www.sciencesignaling.org/cgi/content/full/2/72/ra25/DC1 Supplementary Materials for Bruton’s Tyrosine Kinase Revealed as a Negative Regulator of Wnt–β- Catenin Signaling Richard G. James, Travis L. Biechele, William H. Conrad, Nathan D. Camp, Daniel M. Fass, Michael B. Major, Karen Sommer, XianHua Yi, Brian S. Roberts, Michele A. Cleary, William T. Arthur, Michael MacCoss, David J. Rawlings, Stephen J. Haggarty, Randall T. Moon* *To whom correspondence should be addressed. E-mail: [email protected]Published 26 May 2009, Sci. Signal. 2, ra25 (2009) DOI: 10.1126/scisignal.2000230 This PDF file includes: Fig. S1. Bioinformatic overlap of independent small-molecule and siRNA screens reveals previously unidentified regulators of Wnt–β-catenin signaling. Fig. S2. Zebrafish expression and loss-of-function data. Fig. S3. Protein interaction networks for BTK and CDC73. Fig. S4. Context-dependent regulation of Wnt–β-catenin signaling by CDC73. Table S1. Sequences of siRNA sense strands. Table S2. Descriptions of supplementary database files. Other Supplementary Material for this manuscript includes the following: (available at www.sciencesignaling.org/cgi/content/full/2/72/ra25/DC1) Databases S1 to S12, S16 to S18 (Microsoft Excel format) Databases S13 to S15 (cys format)
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Reported interactions of siRNA screen hits Reported interactions of small molecule screen hits
Up in small molecule screen
Chemical interactor not present in small molecule screen
TEC
Supplemental Figure 1. Bioinformatic overlap of independent small-molecule and siRNA screens reveals previo sly of Wnt/β-catening signaling.unidentified regulators
u
Supplemental Figure 1. Bioinformatic overlap of independent small-molecule and siRNA screens reveals prev- β-catenin signaling. Integration of the protein interaction network generated
direct targets of siRNAs considered hits in the siRNA screen. Proteins in the network are represented by
pathway or small molecules that inhibited the pathway. Green nodes represent proteins the pos-pathway: siRNA targets that when silenced signaling was decreased. Grey nodes represent
molecules that were not present in the small-molecule screen.
iously unidentified regulators of Wnt/through a STRING analysis of targets of the siRNA screen hits totaling 1855 nodes (Left) and the small molecule and protein interaction network generated through a STITCH analysis of the small-molecule screen hits totaling 292 nodes (Right). Proteins common in each network represent the bioinformatic overlap totaling 108 nodes (Center). Of the overlap nodes, 34 were rounded square nodes and small molecules are represented by diamond nodes. Edges represent an interaction bet-ween two nodes. Red nodes represent negative regulators of the pathway inhibitors: siRNA targets that when
proteins not considered positive in the siRNA screen, and white nodes represent proteins that were not targeted in the siRNA screen or small
silenced activated the itively contribute to the
Supplemental Figure 2. Zebrafish expression and loss of function data.
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Supplemental Figure 2. Zebrafish mRNA expression and loss of function data. (A-C) Prior to gastrulation, btk is expressed ubiquitously, after which it is restricted to hematopoietic precursors. (A) Zebrafish embryos were collected, fixed, and stained by in situ hybridization for anti-sense and sense btk transcript at several stages: shield, 14 hours post fertilization (hpf), 21 hpf, and 48 hpf. These data are representative of probes synthesized from two distinct regions of the btk open reading frame. (B-C) cDNA was purified from zebrafish embryos, and btk and actin was amplified by PCR. The results were run on a gel (B), and the bands were quantified by optical densitometry and then plotted as a ratio of btk to actin (C). (D) Attenuation of btk in zebrafish potentiates Wnt/β−catenin signaling. Injection of wnt8 mRNA into 1-cell stage zebrafish embryos causes anterior truncation in zebrafish by 24 hours post fertilization. Embryos co-injected with wnt8 mRNA (1.5 pg) and the second btk morpholino (denoted concentration) exhibited dose-dependent increases in the number and severity of anterior truncation phenotypes relative to co-injection with control morpholino (D). Note that these data are representative of four independent experiments. (E) To confirm that our btk morpholinos disrupted splicing, zebrafish were injected with 4 ng of morpholino and allowed to develop for 24 hours. At this time, genomic DNA was harvested and the indicated splice sites were amplified by PCR. btk morpholino 1 causes the formation of a second, smaller product (arrow), whereas btk morpholino 2 causes degradation of btk mRNA.
NormalSmallEyeHeadlessDorsalizedRadialized
Supplemental Figure 3. Protein Interaction Networks for BTK and CDC73.
A
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BLNK PLCG1
VAV1
LYN WAS
GRB2
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PLCG2
LRRC55MAP3K4
EIF3B EIF3F
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Supplemental Figure 3. Protein interaction networks for BTK and CDC73. Proteins (nodes) are represented as circles (bait) or squares (prey) and the relationship between proteins (edges) are denoted by the lines between the nodes. This protein-protein interaction network (PIN) for BTK and CDC73 generated with data from the literature (red edges), and primary affinity purification, mass spectrometry data (blue edges). The width of the blue edge is directly correlated to the total number of peptides identified in three independent affinity purification experiments. (A) This PIN represents the total number of proteins that co-purified with BTK from multiple pull-downs from HEK293T cells. BTK interacts with the Wnt/β−catenin regulators CDC73, PAF1, and CSNK2A1. Red nodes represent proteins that have been reported previously to have physical linkage with Wnt/β−catenin signaling downstream of the β−catenin destruction complex. (B) This PIN represents selected proteins that co-purified in multiple independent experiments with either BTK or CDC73 from HEK293T cells. Proteins were chosen based on whether they interacted with both CDC73 and BTK (shaded in blue) or have been previously implicated in Wnt/β-catenin signaling and/or transcriptional elongation. This PIN demonstrates the physical relationship of BTK and CDC73 to the Wnt/β−catenin pathway.
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Supplemental Figure 4. Context-dependent regulation of Wnt/β−catenin signaling by CDC73. RKO B cells (A) or HEK293T cells (B) stably expressing a β−catenin activated reporter (BAR) upstream of firefly luciferase and expressing a constitutive reporter that drives renilla luciferase were transiently transfected with the indicated siRNA oligonucleotides. 48 hours after transfection, the cells were treated with either WNT3A- or control-conditioned medium. The following day, firefly luciferase was quantified, normalized to that of renilla luciferase, and all data were plotted as fold change over activity in untreated control siRNA-treated cells. Error bars represent standard deviation from the mean for 3 replicates. This data is representative of three independent experiments.
Supplemental Table 1. Sequences of siRNA sense strands.
Gene Symbol Sequence (5’>3’) Company
Control Negative control 1 Ambion
AXIN1 GGUGUUGGCAUUAAAGGUGdTdT Invitrogen
AXIN2 GGGAGAAAUGCGUGGAUACdTdT Invitrogen
CTNNB1 GGUGGUGGUUAAUAAGGCUdTdT Invitrogen
BTK #1 CCAGUGAAAUGGAGCAAAUdTdT Invitrogen
BTK #2 CCCUUAUCCCUUCCAGGUUdTdT Invitrogen
BTK #3 GCCAAUGAAUGCAAAUGAUdTdT Invitrogen
CDC73 #1 UUUGUAAGAUAGUUGUUCGUGdTdT Invitrogen
CDC73 #2 CAGCGAUCUACUCAAGUCAAAdTdT Invitrogen
Ctnnb1 CUGUCUGUCUGCUCUAGCAdTdT Invitrogen
Btk517 GCTGAGAAAGCGCTGGATTdTdT Invitrogen
Btk1012 GCACATGACTCGAAGTCAAdTdT Invitrogen
Btk1651 GCTGCTTGAGATGTGCAAAdTdT Invitrogen
Table S2. Descriptions of supplementary database files. Database
# Title File Name
(size in KB) Description
1 Small molecule screen in HT22 cells
2000230s1.xls (837)
Raw data for each small molecule that we screened is listed along with an accompanying Simplified Molecular Input Line Entry Specification String (SMILES), concentration, and source. The data are presented in order of decreasing effect on WNT3A activation.
2 Raw data of siRNA screen in RKO cells
2000230s2.xls (989)
Data from distinct pools of siRNAs targeting 2,518 genes are listed in rows. Each pool was screened in quadruplicate, and mean and median effect of the pool on WNT3A-mediated activation of a β-catenin activated reporter is listed.
3 Median reporter activity of all genes in the siRNA screen
2000230s3.xls (173)
The effect on WNT3A-mediated β-catenin activated reporter activity for each gene was listed as an average of the median reporter activity of all individual pools.
4 Hit list for small molecule screen
2000230s4.xls (36)
Small molecules that increased BAR activity greater than 1.5-fold were listed with their accompanying SMILES string, and fold-change over WNT3A alone.
5 Hit list for siRNA screen
2000230s5.xls (233)
Targets of siRNA that increased BAR activity (negative regulators of Wnt/β-catenin signaling) are highlighted in red and targets that decreased BAR activity (positive contributors to Wnt/β-catenin signaling) are highlighted in green.
6 Binary generated from STITCH search of small molecule screen hits
2000230s6.xls (124)
The results from a STITCH search of the hits in the chemical screen are presented as a binary in which the contents of the adjacent columns are direct interactors.
7 Binary generated from STRING search of siRNA screen hits
2000230s7.xls (398)
The results from a STRING search of the hits in the siRNA screen are presented as a binary in which the contents of the adjacent columns are direct interactors.
8 List of randomly chosen genes that did not hit in the siRNA screen
2000230s8.xls (23)
A random list of genes was chosen as a negative control for association in the bioinformatic overlap. The effect on WNT3A-mediated β-catenin activated reporter activity for each gene was listed as an average of the median reporter activity of all individual pools.
9 Binary generated from STRING search of siRNA screen non-hits
2000230s9.xls (81)
The results from a STRING search of the random list of genes chosen from the siRNA screen are presented as a binary in which the contents of the adjacent columns are direct interactors.
10 List of overlaps between small molecule STITCH search and siRNA STRING search
2000230s10.xls (37)
Presented is a list of 108 protein targets that interact with a small molecule screen hit and interact with a siRNA screen hit. The 108 proteins are grouped in three ways: (1) proteins that interact with a small molecule screen hit and that are among the 445 siRNA screen hits; (2) proteins that interact with a small molecule screen hit and were not present in the siRNA screen; and (3) proteins that interact with a small molecule screen hit and did not hit in the siRNA screen.
11 List of overlaps between small molecule STITCH search and a STRING search of siRNA non-hits
2000230s11.xls (17)
Presented is a short list of proteins that interact with a small molecule screen hit and interact with one of the proteins present in a random list of genes present in our siRNA screen.
12 All attribute files and the Cytoscape binary utilized to generate Fig. 2 and Supplementary Database 13
2000230s12.xls (853)
This Excel file has four worksheets, which each contain a binary used in the construction of the cytoscape visualization of the bioinformatic overlap of the small molecule and siRNA screens. The first worksheet (ScoreAttribute) is an attribute file that shows whether a specific protein or compound increased, decreased, or did not affect BAR. The second worksheet (CompoundAttribute) is a binary that was used as an attribute file to specify which nodes in the Cytoscape file are compounds. The third worksheet (CombinedBinary) is the binary containing all the interaction data present in supplementary databases 6 and 7. Finally, the fourth worksheet (SRI_STI_Overlap_Attribute) is an attribute file used to group interactions according to whether they originated from STITCH or from STRING.
13 Cytoscape file showing all siRNA and small molecule screen hits and their overlap
2000230s13.cys (285)
This Cytoscape interactome is the file presented in fig. S1.
14 Cytoscape file for BTK protein-protein interaction map
2000230s14.cys (22)
This Cytoscape interactome is the file presented in fig. S3A.
15 Cytoscape file for CDC73 and BTK protein-protein interaction map
2000230s15.cys (25)
This Cytoscape interactome is the file presented in fig. S3B.
16 Affinity-purification-mass spectrometry data for BTK
2000230s16.xls (21)
Listed is the raw data from the BTK coimmunopurification mass spectrometry experiments. Prey are listed along the accompanying information showing the total number of peptides identified in all experiments, and the peptides identified in independent experiments (BTK, BTK-1, and BTK-2).
17 Affinity-purification-mass spectrometry data for CDC73
2000230s17.xls (20)
Listed is the raw data from the CDC73 coimmunopurification mass spectrometry experiments. Prey are listed along the accompanying information showing the total number of peptides identified in all experiments, and the peptides identified in independent experiments (eCDC73-Nalm6, GLUe_CDC73_B1, and GLUE_CDC73_D1).
18 Isolation of CDC73 peptides from 60 kD band from silver stain
2000230s18.xls (24)
Listed are the peptides identified from in-gel digestion and mass spectrometry analysis of the prominent 60 kD band observed on silver stains following CDC73 immunoprecipitation.