Identifying transcriptional dichotomies by "stochastic sampling"

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1 Identifying transcriptional dichotomies by "stochastic sampling" Kevin Janes and Joan Brugge Department of Cell Biology Harvard Medical School pakt (S473) keratin 10 DAPI National Cancer Institute (CA105134) American Cancer Society (PF MGO) Nikon Imaging HMS

2 Of Mice and Men for mathematicians (why working with mammalian cells is annoying) 1. Removing or fluorescently tagging an endogenous protein is very time consuming (human >> mouse) 2. Mammalian reporters of transcription actual gene expression 3. Mechanistic power ~ (organismal relevance) -1 But... it s us

3 Heterogeneities during in vitro mammary acinar morphogenesis Laminin 5 GM130 (Golgi) phospho-akt cleaved caspase-3 Debnath and Brugge, Nat. Rev. Cancer (2005) Inner cells Apoptosis Clearance MCF-10A Outer cells P-Akt positive P-Akt negative Mature acinus

4 Heterogeneity within 3D structures K10 pakt (S473) FOXO1/4 vigilin Sudan black autofluorescence MCF10A-5E, day 10 Scale bar: 25 μm

5 Transcriptional profiling with single-cell PCR

6 Identifying molecular dichotomies by stochastic sampling

7 Pick n cells at random B rand (n,p) Iterate for m experiments chance of Calculate average gene expression [(5x3)+(1x7)] x noise(σ) Normalize and calculate histogram counts per bin X(b) 1 b reproducibility histogram bins Fit to the expected normal distribution X(b) b X(b) = Aexp [-(b-1) ] 2σ 2 average of middle four bins Calculated sum of squared error Binomial Control Compare with control gene (p = 0)

8 Optimal stochastic sampling with 10 cells and 16 replicates replicates 10 cells 0.25 SSE from normal fit SSE from normal fit Number of cells Number of experiments 16 replicates 10 cells 1.0 Dichotomy Control Correlation (R) 0.0 Correlation (R) Number of cells Number of experiments 8-fold difference in 20% of the population 25% measurement error

9 Single-cell cdna amplification In-house modifications (part 1) 5' mrna 3' AAAA...AAAAAAA + oligo(dt) TTTT...TTTTTTT oligo(dt) 24 Superscript III, 15 min at 50 o C AMV, MMLV reverse transcriptases, 15 min at 37 o C 5' mrna 3' bp AAAA...AAAAAAA TTTT...TTTTTTT RNAse H TTTT...TTTTTTT terminal transferase datp AAAAAAA...AAAA TTTT...TTTTTTT Replaced Minor improvements Major improvements

10 Single-cell cdna amplification In-house modifications (part 2) AAAAAAA...AAAA TTTT...TTTTTTT 10U AmpliTaq, ThermoPol Buffer 4 PCR cycles (94 o C,37 o C,72 o C) 3x sample split AL1 primer: AL1 seq TTTT...TTTT U AmpliTaq, PCR Buffer II 50 PCR cycles (94 o C,42 o C,72 o C) AAAA...AAAA TTTT...TTTT AL1 seq TTTT...TTTT AAAA...AAAA 21 PCR cycles (94 o C,42 o C,72 o C) AL1 seq AL1 seq' AAAA...AAAA TTTT...TTTT AL1 seq TTTT...TTTT AAAA...AAAA AL1 seq AL1 seq' Pool sample splits 5 PCR cycles (94 o C,42 o C,72 o C) Detect PCR fragments Replaced Minor improvements Major improvements

11 Quantitative amplification of small cell populations Cycle threshold n.d gapdh m = -3.9 m p = -3.5 Cycle threshold n.d prdx6 m = -3.5 m p = -3.7 Cycle threshold n.d btg1 m = -3.8 m p = -3.1 Cells Cells Cells Cycle threshold n.d fbxo32 m = -2.9 m p = -3.6 Cycle threshold n.d cav1 m = -3.4 m p = -3.5 Cycle threshold n.d map2k2 m = -2.9 m p = -3.4 Cells Cells Cells Cycle threshold n.d ccnb1 m = -3.9 m p = -3.0 Cycle threshold n.d runx1 m = -3.3 m p = -3.2 Frequency cell replicates = 0.36 Cells Cells Mean-centered C T

12 Heterogeneities during in vitro mammary acinar morphogenesis Laminin 5 GM130 (Golgi) phospho-akt cleaved caspase-3 Debnath and Brugge, Nat. Rev. Cancer (2005) Inner cells Apoptosis Clearance MCF-10A Outer cells P-Akt positive P-Akt negative Mature acinus

13 The FOXO regulatory network Akt FOXO Metabolism Growth arrest Transcription Signaling Sesn1 Sox4 Cav1 Hdlbp Cdkn1a Fbxo32 Sod2 Btg1 Sema3c

14 Dichotomous nucleocytoplasmic localization of FOXO-family members FOXO1/4 FOXO3a FOXO1/4 E-cadherin FOXO3a E-cadherin MCF10A-5E, day 10 Scale bar: 25 m

15 The FOXO regulatory network Akt FOXO Metabolism Growth arrest Transcription Signaling Sesn1 Sox4 Cav1 Hdlbp Cdkn1a Fbxo32 Sod2 Btg1 Sema3c

16 The FOXO regulatory network Transcription factor Transcriptional coactivator Unknown IKK Akt SGK Cdk2 JNK FOXO Metabolism Growth arrest Transcription Signaling Sesn1 Sox4 Cav1 Hdlbp Cdkn1a Fbxo32 Sod2 Btg1 Sema3c p53 Ets STAT AP-2 GATA-6 PR SREBP-1 C/EBP Sp3 Sp1 NF-κB PPARγ c-myc

17 Selective time-dependent induction of FOXO targets during morphogenesis Fold induction (log 10 ) prdx6 s100a hint1 cdkn1c sema3c fbxo32 btg1 sod2 sesn1 krt10 sox4 cav1 gapdh ccnb1 ubc cdkn1a Day of morphogenesis Known FOXO target Reported FOXO target Loading controls Other

18 Identifying heterogeneously expressed FOXO targets Sectioning Microdissection Amplification FOXO active Akt active FOXO inactive e.g., GAPDH

19 LCM cap Section Slide Infrared laser Polymer wetting Captured cells Arcturus Bioscience

20 Localized microdissection of outer cells Before After Scale bar: 20 μm

21 Stochastic sampling of matrix-attached cells at d10 of morphogenesis 8 ccnb1 4 sox4 sesn1 cdkn1c Group 1 Fold change from mean btg1 sema3c prdx6 gapdh hint1 s100a6 ubc cdkn1a cav1 Group fbxo32 sod Repeated 10-cell samplings krt10 Known FOXO target Reported FOXO target Loading controls Other

22 Fluctuations in stochastic samplings are not due to measurement noise 8 ccnb1 ccnb1 4 sox4 sesn1 btg1 s100a6 Fold change from mean cdkn1c btg1 sema3c prdx6 gapdh hint1 s100a6 ubc cdkn1a prdx6 cdkn1a ubc sema3c sod2 fbxo32 hint1 sox4 cav1 cav1 krt fbxo32 sod2 gapdh sesn Stochastic samplings krt10 cdkn1c Measurement replicates Known FOXO target Reported FOXO target Loading controls Other

23 Multicolor RNA fluorescence in situ hybridization sensitivity vigilin-dig Alexa 488 MnSOD Alexa 555 Merge Scale bar: 25 m

24 Image segmentation and quantification

25 Independent single-cell regulation of fbxo32 and sod2 expression Time course fbxo32-dnp Alexa 488 sod2-dig Alexa 555 Stochastic sampling Loading Alexa 647 Loading-normalized sod R = Loading-normalized fbxo32 Scale bar: 25 m

26 Time course Single-cell coregulation of fbxo32 and cav1 expression fbxo32-dnp Alexa 488 cav1-dig Alexa 568 Stochastic sampling Loading Alexa 647 Loading-normalized cav R = Loading-normalized fbxo Scale bar: 25 m

27 Single-cell coregulation among genes within FOXO subclusters identified by stochastic sampling ccnb1 sox4 sesn1 cdkn1c btg1 sema3c hint1 s100a6 prdx6 gapdh ubc cdkn1a cav1 fbxo32 sod2 krt10 Single-cell pairwise correlation Strong (R > 0.6) Weak (0.4 < R 0.6) None (R 0.4) n = cells FOXO subclusters

28 Normal probability plots of RNA FISH data 0.6 fbxo32-cav1-cdkn1c cluster 0.6 btg1-cdkn1c-sox4-sesn1-sema3c cluster Log-transformed quantiles p > 0.5 (J-B test) n = Standard Normal Quantile Log-transformed quantiles p < 0.05 (J-B test) n = Standard Normal Quantile

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30 Single-cell cdna reamplification In-house modifications (part 3) AL1 seq' AAAA...AAAA TTTT...TTTT AL1 seq TTTT...TTTT AAAA...AAAA AL1 seq AL1 seq' 3.5 U High Fidelity polymerase PCR cycles (94 o C,42 o C,72 o C) H Aminoallyl-dUTP: U C=C NH 2 H H HC=C NH 2 AL1 seq' AAAA...AAAA U TTTT...TTTT AL1 seq TTTT...TTTT U AAAA...AAAA HC=C NH 2 H Alexa Fluor 555 succinimidyl ester H O HC=C N C AL1 seq' AAAA...AAAA U TTTT...TTTT AL1 seq TTTT...TTTT U AAAA...AAAA HC=C N C H O = = : O = L O C AL1 seq AL1 seq' AL1 seq AL1 seq' Replaced Minor improvements Major improvements

31 Validated stochastic profiling of 10-cell samples with Illumina gene chips Replicate reamplification-labeling-hybridization Replicate measurement Reamplification replicate # R 2 = Measurement replicate # R 2 = Reamplification replicate # Measurement replicate #1 Kristin Cabral Children s Hospital

32 Stochastic profiling of matrix-attached cells 3 targets 1 ligand 1 receptor 1 receptor 1 TF 3 targets 18 translation genes 1 transcription factor Covarying genes among random samplings Random samplings of 10 cells

33 Novel dichotomies identified by stochastic profiling of matrix-attached cells Receptors Cytoplasmic proteins Transcription factors Control JNK1 Day 10 Scale bar: 25 m

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35 Heterogeneous multiacinar formation upon inducible activation of ErbB2 ErbB2 homodimers No dimerizer With dimerizer Muthuswamy et al., Nat Cell Biol 3:785 (2001) Normal acini: Ductal carcinoma in situ:

36 Single-cell heterogeneity in cancer Irish et al., Cell 118:217 (2004) "Lung cancer heterogeneity. Prognostic implications." Cancer 60:370 (1987). "Gastric cancer heterogeneity." Cancer 63:791 (1989). "Human breast cancer: heterogeneity of estrogen binding sites." Cancer 48:1791 (1981).... Shipitsin et al., Cancer Cell 11:259 (2007)

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