SUPPLEMENTARY INFORMATION

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1 (Supplementary Methods and Materials) GST pull-down assay GST-fusion proteins Fe , and Fe were expressed in BL21 bacterial cells and purified with glutathione-agarose beads (Sigma). For Fe65 fusion proteins, the eluates were pooled and concentrated using an Amicon Ultra 30 kda MWC spin concentrator (Millipore). Approximately 20ug GST fusion proteins were incubated with 2ug recombinant H2AX protein (Millipore) or HeLa histones (Millipore) for 1 hour and protein complexes were collected on glutathione-agarose beads and evaluated by western blotting. RNA Isolation and RT-PCR Cells were rinsed in PBS and RNA was isolated following the manufacturer protocol for the RNeasy Kit (QIAGEN). First-strand cdna synthesis from total RNA template was performed with Invitrogen Superscript III cdna Synthesis System, followed by SYBR green qpcr amplification. Normalization was performed using specific amplification of GAPDH, and PCR reactions were performed in triplicate for each biological duplicate experiment. PpoI DSB ChIP protocol The inducible ER-I-PpoI expression plasmid [20] was transfected into HEK293 cells using Lipofectamine 2000 (Invitrogen). Cells were cultured for 48 hours before adding 4- OHT (Sigma, final concentration at 1µM) to induce the nuclear localization of ER-I-PpoI protein. Cells were then collected at indicated time points (0, 3.5, 6, and 10 hours of 4- OHT treatment) for ChIP assay. The antibodies used for ChIP are anti-!h2ax (Upstate) and anti-eya3 1

2 Supplemental Figure 1. TUNEL staining of transverse sections from Eya1 -/- and Eya1 +/+ e10.5 mouse embryos demonstrates increased apoptotic nuclei specifically in knockout animals. Supplemental Figure 2. sirna knockdown of Eya1, Eya3, and Fe65 in 293T cells as assessed by quantitative RT-PCR demonstrates effective loss of message in comparison to a nonspecific control sirna. 2

3 Supplemental Figure 3 293T human embryonic kidney cells depleted for Eya1/3 using specific sirnas displayed increased apoptotic response to 5Gy IR by TUNEL staining. Cell counts were performed on TUNEL stained cells co-stained with DAPI in triplicate to identify the proportion of TUNEL-positive nuclei. Basal level of apoptosis under these conditions was 1.0% TUNEL-positive/total nuclei. Bar graphs represent mean +/- SEM of fold apoptotic cells normalized to control sirna from triplicate samples. * p-<.05. Supplemental Figure 4 HEK293 cells were transfected with ER-I-PpoI, and 48 h after transfection cells were treated with 4-OHT for the indicated times, then fixed and used for ChIP analyses using the indicated antibodies and primers approximately 8.9Kb downstream to the I-PpoI cut site on human chromosome 1. Line graphs represent mean +/- SEM of percent input from triplicate samples. * p- <.05. 3

4 Supplemental Figure 5 Tyrosine-phosphorylated H2AX exists in 293T cells and bovine histone extract. H2AX protein was immuoprecipitated and subjected to western blotting with an antiphosphotyrosine antibody. Supplemental Figure 6. H2AX tyrosine phosphorylation shows no significant change in response to Eya knockdown in untreated 293T cells. Cells were transfected with specific sirna and then harvested for immunoprecipitation of H2AX followed by western blot with anti-phosphotyrosine (bottom panel) or anti-h2ax (top panel). Lane designations: 1) untransfected, 2) control sirna, 3) sieya1, 4) sieya3, 5) sieya1 + sieya3. 4

5 Supplemental Figure 7 Rescue of endogenous Eya1 function by co-transfection of human sirna and murine wild type or enzymatically inactive muant Eya1 constructs for 48h in 293T human embryonic kidney cells reveals that loss of H2AX phosphotyrosine mark in response to DNA damage (5Gy IR) is dependent on Eya activity. Supplemental Figure 8. Time course comparing level of serine phosphorylation of FLAG-H2AX with FLAG-H2AX Y142F in 293T cells treated with 10Gy IR. Immunoprecipitation with anti-flag was performed followed by western blotting with anti-!-h2ax. Percentages represent band intensity for H2AX Y142F over H2AX wild type for each time point. 5

6 Supplemental Figure 9. Peptide pull-down for MDC1. Nuclear extract from irradiated 293T cells was incubated with synthetic peptides of the H2AX tail (AA ) bearing modification or mutation at S139 and/or Y142 immobilized on beads and resulting pulldown products were analyzed by western blot. Lane designations: 1) input, 2) beads only, 3) biotin S*-Y, 4) biotin S*- Y*, 5) biotin S Y, 6) biotin S* - Y142A. * designates phosphorylation. 6

7 Supplemental Figure 10. Peptide competition assays. Synthetic biotinylated peptides of the H2AX tail bearing phosphorylation at S139 or S139 and Y142 were affixed to beads and incubated with nuclear extract from irradiated 293T cells either in the presence or absence of free peptide (0.5mg) bearing identical phosphorylation marks. Resulting pull-down products were analyzed by western blotting. 7

8 Supplemental Figure 11. (A) FLAG-H2AX interacts with MDC1 in response to DNA damage (10Gy IR) in 293T cells by co-immunoprecipitation. (B) Co-transfection of sirna to Eya3 with FLAG H2AX results in a dramatic loss of H2AX/MDC1 interaction in response to DNA damage by co-immunoprecipitation. 8

9 Supplemental Table 1. Nuclear SH2 or PTB-domain containing factors assessed for interaction with the tyrosine-phosphorylated tail of H2AX by peptide pull-down assay. Additionally, the listed nuclear tyrosine kinases were assessed for H2AX-interaction by co-immunoprecipitation. 9

10 Supplemental Figure 12. (A) Myc-tagged PTB2 domain of Fe65 was seen to be sufficient to interact with H2AX in 293T cells treated with 30uM Etoposide to induce apoptosis by co-immuoprecipitation. (B) GST pull-down using H2AX from purified HeLa histones (Millipore) demonstrates a direct interaction between H2AX and the PTB2 domain of Fe65, not the PTB1 domain. Input lane (lane 1) represents 100% of the H2AX protein used for incubations as compared to GST pulled down protein (lanes 2-4). (C) Anti-phosphotyrosine western blot of H2AX from HeLa histones (Millipore) (Lane 1) in comparison to recombinant H2AX (Millipore) (Lane 2). 10