Supplemental Information

Oncogenic ETS proteins mimic activated RAS/MAPK signaling in prostate cells

Peter C. Hollenhorst, Mary W. Ferris, Megan A. Hull, Heejoon Chae, Sun Kim, and Barbara J. Graves

Supplemental Data

Figure S1

Figure S2

Figure S3

Table S1 (Provided as an Excel File)

Table S2

Table S3 (Provided as an Excel File)

Oligonucleotide primers used in this study

Supplemental References

Figure S1.Confirmation of ChIP-seq bound regions. (A) ChIP followed by quantitative PCR of a subset of ChIP-seq bound regions. Note that FLAG-ChIP results only show enrichment when the FLAG-tagged protein is present. ChIP enrichment is the mean and standard error of a ratio of the copy number of the indicated locus over the mean copy number of two negative control genomic regions in two independent ChIP samples. ChIP samples were independent of those used for ChIP-seq and are derived from the indicated cell lines with the indicated antibodies. Target loci are listed below in the format (ChIP-seq dataset/rank order) and oligonucleotide sequences are available in Supplemental Experimental Procedures. A: ETV1/1, B: ETV1/2437, C: ETV1/3506, D: ETV1/8376, E: ERG/1, F: ERG/981, G: ERG/1716, H: ETV4/1, I: ETV4/2, J: ETV4/629, K: ETV4/1984, L: ETV4/1132, M: ETV4/385, N: JUND/539, O: JUND/2176, P: JUND/1251, Q: JUND/3642. (B) Comparison of FLAG-ChIP bound regions in FLAG-ERG expressing RWPE-1 cells with regions identified by Yu et al. using ChIP-seq with an anti-ERG antibody in ERG-expressing RWPE-1 cells (Yu et al. 2010). The parentheses indicate the number of overlapping regions predicted by overlapping equally sized randomly selected regions.

Figure S2.JUND binds regions with ETS/AP-1 sequences. (A) ChIP enrichment of the PLAU enhancer from PC3 cells using the five indicated antibodies to AP-1 subunits. All antibodies were from Santa Cruz Biotechnology. (B) The frequency of distances between the ETS binding sequence AGGAA and the AP-1 binding sequence TGANTCA in regions occupied by both ETV4 and JUND in PC3 cells. The most frequent position is +6 and represents the sequence AGGAANTGANTCA.

Figure S3.Confirmation of microarray results by reverse-transcription followed by quantitative PCR. Expression levels of three genes down-regulated by U0126 (SDCBP2, CLDN1, and ARHGAP29) and two genes up-regulated by U0126 (KRT4 and IGFBP2) was measured in RNA derived from the indicated cell line. U0126 treatment where indicated was for six hours before RNA isolation. Results are the mean and standard error of two biological replicates that are independent from samples used for microarray. Primer sequences are available below.

Table S1. Provided as an Excel file. Provides chromosomal coordiates (hg18) for all bound regions for each ChIP-seq experiement.

Table S2. Genes downregulated by MEK inhibition in cell lines with BRAF mutations are also downregulated by MEK inhibition in RWPE-1 cells.

a Genes significantly downregulated by MEK inhibition in seven melanoma or colon cancer cell lines with V600EBRAF mutations (Pratilas et al. 2009). Of 48 genes reported by Pratilas et al., the 44 shown were assayed by microarray in this study.

b Log2 transformed ratio of the mean expression change in RWPE-1 cells treated with U0126 compared to untreated cells.

Table S3. Provided as an Excel file. Provides mean expression change and P value for each gene assayed by microarray.

Genea Log2 Ratiob

ARID5A - 0.38B4GALT6 - 0.43BXDC2 - 0.97BYSL - 0.90CCND1 - 1.42CD3EAP - 0.55CHSY1 - 0.38DDX21 - 1.25DUSP4 - 3.80DUSP6 - 4.44EGR1 - 0.78ELOVL6 0.19ETV1 - 0.90ETV4 - 0.46ETV5 - 2.03FOS - 1.41FOSL1 - 3.58GEMIN4 - 0.64GNL3 - 0.48GPR3 - 2.23GTPBP4 - 0.32HMGA2 - 2.81

Genea Log2 Ratiob

IER3 - 1.75IL8 - 0.57LIF - 1.28MAFF - 1.76MYC - 2.33PHLDA2 - 1.34PLK3 - 1.47POLR1C - 0.60POLR3G - 0.62PPAN - 1.52PPAT - 0.84PYCRL - 0.92RRS1 - 1.23SLC1A5 - 0.32SLC4A7 - 0.88SPRED2 - 1.38SPRY2 - 1.13SPRY4 - 3.86TNC - 0.76TNFRSF12A - 0.39WDR3 - 1.06YRDC - .39

OLIGONUCLEOTIDE PRIMERS USED IN THIS STUDY

Name Forward Primer Reverse PrimerA GCTCCGTGCCGTGCGTGTC CGGCCAGCGAGTCGATCGGB GGTGGGAGGCACGTAAGACC CGGAGCTCTTTTCCCTCTACC CTGTCATCAGGGATGTCACACC GAGCCTCAGCACACAACTTTGCD CACCTCATCGACAAGCTTCC AACGGCCGTGCATCATGACCE GCAGGCCGGAGGGAAAAGG CCTCAGACTGTCGGCACAACF CTTGGAGGGAAGATGAATCGG GAAATGGGCGACCGCAAGTCG CATTCGTGCTAGTTGGAGC CCTAACATTGCATCAGCTGGGH CCTCCTCAAACTTCCATATCTGC GGATTGCGTGACTCCTGGAGCI GCTGTCATCAGGAACTCATCC GTCAACAACTTCTCATCCCTGGJ CCTACACATAAGAGGAGCCC CCTCTGTAGAATAGGAATGACACK GGACACTTGACATGACACAACC GTATCTCATTAAGTCTGCTTGCCL CATTGTTTTGGAGTGAGCTCACG GCTTGGGATCTGTCTGTTTCCM GTATTATTGAGTGGGAAACTCTCC CAGGGTCTACTTCTAGGGAACCN GTATCTTGGCTCAAGGACAAGC GCATGGCCATGCAGTCTGCO CAGTCACTTCCCATTTCCTGC GTGTATGATTCCATGAAATGTCTCP ACAAGGCTGAGTTATGCTGACC GGTATGACGCACGGGGAAATGGQ CCATCCATTCCATCGATCTGC ATCCTTGAGGAAAGCAGGTCCPLAU ChIP GCTTCATAACGGTCTCCAAACC CCGTCATGATTCATGTTGCTCCPLAU expression

CTACACAAGGACTACAGCGC GCAGATGGTCTGTATAGTCCG

SDCBP2 GACGGGCAGAATGTTATCGG GATGGTCAGGGTGACAACGCLDN1 GCATGGTATGGCAATAGAATCG CAGTGAACAGAGCCTGACCARHGAP29

GGAATCAGAACGCAAGCAAAATGCG GGGATGCTGATTCAGCCTCTTGG

KRT4 GAGATCGAGAACATCAAGAAGC CTCACCTCGCTGCTCTGCIGFBP3 CTTCTCCTCCGAGTCCAAGC CTTCAGGTGATTCAGTGTGTCETV1 cloning

AGACTACCGGTATGGACTACAAGGACGACGACGACAAGGATGGATTTTATGACCAGCAAGTG

GTGACATTAATTAATTAATACACGTAGCCTTCGTTGTAGG

ETV5 cloning

AGACTACCGGTATGGACTACAAGGACGACGACGACAAGGACGGGTTTTATGATCAGCAAGTC

GTGACATTAATTAATTAGTAAGCAAAGCCTTCGGC

FLI1 cloning

AGACTACCGGTATGGACTACAAGGACGACGACGACAAGGACGGGACTATTAAGGAGGC

GTGACATTAATTAACTAGTAGTAGCTGCCTAAGTGTG

ERG cloning

AGACTGCGGCCGCATGGACTACAAGGACGACGACGACAAGGCCAGCACTATTAAGGAAGCC

GTGACATTAATTAATTAGTAGTAAGTGCCCAGATGAG

FEV cloning

AGACTACCGGTATGGACTACAAGGACGACGACGACAAGAGACAGAGCGGCGCCTCCCAGCC

GTGACATTAATTAACTAGTGGTAATGGCCCCCCAAGTG

ETS1 cloning

AGACTACCGGTATGGACTACAAGGACGACGACGACAAGAAGGCGGCCGTCGATCTCAAGC

GTGACATTAATTAATCACTCGTCGGCATCTGGCTTGACG

ETS2 cloning

AGACTACCGGTATGGACTACAAGGACGACGACGACAAGAATGATTTCGGAATCAAGAATATGGAC

GTGACATTAATTAATCAGTCCTCCGTGTCGGGCTGG

SPDEF cloning

AGACTACCGGTATGGACTACAAGGACGACGACGACAAGGGCAGCGCCAGCCCGGGTCTG

GTGACATTAATTAATCAGATGGGGTGCACGAACTGG

SUPPLEMENTAL REFERENCES

Pratilas CA, Taylor BS, Ye Q, Viale A, Sander C, Solit DB, Rosen N. 2009. (V600E)BRAF is associated with disabled feedback inhibition of RAF-MEK signaling and elevated transcriptional output of the pathway. Proceedings of the National Academy of Sciences of the United States of America 106: 4519-4524.

Yu J, Mani RS, Cao Q, Brenner CJ, Cao X, Wang X, Wu L, Li J, Hu M, Gong Y et al. 2010. An integrated network of androgen receptor, polycomb, and TMPRSS2-ERG gene fusions in prostate cancer progression. Cancer Cell 17: 443-454.