funconal)genomicscreens) in)the)rnai)pla7orm · 2013. 4. 5. · -5 5 15 25 35 45 55 tnip1 csf1r...

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


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


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





biology


screens


William Harrington

Rakela Lubonja

Nancy Tran

Xiaoping Yang Glen Munson Cindy Nguyen Thu Nguyen Tenchoe Nyima Pema Tenzing





biology


screens


Mukta Bagul

John Doench Daniel Lam Thomas Nieland Michael Okamoto Federica Piccioni


Admin: Emmi Snyder





biology


screens


Levi Ali Simon Aoyama

Glenn Cowley Bill Gerath Jessica Hsiao Guozhi Jiang Justine Scott


Admin: Emmi Snyder





biology


screens


Douglas Alan

Adam Brown Thomas Green

Matthew Greene Mark Tomko John Sullivan


Admin: Emmi Snyder





biology


screens


Ella Hartenian Peyton Greenside Jen Grenier Mudra Hegde Cong Zhu


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/

funconal)genomicscreens) in)the)rnai)pla7orm · 2013. 4. 5. · -5 5 15 25 35 45 55 tnip1 csf1r...

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