R.et al.(2009)"Scoring diverse cellular morphologies in image-‐based screens with itera*ve feedback and machine learning.“PNAS106:1826-‐31. Kagey, M.H. et al. (2010) “Mediator and cohesin connect gene expression and chroma*n architecture.” Nature 467: 430–435. 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: 1283-‐98. Oberdoerffer, S. et al. (2008) "Regula*on of CD45 alterna*ve splicing by heterogeneous ribonucleoprotein, hnRNPLL." Science 321: 686-‐91. Pagliarini, D. J. et al. (2008) "A mitochondrial protein compendium elucidates complex I disease biology." Cell 134: 112-‐23. 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.” Cell143:122-‐133. Perocchi F. et al. (2010) “MICU1 encodes a mitochondrial EF hand protein required for Ca2+ uptake.” Nature, 467: 291-‐6. Root, D. E. et al. (2006) "Genome-‐scale loss-‐of-‐func*on screening with a len*viral RNAi library." Nat Methods 3: 715-‐9. Sancak, Y. et al. (2007) "PRAS40 is an insulin-‐regulated inhibitor of the mTORC1 protein kinase." Mol Cell 25: 903-‐15. 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: 544-‐55. 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: 1037-‐48. Scholl,C.etal.(2009)"Synthe*c lethal interac*on between oncogenic KRAS dependency and STK33 suppression in human cancer cells.“Cell137:821-‐34. 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: 2654-‐2665. Sugiana, C.et al.(2008) “Muta*on of C20orf7 disrupts complex I assembly and causes lethal neonatal mitochondrial disease."AmJHumGenet 83:468-‐78 Vasudevan, K. M. et al. (2009) "AKT-‐independent signaling downstream of oncogenic PIK3CA muta*ons in human cancer." Cancer Cell 16: 21-‐32. Vicent, S. et al.(2010)“Wilms tumor 1 regulates KRAS-‐driven oncogenesis and senescence in mouse and human models.”J Clin Invest120:3940–3952. 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): 413-‐24.
• Genomic screening projects – fct. genomics
exper5se
• R & D in func5onal genomics tools, strategies • Infrastructure for maintaining, distribu5ng,
employing gene perturba5on reagents
• Libraries, reagents crea5on
RNAi Pla7orm – what do we do?
The Broad RNAi Platform
Informatics and computational biology
Screening collaborations
Production of shRNA + ORF libraries; others
Research on functional genomics methods; design
of new reagents
~35 People
Researchers in Broad community T The research world
Director: David Root
Admin: Emmi Snyder
Production: Virus, DNA, Process Development
Screening: Guide and support collaborations
Informatics: Software engineering and support
R & D: Vector, tools, computational
biology
Achilles: Set up, execute proliferation
screens
Platform Organization
Director:
David Root
Admin: Emmi Snyder
Production: Virus, DNA, Process Development
Screening: Guide and support collaborations
Informatics: Software engineering and support
R & D: Vector, tools, computational
biology
Achilles: Set up, execute proliferation
screens
Platform Organization
William Harrington
Rakela Lubonja
Nancy Tran
Xiaoping Yang Glen Munson Cindy Nguyen Thu Nguyen Tenchoe Nyima Pema Tenzing
Production: Virus, DNA, Process Development
Screening: Guide and support collaborations
Informatics: Software engineering and support
R & D: Vector, tools, computational
biology
Achilles: Set up, execute proliferation
screens
Platform Organization
Mukta Bagul
John Doench Daniel Lam Thomas Nieland Michael Okamoto Federica Piccioni
Director: David Root
Admin: Emmi Snyder
Production: Virus, DNA, Process Development
Screening: Guide and support collaborations
Informatics: Software engineering and support
R & D: Vector, tools, computational
biology
Achilles: Set up, execute proliferation
screens
Platform Organization
Levi Ali Simon Aoyama
Glenn Cowley Bill Gerath Jessica Hsiao Guozhi Jiang Justine Scott
Director: David Root
Admin: Emmi Snyder
Production: Virus, DNA, Process Development
Screening: Guide and support collaborations
Informatics: Software engineering and support
R & D: Vector, tools, computational
biology
Achilles: Set up, execute proliferation
screens
Platform Organization
Douglas Alan
Adam Brown Thomas Green
Matthew Greene Mark Tomko John Sullivan
Director: David Root
Admin: Emmi Snyder
Production: Virus, DNA, Process Development
Screening: Guide and support collaborations
Informatics: Software engineering and support
R & D: Vector, tools, computational
biology
Achilles: Set up, execute proliferation
screens
Platform Organization
Ella Hartenian Peyton Greenside Jen Grenier Mudra Hegde Cong Zhu
Director: David Root
Admin: Emmi Snyder
RNAi Pla7orm
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
Get information at our Broad site: https://iwww.broadinstitute.org/rnai/db/
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