2003

Inferring Connection Maps from AfCS Experimental Data and

Legacy Data

2003

COMPONENTS

Parts-List

INTERACTIONS

AND NETWORKS

COMPUTATIONAL

MODELS

Alliance for Cellular Signaling

Context-Specific

2003

2003

Our experiments measure genes, proteins and key metabolites. What are the underlying biological relationships amongst these entities? The cell functions as an integrated system involving all these players. How can we analyze our data to reveal this interconnectedness?

Data Analysis

2003

2003

Reconstructing Networks

Legacy Data AfCS Data

Protein Interactions Microarray Data

Biochemical Pathways Yeast Two-Hybrid Data

RNAi Data

Protein Data

Perturbation Data

Microscopy Data

Published Literature

Reconstructing Networks

2003

Signal Transduction in a Cell

from Downward, Nature, August (2001)

2003

Significance analysis of microarrays* (SAM)(R. Tibshirani, G. Chu 2002)

Objective: The replicated expression for each gene is taken for the 4hr time condition (untreated vs ligand) to determine whether the gene is statistically

differentially up- or down- regulated.

The t-statistics for all the genes are ordered and noted. The labels are then permutated and the t-statistic is calculated again. After many iterations, the cumulative t-statistics is averaged for each gene. Finally, for a given false positive rate, [called “False Discovery Rate” or FDR], the significant genes are selected.

For each gene, define the adjusted “t-statistic” as follows:

treated - untreated

+ adjustment factor

mean of replicates

standard deviation for the gene

2003

Differentially expressed genes for ligands vs UNTREATED @ 4hr [ SAM ; False Discovery Rate ( ) ]

ligand (4hr)

40L

(1%

)

LPS

(1%

)

AIG

(1%

)

IL4

(1%

)

CP

G (

1%)

IFB

(1.

5%)

GR

H (

1%)

2MA

(18

%)

LPA

(17

%)

CG

S (

2.9%

)

BO

M (

35%

)

IGF

(8%

)

S1P

(38

%)

PA

F (

2.4%

)

70L

(6%

)

NP

Y (

10%

)

DIM

(9%

)

LB4

(23%

)

M3A

(3.

5%)

FM

L (1

1%)

TG

F (

2.5%

)

TE

R (

35%

)

IL10

(20

%)

ELC

(26

%)

PG

E (

11%

)

BA

FF

(11

%)

BLC

(57

%)

NG

F (

42%

)

TN

F (

33%

)

SD

F (

20%

)

IFG

(25

%)

NE

B (

25%

)

SLC

(N

A)

num

ber

of g

enes

(pr

obes

)di

ffere

ntia

lly e

xpre

ssed

0

50

100

150

200

500

600

700

800

900

1000

1100

down-regulated up-regulated

2003

“mitogenic” ligands FDR = 1%

FDR = 35%FDR = 18%

FDR = 1%- 3%

Two-way dendrogram using significantly expressed genes (4hr)

2670 unique genes

2003

Concordance of significantly up (+) or down (-) regulated genes mitogenic ligands (FDR = 1%)

756 (-)

1082 (+)

337 (-)135 (-) 553 (-)

147 (-)

“down-regulated” matches

“up-regulated” matches

3 (-)

446 (-)

887 (+)

96 (-)

Mosaic plot

578 (+)

73 (+)

597 (+)

117 (+)

47 (+)

477 (+)

117 (+)

4 (+) 6 (+) 3 (+)

796 (-)

854 (+)

5 (+) 4 (+)

3 (-)

10 (+)

1 (-)

3 (-)

2 (-)

3 (-)

72 (+)

18 (+)

341 (-)

143 (-)

152(-)

80(+)

108 (+)

171 (-)

163 (+)

151 (-)

119 (-)

Discordance matrix

Example: CD40L had 756 down-regulated and 1082 up-regulated genes.

Those which were similarly regulated in AIG:

337 down

578 up.

72 (-)

40L AIG CPG LPS IL4 IL1040L - 17 0 0 9 0AIG 4 - 0 1 3 0CPG 0 6 - 0 2 0LPS 1 17 0 - 11 1IL4 3 3 0 0 - 0IL10 0 0 0 0 0 -

2003

Beyond Clustering

• How can we obtain biological information from array data at the level of individual genes and correlations in expression between genes?

• Can we use the correlations to build a connection network that reflects correlations in expression? Is there biological significance to this?

2003

Two-way hierarchical cluster:

mean ratio (vs control) of phosphoprotein levels and ligand

Note: the ligands that elicit an ERK response (chemokines + AIG, CD40L) clustered together. A correspondence plot below also showed the grouping.

2003

Similarity measures between genes under different conditions with respect to expression levels for…

… groups of genes clustering methods

… pairs of genes correlation methods

Covariance = Nk=1 {el(x(k)) – xmean)}{el(y(k)) – ymean) = r

xy

Correlation = r xy/(x y

Where, el(x(k)) indicates the expression level of gene x under condition k. xmean is the expression level of gene x over N different conditions. x is the standard deviation for gene x.

2003

Transcription factor encoded by fos is stabilized by ERK and continues to affect other IE genes such as jun

from Nature Cell Biology August 2002

2003

Jun Fos Ap1b1 Ap1g1 Ap1m2 UnknownAFCS_ID GeneSymbol Jun Fos Ap1b1 Ap1g1 Ap1m2 UnknownA001300 Jun 1.00A000404 Fos 0.70 1.00A000284 Ap1b1 0.11 -0.03 1.00A000285 Ap1g1 0.54 0.40 -0.05 1.00A000288 Ap1m2 0.09 0.09 0.42 0.31 1.00

Unknown 0.35 0.10 0.43 0.42 0.37 1.00

Schematic interpretation of ERK signal duration for IE gene product for fos

AFCS_ID A001300 A000404 A000284 A000285 A000288AFCS_ID Jun Fos Ap1b1 Ap1g1 Ap1m2 UnknownA001300 Jun 1.00A000404 Fos 0.14 1.00A000284 Ap1b1 0.04 0.08 1.00A000285 Ap1g1 0.05 0.01 0.41 1.00A000288 Ap1m2 0.10 0.18 0.26 0.30 1.00

Unknown 0.28 0.31 0.16 0.20 0.41 1.00

Cross-correlation matrices

Transcription response from “non-ERK” ligands

Transcription response from “ERK” response ligands

2003

Microarray analysis model using gene expression profiles

DNA Gene A Gene B Gene C Gene D

Protein

mR

NA

mR

NA

P

mR

NA

Pm

RN

A

Signal transduction is most likely regulated on the protein level, but the downstream signal on the transcriptional level is the resultant output from the upstream (outside the nucleus) signal input. The signal information processing complexity is now increased on the transcription level but some information flows upstream and oscillates in an input/output fashion.

2003

Beyond Clustering

• Mechanisms for inducing high correlation between genes in their expression profiles– A direct interaction

– An indirect interaction (the regulatory information of gene A product is transferred through the expressions of some other genes to induce the expression of gene B)

– Regulation by a common gene (the expression of genes A and B are regulated by a common gene)

2003

Mitogen-Activated Protein Kinase Pathways Mediated by ERK, JNK, and p38 Protein Kinases

G. L. Johnson and R. Lapadat Science 2002 December 6; 298: 1911-1912. (in Review)

2003

Transcriptional effects downstream from proteins recruited in MAPK cascades (Hazzalin, et al ,Nature Cell Biology (2002)

2003

“marginal correlation”

“marginal” global correlation (for ligand j )

difference in correlation = r2

all xy - r2 all xy except ligand j

Red indicates positive influence on the gene upon removing ligand j

Green indicates negative influence on the gene upon removing ligand j

2003

“Marginal” correlation

IE genes downstream from MAPK

Ligand n=33

Idea: indicates the “leverage” on the global correlation coefficient for a gene for the particulat ligand

2003

Marginal Correlations between Genes

• Provides a “biologically”-driven approach to discriminating ligand responses at the gene and gene-product level.

• Serves as a pathway driven hypothesis generation method for QRTPCR.

• Suggests ideal double ligand experiments to explore major signaling pathways that lead to downstream gene expression changes.

2003

“Marginal” correlation signatures

IE genes downstream from MAPK

Ligand n=33

Correlation coefficient

green = negative

red = positive

Mitogenic ligand

2003

“Marginal” correlation signatures

IE genes downstream from MAPK

Ligand n=33

Correlation coefficient

green = negative

red = positive

chemokines

No obvious pattern so consider data reduction

2003

mitogenic chemokines

2003

For the case of ligand 2MA…

cAMP responsive element modulator

2003

Marginal Correlations averaged over Pathway-Specific Genes

2003

Marginal Correlations averaged over Pathway-Specific Genes

2003

Marginal Correlations averaged over Pathway-Specific Genes

2003

Marginal Correlations averaged over Pathway-Specific Genes

2003

transcription factor binding sites immediately upstream from “immediate-

early” genes fos & jun (Hazzalin, et al ,Nature Cell Biology (2002)

= expression measured indirectly in ligand AfCS experiment

2003

"ERK" minus "nonERK" ETS MEF2 CREM CCAAT CRE3 CREM.. junD ETS. SRE MEF2. AP-2b CRE1.. AP-2a. c-jun c-fosETSMEF2 0.05CREM -0.15 0.45CCAAT 0.15 -0.08 -0.27CRE3 -0.29 0.28 0.29 -0.39CREM.. -0.32 0.27 0.19 0.02 0.20junD -0.28 0.27 0.17 -0.25 0.17 -0.01ETS. 0.00 -0.12 0.27 -0.48 0.55 0.11 0.21SRE 0.26 0.56 0.38 -0.02 -0.04 0.38 0.21 -0.13MEF2. -0.19 0.29 0.35 -0.06 0.24 0.03 0.13 0.29 0.38AP-2b 0.08 0.53 0.12 -0.05 0.50 0.28 0.20 0.13 0.23 0.20CRE1.. -0.39 0.29 0.37 -0.25 0.41 0.07 0.09 0.66 0.17 0.13 0.17AP-2a. 0.09 -0.10 -0.06 -0.19 0.06 0.15 0.15 -0.07 -0.18 0.13 -0.13 0.26c-jun 0.10 0.09 0.07 -0.13 -0.03 0.09 0.08 -0.39 0.16 0.39 0.17 0.21 -0.20c-fos -0.18 0.19 0.14 -0.12 0.11 0.07 0.07 0.09 0.11 0.06 0.13 0.06 0.09 0.38SP1cofactor3 -0.50 -0.11 -0.23 -0.08 -0.02 -0.28 -0.23 -0.02 0.14 -0.28 -0.02 -0.18 0.29 0.29 -0.12Octa-2 -0.29 -0.46 -0.23 0.08 -0.17 -0.23 -0.03 0.16 0.07 -0.26 -0.35 -0.27 0.15 -0.21 -0.09

Difference in IE gene cross-correlations from ligands that involve ERK pathway

Critical level p =0.00001

Partial correlations

Ligands that stimulate ERK

ETS

MEF2

CREM

CCAAT

CRE3CREM..

junD

ETS.

SRE

MEF2.

AP-2b

CRE1..

AP-2a.c-jun

c-fos

SP1cofactor3

Octa-2

a

b

c

d

ef

g

h

i

j

k

l

mn

o

p

q

Note: junB expression wasn’t detected

transcription factor AP-2, alphatranscription factor AP-2 betaserum response factor(element)non-POU-domain-containing, octamer binding proteinmyocyte enhancer factor 2Cmyocyte enhancer factor 2CJun-B oncogeneJun proto-oncogene related gene d1 jund1Jun oncogene jun cFBJ osteosarcoma oncogeneets variant gene 5ets homologous factorcofactor required for Sp1 transcriptional activation subunit 2CCAAT/enhancer binding protein alpha (C/EBP), related sequence 1cAMP responsive element modulatorcAMP responsive element binding protein 3cAMP responsive element binding protein 1

2003

"ERK" minus "nonERK" ETS MEF2 CREM CCAAT CRE3 CREM.. junD ETS. SRE MEF2. AP-2b CRE1.. AP-2a. c-jun c-fosETSMEF2 0.05CREM -0.15 0.45CCAAT 0.15 -0.08 -0.27CRE3 -0.29 0.28 0.29 -0.39CREM.. -0.32 0.27 0.19 0.02 0.20junD -0.28 0.27 0.17 -0.25 0.17 -0.01ETS. 0.00 -0.12 0.27 -0.48 0.55 0.11 0.21SRE 0.26 0.56 0.38 -0.02 -0.04 0.38 0.21 -0.13MEF2. -0.19 0.29 0.35 -0.06 0.24 0.03 0.13 0.29 0.38AP-2b 0.08 0.53 0.12 -0.05 0.50 0.28 0.20 0.13 0.23 0.20CRE1.. -0.39 0.29 0.37 -0.25 0.41 0.07 0.09 0.66 0.17 0.13 0.17AP-2a. 0.09 -0.10 -0.06 -0.19 0.06 0.15 0.15 -0.07 -0.18 0.13 -0.13 0.26c-jun 0.10 0.09 0.07 -0.13 -0.03 0.09 0.08 -0.39 0.16 0.39 0.17 0.21 -0.20c-fos -0.18 0.19 0.14 -0.12 0.11 0.07 0.07 0.09 0.11 0.06 0.13 0.06 0.09 0.38SP1cofactor3 -0.50 -0.11 -0.23 -0.08 -0.02 -0.28 -0.23 -0.02 0.14 -0.28 -0.02 -0.18 0.29 0.29 -0.12Octa-2 -0.29 -0.46 -0.23 0.08 -0.17 -0.23 -0.03 0.16 0.07 -0.26 -0.35 -0.27 0.15 -0.21 -0.09

Difference in IE gene cross-correlations from ligands that involve ERK pathway

Critical level p =0.00001

Partial correlations

ERK

CREM

a h

k

Possible interpretation of a gene regulatory network

2003

CRSP3

MEF2C

CRSP2

EHF

CREB1JUND

CREB3

NONO

ETV5

ETV6

JUN

CREM

FOS

JUNB

SRF

J Biol Chem 1998 Nov 20;273(47):31327-36

The transcription factors ID{-3321=Elk-1} and ID{-11291=Serum Response Factor} are necessary for GH-stimulated transcription of ID{-3796=c-fos} through the Serum Response Element (SRE).

Proc Natl Acad Sci U S A 1991 Jun 15;88(12):5448-52

Furthermore, expression of antisense ID{2352=CREM} enhances ID{-3796=c-fos} basal and cAMP-induced transcription.

Neurol Res 2000 Mar;22(2):138-44In the non-trauma patients 36% expressed ID{-3796=c-fos} and 73% expressed ID{-6204=c-jun} mRNA, with all patients studied expressing ID{-3796=c-Fos} and ID{-6204=c-Jun} proteins.

Mol Cell Biol 1991 Jan;11(1):192-201We observe that the expression of endogenous ID{-6204=c-jun} and ID{-6205=jun B} genes is induced by E1A, which directly transactivates the promoters of ID{-3796=c-fos}, ID{-6204=c-jun}, and ID{-6205=jun B}.

Genes Correlated by Gene Expression from Legacy Data extracted from Pathway Assist (Stratagene Database)

2003

CRSP3

MEF2C

CRSP2

EHF

CREB1

CREB3

NONO

ETV5

ETV6

FOS

SRF

JUNB

JUN

JUND

CREM

Connections at the Protein Level from Legacy Data extracted using Pathway Assist (Stratagene)

2003

2003

Full view of two-way dendrogram

Two-Way Dendrogram from AfCS ligand screen using the probes (genes) relating to the “immediate-early” genes (with additional genes that encode MAPK proteins involved in the cascade).

Summary: The transcription profiles of these selected genes distinguished the “mitogenic” ligands (AIG, CPG, CD40L, IL-4, IL10, LPS) from the “non-mitogenic” at the 2hr / 4hr time period. Since the upstream MAPK-ERK pathway is involved in cell proliferation this would be expected under ideal experimental conditions. The fact that a distinct two-way “bicluster” (mitogenic ligands are clustered to the IE genes from MAPK-ERK) as a first-pass result of the microarray experiment is highly encouraging. This “semi-supervised” approach indicates our expression data is biologically informative.

2003

Kohn’s Mammalian Cell Cycle Map (with AfCS genes)

2003

Kohn’s Mammalian Cell Cycle Map (with AfCS genes)

Mad3 Ndr3 Oazin Mga Mina Ndr1 Cdh2 Rbbp9 Ccnd2 Rb1 Mad Rb12900002K07Rik2900002K07RikMina Cdh11 Nmyc12310005B10RikCdh13 Rbbp4 Mad4 Ap2a2 Rbbp7Mad3 1.000Ndr3 0.039 1.000Oazin -0.303 0.080 1.000Mga -0.315 -0.025 0.293 1.000Mina -0.606 -0.004 0.376 0.209 1.000Ndr1 -0.177 0.129 0.261 0.002 0.303 1.000Cdh2 0.552 0.095 0.017 -0.306 -0.254 -0.022 1.000Rbbp9 0.197 0.020 0.017 -0.230 -0.056 0.119 0.243 1.000Ccnd2 0.061 0.192 0.178 -0.136 0.152 0.167 0.093 -0.025 1.000Rb1 -0.423 0.021 0.171 0.010 0.322 0.012 -0.116 0.115 0.021 1.000Mad -0.151 0.337 0.261 0.023 0.214 0.254 0.033 0.077 0.138 0.194 1.000Rb1 0.077 0.186 0.031 -0.094 -0.073 0.126 0.085 0.040 0.120 0.046 0.478 1.0002900002K07Rik -0.086 0.001 -0.045 -0.240 -0.129 0.051 -0.198 0.196 -0.232 0.068 -0.075 0.072 1.0002900002K07Rik -0.122 -0.022 -0.143 -0.165 -0.060 -0.021 -0.247 0.197 -0.393 0.099 -0.104 0.003 0.847 1.000Mina -0.201 0.264 0.240 -0.056 0.175 0.180 0.064 0.117 0.095 0.250 0.586 0.139 0.003 -0.033 1.000Cdh11 -0.460 -0.042 0.254 0.707 0.339 0.138 -0.243 -0.271 -0.088 0.011 0.050 -0.052 -0.204 -0.123 -0.043 1.000Nmyc1 -0.015 -0.016 -0.069 0.090 -0.079 -0.085 -0.172 -0.179 0.149 0.179 -0.126 0.138 0.062 0.073 -0.043 0.091 1.0002310005B10Rik -0.247 0.062 0.165 0.194 0.139 0.085 -0.241 -0.067 0.393 0.185 0.032 -0.004 -0.012 -0.057 0.062 0.125 0.335 1.000Cdh13 0.237 0.073 0.032 -0.052 -0.336 -0.010 0.258 0.308 0.038 -0.002 0.133 0.142 0.203 0.099 -0.001 -0.093 -0.113 -0.144 1.000Rbbp4 -0.105 0.062 0.014 -0.110 0.143 0.091 -0.057 -0.239 0.237 0.014 0.088 0.073 -0.129 -0.117 0.133 -0.130 0.096 0.096 -0.243 1.000Mad4 0.065 0.028 0.013 0.400 -0.265 0.102 -0.226 -0.002 0.094 -0.255 -0.148 -0.045 0.003 -0.041 -0.235 0.347 0.111 0.240 0.243 -0.175 1.000Ap2a2 -0.078 -0.194 -0.143 0.383 -0.058 -0.119 -0.058 -0.333 -0.609 -0.195 -0.121 -0.093 -0.069 0.053 -0.049 0.399 -0.111 -0.377 -0.039 -0.078 -0.039 1.000Rbbp7 -0.385 0.008 0.255 -0.011 0.314 0.034 -0.209 -0.004 0.211 0.467 0.103 0.025 0.152 0.100 0.144 -0.132 0.211 0.500 -0.090 0.244 -0.157 -0.336 1.000Rbl1 0.392 0.077 -0.138 -0.608 -0.132 -0.010 0.562 0.409 0.154 0.089 0.114 0.177 0.180 0.166 0.150 -0.514 -0.092 -0.188 0.194 -0.003 -0.322 -0.346 0.061Pcdh18 0.540 -0.039 -0.345 -0.026 -0.493 -0.227 0.271 -0.198 -0.358 -0.477 -0.202 -0.103 -0.187 -0.117 -0.169 0.001 -0.131 -0.420 -0.017 -0.112 0.069 0.507 -0.613Rbl1 0.241 0.062 -0.096 -0.646 -0.085 -0.038 0.272 0.464 0.279 0.223 0.023 0.070 0.283 0.238 0.118 -0.660 -0.007 -0.010 0.158 0.016 -0.302 -0.667 0.247Rbbp9 0.724 0.052 -0.137 -0.406 -0.394 -0.087 0.601 0.524 0.057 -0.182 -0.084 0.024 0.047 0.055 -0.084 -0.520 -0.043 -0.156 0.299 -0.118 -0.025 -0.299 -0.144Mycbp -0.072 -0.116 -0.009 0.214 -0.040 -0.146 0.126 -0.184 -0.598 0.034 0.108 -0.069 -0.292 -0.138 0.106 0.180 -0.287 -0.347 -0.045 -0.077 -0.140 0.588 -0.2091700018O18Rik -0.032 0.100 0.256 -0.024 0.291 0.181 0.028 0.158 0.777 0.069 0.136 0.081 -0.143 -0.283 0.095 -0.069 0.147 0.496 -0.079 0.253 0.094 -0.603 0.308Ccnd2 0.132 -0.230 0.016 0.058 -0.137 -0.048 -0.027 0.029 -0.154 -0.179 -0.157 0.062 0.037 0.090 -0.437 0.129 0.027 -0.151 0.056 -0.120 0.137 0.084 -0.205Oaz1 0.002 0.060 -0.073 -0.163 -0.213 -0.057 -0.080 0.068 -0.016 0.044 -0.083 -0.016 0.191 0.145 -0.070 -0.184 0.126 0.003 0.013 0.070 0.010 -0.173 0.050Ahcy -0.128 -0.003 0.022 -0.110 0.068 -0.020 -0.157 -0.218 0.238 0.184 0.013 0.141 -0.106 -0.125 0.022 -0.091 0.215 0.205 -0.193 0.233 -0.112 -0.186 0.224Cdh23 -0.151 0.204 0.352 0.054 0.239 0.384 0.031 0.098 0.243 0.076 0.632 0.327 -0.065 -0.124 0.554 0.082 -0.133 0.125 0.029 0.088 0.012 -0.177 0.128Tcfap2b -0.359 -0.017 0.285 0.013 0.544 0.239 0.151 0.352 -0.047 0.366 0.157 -0.152 -0.071 0.025 0.155 0.206 -0.277 0.044 0.057 -0.170 -0.137 -0.132 0.130Cdh11 -0.065 -0.005 -0.033 0.371 -0.031 -0.165 -0.167 -0.346 -0.014 -0.086 -0.071 0.021 -0.178 -0.159 -0.217 0.296 0.201 0.144 -0.070 -0.080 0.321 0.161 0.008Cdh11 -0.583 -0.009 0.252 0.569 0.457 0.180 -0.322 -0.363 -0.037 0.053 0.092 -0.057 -0.176 -0.088 0.012 0.843 -0.004 0.156 -0.195 0.011 0.200 0.380 0.002Cdh23 0.548 0.007 -0.359 -0.310 -0.279 -0.283 0.312 0.069 -0.017 -0.308 -0.192 0.023 -0.055 -0.073 -0.221 -0.399 -0.192 -0.221 -0.024 -0.052 -0.115 0.026 -0.3575730443C07UnK -0.301 0.045 0.277 0.085 0.321 0.049 -0.140 0.176 0.045 0.350 0.083 -0.047 0.048 0.101 0.103 -0.070 -0.016 0.167 -0.008 0.049 -0.034 -0.245 0.339Ap2a2 -0.294 -0.164 -0.092 0.134 0.077 -0.029 -0.077 -0.063 -0.209 -0.012 -0.042 -0.033 0.038 0.020 -0.104 0.311 -0.134 -0.100 0.096 0.056 0.051 0.299 0.034Pcdhb14 -0.056 -0.149 -0.295 -0.048 -0.294 -0.232 -0.362 -0.254 -0.126 -0.141 -0.210 -0.177 0.205 0.169 -0.157 -0.029 0.031 0.161 -0.061 -0.012 0.152 0.196 0.027Cdh11 -0.498 -0.084 0.205 0.586 0.350 0.096 -0.261 -0.508 -0.032 0.003 0.018 -0.092 -0.345 -0.307 -0.046 0.747 -0.022 0.224 -0.304 0.127 0.122 0.417 -0.011Mga -0.235 0.024 0.244 0.153 -0.021 0.002 -0.260 -0.300 0.038 -0.028 0.007 0.029 -0.044 -0.138 -0.046 0.167 0.058 0.189 -0.107 0.142 0.017 0.007 0.162Tcfap2a -0.087 0.054 0.286 -0.118 0.548 0.184 0.232 0.108 0.266 0.090 0.152 -0.086 -0.315 -0.277 0.128 -0.039 -0.202 0.120 -0.294 0.166 -0.308 -0.285 0.120Mina 0.122 0.131 0.219 -0.187 0.137 0.068 0.270 0.394 0.500 0.121 0.153 0.073 -0.079 -0.178 0.118 -0.355 -0.022 0.388 0.066 0.098 -0.078 -0.687 0.362Max 0.507 -0.031 -0.214 -0.485 -0.258 -0.156 0.474 0.301 0.189 -0.073 -0.055 0.077 0.032 -0.004 -0.010 -0.423 0.040 -0.217 0.203 -0.085 -0.219 -0.290 -0.126Mycbp -0.062 -0.057 -0.028 0.304 -0.146 -0.122 -0.049 -0.062 -0.653 -0.039 0.014 -0.173 -0.155 -0.002 -0.007 0.265 -0.245 -0.262 0.013 -0.237 0.160 0.500 -0.240Cdh13 -0.150 -0.098 0.040 0.163 0.004 -0.135 -0.200 -0.094 -0.018 0.055 -0.008 0.013 -0.068 -0.022 -0.179 0.200 0.073 0.055 -0.066 -0.094 0.094 0.007 0.025Oaz3 0.196 0.086 0.014 -0.389 0.192 -0.008 0.520 0.243 0.185 0.036 0.116 -0.038 -0.123 -0.151 0.126 -0.329 -0.308 -0.174 0.157 0.024 -0.330 -0.223 0.014Oaz1 -0.132 0.025 -0.055 -0.237 -0.216 -0.012 -0.381 -0.035 -0.070 0.025 -0.174 -0.083 0.320 0.278 -0.075 -0.228 0.074 0.037 -0.087 -0.044 0.061 -0.239 0.069Oaz1 -0.158 -0.038 -0.077 -0.317 -0.090 -0.025 -0.347 0.034 -0.124 0.055 -0.159 -0.081 0.319 0.307 -0.088 -0.233 0.020 -0.052 -0.131 -0.115 -0.075 -0.253 0.049Ap2a1 0.257 0.093 0.031 -0.323 -0.081 0.178 0.123 0.333 0.181 0.035 0.054 0.118 0.019 0.004 0.120 -0.439 0.003 -0.013 0.208 -0.037 -0.003 -0.353 0.065Cdh3 0.328 0.107 0.060 -0.323 -0.080 0.030 0.374 0.365 0.126 -0.113 0.007 -0.075 -0.029 -0.077 0.151 -0.384 -0.297 -0.058 0.061 0.005 -0.109 -0.281 -0.0534631427C17Rik -0.032 0.148 0.298 -0.135 0.349 0.162 0.297 0.250 0.275 0.128 0.270 0.203 -0.118 -0.205 0.152 -0.188 -0.203 0.070 -0.093 0.214 -0.290 -0.410 0.194Tcfap2a 0.560 0.161 -0.081 -0.532 -0.124 -0.031 0.612 0.469 0.092 -0.074 0.028 -0.070 -0.018 0.000 0.089 -0.530 -0.233 -0.180 0.109 -0.052 -0.244 -0.305 -0.116Pcdh8 -0.136 0.111 0.411 -0.026 0.172 0.109 0.023 0.256 0.127 0.227 0.195 0.049 -0.006 -0.062 0.154 -0.104 -0.107 0.099 0.063 0.004 -0.097 -0.292 0.2822310005B10Rik 0.143 0.103 0.229 0.074 0.088 0.142 0.163 0.120 0.575 -0.182 0.043 0.043 -0.237 -0.376 -0.054 -0.048 -0.002 0.428 -0.048 0.127 0.179 -0.514 0.1062900002K07Rik 0.125 -0.137 -0.146 -0.139 -0.288 -0.205 -0.135 -0.088 -0.189 -0.160 -0.253 -0.002 0.362 0.446 -0.112 -0.146 0.277 -0.072 -0.010 -0.008 -0.009 0.095 -0.048Ndr2 0.123 0.137 0.000 -0.107 -0.138 0.141 0.123 0.221 0.084 -0.167 0.148 0.192 0.059 -0.045 0.178 -0.089 -0.105 -0.105 0.186 -0.068 0.181 -0.173 -0.1562310005B10Rik -0.161 0.068 0.208 0.074 0.273 0.152 -0.103 0.003 0.344 0.062 0.107 -0.012 -0.016 -0.057 0.064 -0.022 0.095 0.499 -0.136 0.255 0.060 -0.289 0.325

2003

Kohn’s Mammalian Cell Cycle Map (with AfCS genes)

2003

Subcluster of“mitogenic” ligand(late time periods)

Myc box genes (cell cycle)

2003

mito Mina 2900002K07Rik2900002K07RikMycbp1700018O18Rik Mina Mycbp2310005B10Rik Mga Mad4 Max Tcfap2a Ap2a1 Cdh11 Cdh11 Rbbp9 Rbl1Mina 1.002900002K07Rik 0.17 1.002900002K07Rik 0.13 0.86 1.00Mycbp 0.23 0.07 0.17 1.00

1700018O18Rik 0.21 -0.05 -0.28 -0.58 1.00

Mina 0.31 -0.03 -0.20 -0.43 0.90 1.00

Mycbp 0.33 0.13 0.17 0.94 -0.51 -0.31 1.00

2310005B10Rik 0.38 0.04 -0.24 -0.47 0.90 0.92 -0.30 1.00

Mga 0.51 0.33 0.17 0.25 0.10 0.21 0.42 0.31 1.00Mad4 0.24 0.33 0.10 0.38 0.11 0.20 0.58 0.27 0.59 1.00Max -0.04 0.20 0.13 0.37 0.21 0.31 0.31 0.19 -0.02 0.29 1.00Tcfap2a 0.74 0.08 0.02 0.25 0.39 0.55 0.32 0.48 0.31 0.33 0.34 1.00Ap2a1 0.01 -0.08 -0.03 0.33 0.12 0.29 0.31 0.06 -0.18 0.25 0.60 0.26 1.00Cdh11 0.61 0.43 0.23 0.36 0.07 0.17 0.55 0.34 0.76 0.78 0.07 0.45 -0.03 1.00Cdh11 0.73 0.17 0.08 0.35 0.11 0.21 0.48 0.26 0.62 0.55 -0.04 0.63 0.05 0.82 1.00Rbbp9 0.18 0.20 0.26 0.23 0.11 0.18 0.24 0.05 0.35 0.28 0.34 0.06 0.31 0.28 0.36 1.00Rbl1 -0.23 0.03 0.19 0.21 0.03 0.10 0.11 -0.11 -0.47 0.15 0.58 0.13 0.65 -0.17 -0.11 0.31 1.00Rbl1 -0.02 0.13 0.11 -0.18 0.45 0.58 -0.21 0.39 -0.19 -0.01 0.59 0.26 0.62 -0.19 -0.24 0.21 0.54Rbbp9 -0.27 0.01 0.03 0.19 0.08 0.18 0.16 0.10 -0.18 0.34 0.62 -0.02 0.53 -0.01 -0.22 0.27 0.69

non-mito1 Mina 2900002K07Rik2900002K07RikMycbp1700018O18Rik Mina Mycbp2310005B10Rik Mga Mad4 Max Tcfap2a Ap2a1 Cdh11 Cdh11 Rbbp9 Rbl1Mina 1.002900002K07Rik 0.15 1.002900002K07Rik 0.30 0.77 1.00Mycbp 0.30 0.34 0.41 1.00

1700018O18Rik 0.41 0.61 0.67 0.17 1.00

Mina 0.12 0.79 0.77 0.48 0.70 1.00

Mycbp 0.37 0.48 0.55 0.94 0.38 0.62 1.002310005B10Rik 0.38 0.64 0.75 0.74 0.61 0.82 0.88 1.00

Mga 0.59 0.30 0.40 0.77 0.33 0.36 0.86 0.72 1.00Mad4 0.21 0.63 0.72 0.75 0.51 0.72 0.85 0.83 0.61 1.00Max 0.03 0.55 0.66 0.32 0.35 0.50 0.28 0.39 0.05 0.48 1.00Tcfap2a 0.66 0.40 0.36 0.32 0.58 0.43 0.36 0.40 0.37 0.43 0.27 1.00Ap2a1 0.03 0.51 0.70 0.46 0.42 0.59 0.52 0.63 0.29 0.70 0.69 0.10 1.00Cdh11 0.68 -0.07 0.18 0.40 0.31 -0.05 0.51 0.41 0.74 0.33 -0.12 0.36 0.10 1.00Cdh11 0.77 0.24 0.26 0.53 0.40 0.18 0.62 0.54 0.71 0.41 -0.10 0.53 0.10 0.74 1.00Rbbp9 0.18 0.33 0.54 0.00 0.26 0.35 0.17 0.33 0.04 0.32 0.25 0.04 0.17 -0.02 0.05 1.00Rbl1 0.05 0.50 0.54 0.08 0.44 0.52 0.05 0.26 -0.20 0.27 0.71 0.36 0.54 -0.34 -0.05 0.08 1.00Rbl1 -0.19 0.51 0.45 -0.23 0.36 0.47 -0.20 0.03 -0.44 0.08 0.55 0.15 0.25 -0.62 -0.34 0.32 0.80Rbbp9 -0.16 0.44 0.48 0.41 0.27 0.47 0.35 0.37 0.05 0.54 0.74 0.18 0.66 -0.10 -0.05 0.00 0.68

Non-mitogenic ligand response gene correlations

Mitogenic ligand response gene correlations

2003

MYC Box and related genes

ProbeName(3-03) GeneName(3-03) Name AFCS_ID(3-03) AFCS_Name(3-03)2410057H13 myc induced nuclear antigen Mina2900002K07 B-MYC TRANSFORMING PROTEIN (FRAGMENT) homolog [Rattus norvegicus] 2900002K07Rik2900074D10 B-MYC TRANSFORMING PROTEIN (FRAGMENT) homolog [Rattus norvegicus] 2900002K07Rik5830401O17 c-myc binding protein Mycbp1700018O18 hypothetical Myc-type, helix-loop-helix dimerization domain containing protein 1700018O18Rik3830408E23 myc induced nuclear antigen Mina5730488M09 c-myc binding protein Mycbp2310005B10 DEAD/H (ASP-GLU-ALA-ASP/HIS) BOX POLYPEPTIDE 18 (MYC-REGULATED) homolog [Homo sapiens] 2310005B10Rik2310022K02 MAX gene associated Mga1810048H04 Max dimerization protein 4 Mad42310020E07 Max protein Max A001480 Max3010026L22 transcription factor AP-2, alpha Tcfap2aH3018B01 adaptor protein complex AP-2, alpha 1 subunit Ap2a1 A000290 AP-2 alphaA5730543A01 cadherin 11 Cdh11 A000407 Cadherin 112810038F24 cadherin 11 Cdh11 A000407 Cadherin 114930568E03 retinoblastoma binding protein 9 Rbbp92700054A06 retinoblastoma-like 1 (p107) Rbl1H3116E08 retinoblastoma-like 1 (p107) Rbl1 A002037 Rbl15730432F05 retinoblastoma binding protein 9 Rbbp9

2003

MYC Connection Map

Ap2a1Ap2a2Arhgef6

Cdc16

Cdc42ep3

Cdc42ep4.

Cdc42ep5

Cdgap

Cdh11

Cks1

Clk

Clk2Clk3 Clk4 Mad4

Max

Mga

Mina.

Mycbp

Rb1

Rbbp9.

Rbl1

Spec1

Srprb..

Tcfap2aTcfap2b

abc

d

e

f

g

h

i

j

k

lm n o

p

q

r

s

t

u

v

w

x

yz

Genetic regulatory module generated by partial correlations critical value = 10-6

2003

Literature-derived expression-based connection maps for all AfCS proteins

AfCS proteins with no known connections

2003

20032003

20032003