Structure/Function Analysis of Recurrent Mutations in SETD2 Protein Reveals a Critical and Conserved Role for a SET Domain Residue in Maintaining Protein Stability and Histone H3 Lys-36 Trimethylation*

The yeast Set2 histone methyltransferase is a critical enzyme that plays a number of key roles in gene transcription and DNA repair. Recently, the human homologue, SETD2, was found to be recurrently mutated in a significant percentage of renal cell carcinomas, raising the possibility that the activity of SETD2 is tumor-suppressive. Using budding yeast and human cell line model systems, we examined the functional significance of two evolutionarily conserved residues in SETD2 that are recurrently mutated in human cancers. Whereas one of these mutations (R2510H), located in the Set2 Rpb1 interaction domain, did not result in an observable defect in SETD2 enzymatic function, a second mutation in the catalytic domain of this enzyme (R1625C) resulted in a complete loss of histone H3 Lys-36 trimethylation (H3K36me3). This mutant showed unchanged thermal stability as compared with the wild type protein but diminished binding to the histone H3 tail. Surprisingly, mutation of the conserved residue in Set2 (R195C) similarly resulted in a complete loss of H3K36me3 but did not affect dimethylated histone H3 Lys-36 (H3K36me2) or functions associated with H3K36me2 in yeast. Collectively, these data imply a critical role for Arg-1625 in maintaining the protein interaction with H3 and specific H3K36me3 function of this enzyme, which is conserved from yeast to humans. They also may provide a refined biochemical explanation for how H3K36me3 loss leads to genomic instability and cancer.

The structural and functional similarities between SETD2 and Set2 provide an exceptional opportunity in which existing assays in Saccharomyces cerevisiae can be applied to investigate the functional consequences of SETD2 mutations reported in human cancer. In this work, we characterized cancer-associated SETD2 mutations that occur at evolutionarily conserved residues in functionally important domains (i.e. the SET and SRI domains). We discovered that a missense mutation in the SET domain of SETD2 (R1625C) altered the capacity of this mutant to engage H3, leading to reduced protein stability, and a complete loss of H3K36me3. Strikingly, the same mutation in yeast Set2 (R195C) resulted in an identical effect on H3K36me3 but not H3K36me1 or H3K36me2 levels (or biological outcomes associated with these lower methylation states). Further biological studies in human cells revealed that loss of H3K36me3 in the R1625C mutant leads to DNA repair defects, thereby revealing a greater understanding of how this recurrent mutation probably leads to a loss of SETD2 tumor-suppressive activity.

SETD2 and Set2 Share a High Degree of Structural and Sequence Homology at Their SET and SRI Domains-SETD2
and Set2 share significant structural and functional homology. SETD2 demonstrates strong sequence conservation at all of the annotated functional domains present in yeast Set2: AWS (associated with SET), 42%; SET (Su(var)3-9, Enhancer-ofzeste, Trithorax), 56%; PS (post-SET), 59%; coiled-coil, 33%; WW, 26%; and SRI (Set2 Rpb1-interacting), 35% (Fig. 1A). Given this similarity, we compared the structure of the SETD2 and Set2 SET domains to identify highly conserved residues for further study. The structure of the SET domain in SETD2 has been solved by crystallography (34), whereas the SET domain of Set2 was predicted here using I-TASSER (35)(36)(37)(38). When the predicted structure of the Set2 SET domain was aligned with the crystal structure of the SETD2 SET domain, the structures were strikingly similar (Fig. 1B). We then examined the conservation of amino acids previously reported to be mutated in human ccRCC (1,4,11,12) across six organisms (Homo sapiens, Drosophila melanogaster, S. cerevisiae, Mus musculus, Xenopus tropicalis, and Danio rerio). Seven of the nine ccRCC mutations occur at residues that are conserved across all model organisms (Fig. 1C). Additionally, three of these seven mutations occur in a region previously identified to act as the catalytic site for lysine methylation (13). One of these mutations, R1625C, is found in a location that is adjacent to the S-adenosyl-L-methionine (SAM) binding site in the structure and thus would be predicted to impact catalytic activity (Fig. 1B). This residue is the most common site of missense mutation reported in both CBioPortal (39,40) and COSMIC (41). The specific arginine to cysteine mutation is found in both glioma (5) and ccRCC (1). Significantly, mutation of the corresponding residue in S. cerevisiae is known to affect Set2 catalytic activity (42). Given its location and mutation frequency, we chose this mutation for further analysis.
We then examined the sequence and structural conservation of the SRI domain and the location of ccRCC-associated missense mutations. In contrast to the SET domain, primary sequence of the SRI domain is less conserved across model organisms (Fig. 1D). However, the aligned crystal structures of yeast (43) and human (44) SRI domains display structural conservation (Fig. 1E). In particular, the predicted site of SETD2 and RNAPII interaction was previously suggested to be the concave surface between ␣ helix 1 and ␣ helix 2 (44). The physical relationship of these helices appears conserved between Set2 and SETD2. We therefore selected the Arg-2510 residue for further study because this amino acid is recurrently mutated (R2510H and R2510L) in ccRCC (1,12) and is predicted to be important for SETD2-RNAPII interaction by in vitro peptide interaction assays (44).
SET Domain Mutation Destabilizes SETD2 in Cells-To establish a human cell system in which to study the function of SETD2 mutants, we generated SETD2-deficient cells (SETD2⌬). TAL effector nucleases (TALENs) (45,46) targeting exon 3 of SETD2 were introduced into two immortalized kidney cell lines (human SV40 immortalized proximal tubule kidney cells (HKC) (47) and 293T). Individual clones of TALENtreated cells were isolated, and loss of H3K36me3 was demonstrated by immunocytochemistry ( Fig. 2A). We verified inactivation of both alleles of SETD2 via Sanger sequencing. Representative allelic sequencing is shown (Fig. 2B).
We then exogenously expressed a truncated wild-type FLAG-tagged form of SETD2 (amino acids 1323-2564; tSETD2), which includes all known functional domains. The R1625C or R2510H mutants were generated in tSETD2. Relative to tSETD2 and R2510H, R1625C protein levels were reduced (Fig. 2C). R1625C mutant mRNA levels were also less abundant (Fig. 2D). We examined protein stability after treatment with the protein synthesis inhibitor cycloheximide. The R1625C protein demonstrated a significantly shorter half-life compared with that of wild type (Fig. 2E). In contrast, the halflife of the R2510H mutant was unchanged (Fig. 2E). These data suggest that the decreased protein level of the R1625C SET domain mutant results from both decreased RNA and a shortened protein half-life.
Histone H3 Lysine 36 Trimethylation Is Linked to SETD2 Mutational Status-We interrogated H3K36 methylation status in cells transiently transfected with either tSETD2 or the mutants R1625C and R2510H. Using immunocytochemistry, we found that transfection of tSETD2 resulted in global restoration of H3K36me3 (Fig. 2F), demonstrating that the N terminus is not required for catalytic activity of SETD2. Transfection of the R1625C (SET domain) mutant construct failed to restore H3K36me3. In contrast, expression of the R2510H SRI mutant globally restored H3K36me3.
We next examined the H3K36 methylation status by Western blotting analysis. Consistent with findings from immunocytochemistry, SETD2⌬ cells show complete loss of H3K36me3. Trimethylation was restored to wild-type levels by expression of either the tSETD2 or the SRI mutant. In contrast, the SET domain mutant failed to trimethylate H3K36 (Fig. 2G). Monomethylation (H3K36me1) and dimethylation (H3K36me2) were unaffected by SETD2 loss or expression of SETD2 variants (Fig. 2G). These results are in agreement with the findings that SETD2 is the exclusive H3K36 trimethyltransferase in mammalian cells.
We then asked whether expression of either tSETD2 or R2510H restored H3K36me3 to levels similar to those of wildtype cells at specific loci. H3K36me3 levels have been shown to increase along the gene body with preference for exons (48). Using ChIP-qPCR, we examined the H3K36me3 levels at multiple exons of two genes, CDK2 and MYC, which had previously been described (49). As expected, SETD2⌬ cells displayed low H3K36me3 levels at all sites (Fig. 2H). Expression of tSETD2 recapitulated the previously described pattern for H3K36me3 in wild-type cells (49) at both CDK2 and MYC, with higher signal at exons 5 and 6 relative to exon 1 in CDK2, and in exons 2 and 3 relative to exon 1 in MYC. Cells expressing the R1625C SET domain mutant displayed loss of H3K36me3 at levels similar to that of SETD2⌬ cells. Finally, expression of the R2510H mutant also showed greater signal at later exons, indicating that this point mutation restores not only the levels of methylation but also the spatial placement of these methyl marks on actively transcribed genes.
The SETD2 R1625C Variant Is Enzymatically Inactive in Vitro and Has Diminished Substrate Binding-Given that the R1625C SETD2 variant is associated with loss of H3K36me3 in cells, we asked whether the R1625C mutation disrupts the methyltransferase activity of SETD2 in vitro. To do this, we expressed and purified from bacteria a wild-type or R1625C mutated fragment of SETD2 (residues 1345-1711) containing the SET domain. Both the wild-type and the R1625C SET domain constructs yielded soluble proteins that were Ͼ90%  (yellow). The N terminus is marked in green, the C terminus is marked in pink, and residues mutated are shown as sticks. C, partial SET domain sequence alignment across multiple species. Amino acids 1612-1673 of the human SET domain (amino acids 1550 -1667) are shown. Residues mutated in ccRCC are shown in red and marked with an asterisk. The arrow indicates Arg-1625, the residue mutated for study. The black box indicates residues previously shown to be an important catalytic site. Residues that are conserved across species are indicated in green. D, SRI domain sequence alignment across multiple species. Residues mutated in ccRCC are in red and marked with an asterisk. The arrow indicates Arg-2510, the residue mutated for study. Residues that are conserved across species are indicated in green. E, alignment of the human SRI domain crystal structure (blue) with the yeast SRI domain crystal structure (yellow). The N terminus is marked in green, the C terminus is marked in pink, and residues mutated are shown as sticks.
pure as assessed by SDS-PAGE (Fig. 3A). Methyltransferase activity was then assessed using a radiometric assay with chicken oligonucleosomes as the substrate. Whereas wild-type SETD2 displayed robust activity, the R1625C variant displayed little enzymatic activity over the no enzyme control (Fig. 3B).
We next sought to determine why the R1625C variant is catalytically inactive. We first considered whether this mutation results in a misfolded protein, thereby inactivating the SET domain. We compared the CD spectra of the wild-type SETD2 with the R1625C variant. The CD spectra in the low UV range (185-260 nm) of the wild type and the R1625C variant were nearly indistinguishable, suggesting that the R1625C substitution does not alter the secondary structure of the SET domain (Fig. 3C). To determine whether the R1625C variant alters the thermal stability of the SET domain, we monitored the CD signal at the 207-nm peak over a temperature range from 20 to 95°C. Both the wild type and the R1625C variant showed highly similar thermal melt curves with a melting temperature (T m ) of ϳ55°C (Fig. 3D). Together, these results suggest that the loss of catalytic activity observed for the R1625C variant is not due to protein misfolding or reduced thermal stability.
Structural analysis of the SETD2 SET domain shows that Arg-1625 is positioned within the active site, opposite the SAM binding pocket, and is located about 7 Å away from the sulfur group of S-adenosyl-L-homocysteine (SAH) (Fig. 3E). Although substitution of Arg-1625 with cysteine would not be expected to directly disrupt SAM binding, the Arg-1625 side chain engages in three hydrogen bonds with the backbone carbonyl oxygens of Ala-1617 and Thr-1618 (Fig. 3E). Substituting cysteine for Arg-1625 using in silico mutagenesis showed that every possibly cysteine rotamer would cause steric clashes. The cysteine side chain would not recapitulate the hydrogen bonding network of Arg-1625 when oriented in the same direction as the Arg-1625 side chain observed in the crystal structure (Fig.  3F). Although no structure of the SETD2 SET domain ternary complex containing histone H3 is available, the location of Arg-1625 in close proximity to, but opposite, the SAM binding pocket suggests that Arg-1625 may directly or indirectly engage H3 or may maintain local structural integrity that aids substrate binding.
To determine whether the R1625C variant has altered substrate binding, we performed peptide pull-down experiments

SETD2/Set2 Domain Mutations and H3K36 Methylation
using histone H3 peptides that were unmodified or were methylated at Lys-36. The pull-down experiments showed that the R1625C variant associated with all of the histone peptides to a lesser degree compared with the wild-type SETD2 SET domain, suggesting that the R1625C substitution weakens substrate binding (Fig. 3G). Taken together, our results suggest that the R1625C substitution impairs enzymatic activity by reducing substrate binding, which is probably a consequence of fine structural disturbances induced by loss of the Arg-1625 hydrogen bonding network within the active site.
Domain-specific Mutations in Yeast Set2 Separate Roles of H3K36 Methylation States-To further explore the functional significance of ccRCC-associated mutations, we took advantage of several well characterized phenotypic assays in S. cerevisiae. Using set2⌬ cells, which are devoid of all H3K36 methylation (42), we created strains that either contained vector alone or exogenously expressed either wild-type or mutated forms of Set2. Mutant forms of Set2 included the homologous SETD2 SET domain mutant (R195C), the homologous SETD2 SRI mutant (K663L), or a control SET domain mutant (H199L) previously characterized to disrupt both tri-and dimethylation while retaining monomethylation activity (42). As expected, Set2 loss resulted in the complete absence of mono-, di-, and trimethylation of H3K36, which was rescued upon the addition of wild-type SET2 (Fig. 4A). As shown previously, the H199L mutant only restored monomethylation. In contrast, whereas the K663L mutant restored all H3K36 methylation states, the R195C only restored H3K36 mono-and dimethylation. Intriguingly, the restoration of H3K36 mono-and dimethylation by the R195C mutant mimics the status of SETD2-deficient human cells (i.e. both have a selective loss of H3K36me3). Because the SETD2 R1625C mutant demonstrated decreased protein stability in human cells, we examined protein levels of the R195C mutant in the yeast cells. Following cycloheximide treatment, we observed decreased protein levels of the R195C mutant relative to wild-type Set2, particularly at 3 h post-treat- . F, in silico mutagenesis analysis (performed in PyMOL, Schrö dinger Inc.)). The distances between the R1625C thiol and the carbonyl oxygens of Ala-1617 and Thr-1618 were measured in PyMOL (yellow dashed lines). G, peptide pull-down assays comparing the binding of the wild type and the R1625C variant with the indicated histone H3 peptides. All peptides were biotinylated at the C terminus and were unmodified or were modified as indicated. Streptavidin-coated magnetic beads without peptide served as the negative control. Short (top) and Long (bottom) refer to exposure length. SEPTEMBER 30, 2016 • VOLUME 291 • NUMBER 40 ment. This effect was rescued by treatment with the proteosome inhibitor MG132 (Fig. 4B). This suggests that, as in humans, the R195C variant is less stable than wild type in yeast cells.

SETD2/Set2 Domain Mutations and H3K36 Methylation
Loss of Set2 has been implicated in various phenotypes in S. cerevisiae, including transcription elongation defects, cryptic initiation, and sensitivity to DNA-damaging agents (42). We asked whether the R195C Set2 mutant would be associated with any of these phenotypes. To examine transcriptional elongation, we performed a spotting assay in the presence of the transcription elongation inhibitor 6-azauracil (6-AU). This assay has been used previously to assay for the presence of transcriptional elongation defects in yeast (16). As expected, wild-type yeast were sensitive to 6-AU (200 g/ml), whereas set2⌬ cells were resistant to this drug (Fig. 4C) (16). Although expression of wild-type SET2 restored sensitivity to 6-AU, the H199L mutant did not. The R195C and K663L mutants behaved similar to wild-type Set2. These data suggest that H3K36me2 is primarily responsible for the sensitivity to inhibitors of transcriptional elongation.
Cryptic initiation has been previously associated with Set2 loss (18). We therefore assessed the effects of our Set2 muta-tions in a cryptic transcription reporter assay. This assay monitors the growth of yeast cells that contain the HIS3 gene with a cryptic start site that exists in the FLO8 gene. Importantly, the cryptic start site is out of frame when the 5Ј promoter is used, and a functional transcript is only produced if the 3Ј cryptic start site is utilized. In this setting, cryptic transcription results in expression of HIS3, which can restore growth in medium lacking histidine. Consistent with previous results (18), loss of Set2 permits growth in the absence of histidine (Fig. 4D). No growth was observed in the cells expressing the R195C or K663L mutants. However, cell growth occurred in the presence of the H199L mutant (Fig. 4D). These data indicate that trimethylation is dispensable for preventing cryptic initiation, whereas dimethylation is required to suppress this phenotype.
Recent studies have demonstrated that yeast lacking Set2 cannot properly activate the DNA damage checkpoint (42,50).
To determine whether the RCC-associated SET domain mutation impacts this phenotype, we assessed the impact of the Set2 point mutants on growth in the presence of phleomycin, a double strand break-inducing agent. As expected, set2⌬ cells dis-

SETD2/Set2 Domain Mutations and H3K36 Methylation
played increased sensitivity to phleomycin relative to Set2 wildtype yeast (Fig. 4E). set2⌬ cells expressing either wild-type Set2 or the R195C or K663L mutants showed sensitivity similar to that of the wild-type rescue. However, yeast expressing the H199L mutant showed a level of sensitivity similar to that of the set2⌬ cells (Fig. 4E). Taken together, these data indicate that the cellular phenotypes associated with Set2 loss in yeast are associated with H3K36me2 and that H3K36me3 is dispensable for these activities (summarized in Fig. 4F).
Human Kidney Cells Display an H3K36 Trimethylationdependent DNA Damage Response-Because of the exclusivity of SETD2 in mediating trimethlyation in human cells, we studied phenotypes similar to those examined in yeast in human cells that express ccRCC-relevant mutants. We first examined the effect of the transcriptional elongation inhibitor 5,6-dichlorobenzimidazole 1-␤-D-ribofuranoside (DRB) on cell survival. DRB inhibits CDK9, which results in premature termination of transcription (51). Assessing viability at 12 h time points for 3 days, we observed that DRB-associated toxicity did not differ between SETD2 wild-type and SETD2⌬ cells (Fig.  5A).
Several recent studies have examined the effect of SETD2 loss in human cells on the response to DNA damage (22)(23)(24)(25)(26). To further explore the role of H3K36me3 in the DNA damage response, we irradiated HKC to 2 Gy and then performed immunofluorescence for ␥H2A.X, a marker of DNA damage. At 30 min postirradiation, ␥H2A.X foci were seen in all cell types at similar levels (Fig. 5B). In untransfected and in control transfected wild-type cells, the number of foci greatly decreased by 1 h and largely resolved by 4 h. However, in SETD2⌬ cells, the number of ␥H2A.X foci remained elevated at both 1 and 4 h. Expression of tSETD2 in SETD2⌬ cells led to resolution of foci at time points similar to wild-type cells. Cells expressing the SRI mutant, R2510H, also showed rapid foci resolution. However, foci persisted in cells expressing the R1625C mutant (Fig. 5B). Quantification of these results demonstrated that both SETD2⌬ cells and R1625C-expressing cells had a significantly higher percentage of cells with greater than 10 foci compared with the other conditions (Fig. 5C).
We quantified ␥H2A.X by immunoblotting, enabling us to account for changes in total protein and histone levels. These studies were performed in 293T cells, as additional validation of results in HKC. As observed with HKC, regardless of SETD2 status, 293T cells showed increased total ␥H2A.X at 30 min postirradiation (Fig. 5D). By 4 h, ␥H2A.X levels returned to baseline in cells with H3K36 trimethylation (WT, tSETD2, and R2510H). However, elevated levels of ␥H2A.X were observed in cells lacking H3K36me3 associated with SETD2 loss or R1625C expression. Finally, we examined the effect of irradiation on viability using a colony formation assay. The fraction of surviving colonies did not differ between SETD2 wild-type and SETD2⌬ cells (Fig. 5E). Overall, these findings demonstrate that SETD2-mediated H3K36me3 is coupled to the efficient resolution of double strand breaks. Corresponding to results in yeast, loss of trimethylation is not associated with enhanced sensitivity due to inhibition of transcriptional elongation or from DNA damage.

Discussion
In an effort to explore the function of missense mutations identified in human cancers, we examined several recurrent mutations that occur at evolutionarily conserved residues in yeast and human cell lines. The striking homology between SETD2 and Set2 creates an opportunity to compare the effects of mutations while taking advantage of the strengths of each model system. Indeed, a recent study (52) also modeled cancer mutations in yeast, highlighting the utility and power of yeast to be a robust model system to aid in human protein analyses. In this paper, we investigated how two highly conserved SETD2 residues that are commonly mutated in cancer affect the functions of this enzyme. Limited studies have explored the potential roles that SETD2 loss may play in cancer development. We found that mutation of the SET domain, but not the SRI domain, resulted in effects in the human and yeast assays. Specifically, we identified R1625C as a critical mutation that impacted SETD2 enzymatic activity and protein stability in cells, an effect also noted when this mutation was modeled in yeast Set2. Loss of H3K36me3 in human cells also led to defects in DNA repair, indicating a potential mechanism by which SETD2 functions as a tumor suppressor. To our knowledge, this study is the first to dissect the impact of cancer-associated mutations in SETD2 and further validate using yeast as a model to complement human cell analyses.
A key discovery emerged from the study of the R1625C mutant. In contrast to the human R1625C variant, which was catalytically inactive in vitro, the homologous substitution in yeast Set2 led to an uncoupling of di-and trimethylating activities. This suggests that this residue may be important for the specific trimethylating activity of the enzyme. Moreover, many substrate binding interactions govern stability. Thus, the reduced protein stability (in the absence of other thermal instability) may reflect a structural role that differentiates mono-, di-, and trimethylating activity. Because of this unique mode of regulation, the R195C mutation allowed us to examine the functions specifically associated with the trimethylated state of H3K36 in cells (i.e. impaired transcriptional elongation, cryptic initiation, and impaired survival in the face of DNA damage). Consistent with other reports that examined cryptic initiation (19,53), we found that H3K36me3 is dispensable, whereas H3K36me1/me2 is required to suppress cryptic initiation as well as for transcription elongation and DNA damage survival phenotypes. In contrast, the SET domain mutation in SETD2 had a similar impact on H3K36me3 levels but resulted in a clear DNA damage response phenotype.
These studies offer a rationale for differences in observed phenotypes in SETD2 deficient human cells associated with Set2 loss in yeast, including cryptic initiation and impaired transcriptional elongation. However, impaired response to DNA damage, as we observed, has been reported for both mammalian systems and Set2 in yeast, linking this feature with H3K36 trimethylation. The ␥H2A.X results suggest that resolution of DNA strand breaks in human cells requires H3K36me3. (Due to the presence of multiple H3K36 dimethylating enzymes in mammalian cells, absence of H3K36me2 is rarely encountered in human models.) H3K36me2 is induced by ionizing radiation and improves association of early DNA repair components with an induced break and improved repair by non-homologous end joining in human cells (27). Although our data show that the resolution of strand breaks, as measured by clearance of ␥H2A.X foci, was delayed in the absence of H3K36me3, our data also shows that H3K36me3 loss does not affect viability after radiation in mammalian cells. Thus, loss of dimethylation may convey a sensitivity to DNA damage that is not present in the absence of SETD2 trimethylating activity. It is important to consider that multiple factors may influence cell death in transformed cells. However, these distinct findings in yeast and mammalian systems indicate a complex level of regulation of DNA repair mediated by the histone code at H3 lysine 36. Multiple studies have concluded that the loss of SETD2 confers a variety of types of genomic instability, ranging from microsatellite instability to impairment of non-homo-  (2 Gy) in HKC wild-type or SETD2-inactivated cells transfected with GFP, tSETD2, R1625C mutant, or R2510H mutant. The nuclei were visualized by DAPI staining. Representative immunofluorescence images are shown. Scale bar, 10 m. At least five fields were taken from each condition, and four independent experiments were performed. C, percentage of HKC with Ͼ10 ␥H2A.X foci/cell at 0, 0.5, 1, and 4 h after irradiation (2 Gy). Error bars, S.E. *, p Ͻ 0.05; **, p Ͻ 0.01 (two-sided t test, comparison with HKC wild type). D, immunoblotting analysis for the expression of ␥H2A.X and H3 (loading control) from the 293 wild-type or SETD2-inactivated cells transfected with GFP, tSETD2, SET domain R1625C mutant, or SRI domain R2510H mutant. The cells were irradiated by 2 Gy, and histones were acid-extracted at various time points. Shown is average quantification of ␥H2A.X/H3 after irradiation in three independent Western blots. Error bars, S.E. *, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.001 (two-sided t test, comparison with 293 wild type ϩ GFP). E, radiation foci formation assay. The surviving fraction represents ratio of treatment (37,75,150, and 300 rads) to 0 rad comparison. Error bars, S.D. of triplicate results.
Through modeling disease-relevant SETD2 mutations, we were able to gain insight into H3K36me3 function and dissect the roles of H3K36 dimethylation and trimethylation. Future studies will further explore the roles of the SETD2 SRI domain and examine the effects of additional mutations and will further define the role of SETD2 loss in the development of kidney cancer and other tumor types.

Experimental Procedures
Modeling SETD2 and Set2-The primary protein sequences of Set2 from S. cerevisiae and SETD2 from H. sapiens were compared via BLAST alignment analysis, and the percentage of homology between annotated domains was determined using the percentage overlap of the BLAST-aligned regions. The primary sequences of the SET and SRI domains of the enzyme responsible for H3K36 methylation from H. sapiens, S. cerevisiae, X. tropicalis, D. melanogaster, D. rerio, and M. musculus were aligned using ClustalOmega (56) and annotated with reported SETD2 mutations in ccRCC (1,4,11,12). The structures of the SETD2 SET domain (34), SETD2 SRI domain (44), and Set2 SRI domain (43) have been reported previously. To predict the structure of the yeast SET domain, the amino acid sequence (UniProtKB, P46995) was submitted to I-TASSER using the default parameters (35)(36)(37)(38). The ribbon structures were aligned using the align command in the PyMOL Molecular Graphics System (57).
Mammalian Cell Lines Transfections and Phenotypic Assays-293T human embryonic kidney cells were generously provided by Dr. Jenny Ting (University of North Carolina, Chapel Hill, NC). The SV40 transformed human renal tubule epithelial cell line (referred to as HKC) was obtained from Dr. Lorraine Racusen (Johns Hopkins University, Baltimore, MD) (47). A pair of vectors containing TALENs targeting exon 3 of SETD2 was generated using the REAL (restriction enzyme and ligation) assembly method. Component plasmids were obtained from Addgene. Briefly, target sites were selected, and TALENs were designed using Zifit, followed by assembly (46). The TALEN target sequences are 5Ј-TCATGTAACATCCAG-GCC-3Ј and 5Ј-ACAGCAGTAGCATCTCCA-3Ј.
An expression construct containing an N-terminal truncated form of SETD2 (amino acids 1323-2564; tSETD2) was sequence-optimized for expression in human cells, tagged with the FLAG sequence on the C terminus, and synthesized by Life Technologies, Inc. tSETD2 was specifically used because it models the yeast protein in domain structure, and expression of full-length SETD2 was technically unfeasible. tSETD2 was subcloned into the pINDUCER20 vector (58). Disease-relevant SETD2 mutations were introduced into the tSETD2 pINDUCER20 construct using the QuikChange II site-directed mutagenesis kit according to the manufacturer's instructions (Agilent Technologies). Mutations were verified through direct DNA sequence analysis.
293T and HKC human renal cells were transfected with the TALEN constructs, tSETD2 construct, and mutation constructs using the Amaxa cell line Nucleofector kit V (Lonza). For the protein stability assay, cycloheximide (100 ng/ml) was applied to cells 72 h post-transfection. For the DRB transcription inhibition assay, 1000 cells/well were plated in triplicate on a 96-well plate and treated with 100 M DRB for 72 h, with viability being measured every 12 h by Cell Titer Glo (Promega).
Immunoblotting Analysis-To isolate mammalian cellular proteins, cells were lysed in mammalian protein extraction reagent (Pierce) supplemented with Complete Mini Protease Inhibitor Mixture (Roche Applied Science). Histones were extracted using an overnight acid extraction protocol (Abcam). For yeast immunoblots, asynchronously grown mid-log (0.6 -0.8 OD) phase cultures were lysed in SUMEB buffer using glass bead methods described on the Gottschling laboratory website.
Immunocytochemistry-Cells were fixed with 4% paraformaldehyde for 15 min and permeabilized using 0.25% Triton X-100 in PBS. Endogenous peroxidase activity was blocked by incubation in 1% H 2 O 2 . Cells were then blocked in 5% bovine serum albumin, followed by incubation in primary antibody. The Vectastain ABC kit (PK6101, Vector Laboratories) was used for secondary antibody and HRP conjugation, followed by the DAB peroxidase substrate kit (SK-3100, Vector Laboratories) and hematoxylin staining.
Quantitative RT-PCR-Total RNA was extracted using the Qiagen RNeasy minikit. cDNA was made from total RNA using random primers and Superscript II reverse transcriptase reagents (Invitrogen). Primers used for RT-PCR are listed in Table 1.
Expression and Purification of Human SETD2-An E. coli codon-optimized synthetic gene corresponding to human SETD2 (UniProtKB ID Q9BYW2) residues 1345-1711 followed by a stop codon was cloned into the pGEX-6P-2 expression vector (GE Healthcare) using standard procedures. The protein was expressed in soluBL21 (DE3) (Amsbio) cells by growing cells in Terrific Broth II medium (MP Biomedicals) at 37°C until an A 600 of ϳ0.6 and then chilling the cells for 30 min at 4°C before inducing them with 1 mM isopropyl 1-thio-␤-Dgalactopyranoside in the presence of 25 M ZnCl 2 for 20 h at 16°C. Cells were harvested by centrifugation, and pellets were flash-frozen in liquid nitrogen. For purification, thawed cell pellets were resuspended in binding buffer (50 mM Tris, pH 7.3, 300 mM NaCl, 4 mM DTT, 10% glycerol, and 1 M ZnCl 2 ) supplemented with 1 Complete mini-EDTA-free protease inhibitor tablet (Roche Applied Science), 0.1 mM PMSF, 0.5 mg/ml chicken egg lysozyme, and 0.2% (v/v) Triton X-100 and incubated on ice for 45 min and then lysed with sonication and clarified by centrifugation. Clarified lysates were diluted 1:2 with binding buffer and applied to a 5-ml glutathione-agarose gravity flow column (pre-equilibrated with 10 column volumes of binding buffer) at a flow rate of ϳ0.5 ml/min at 4°C. The bound protein was washed with 10 column volumes of binding buffer and then eluted from the column with 35 ml of elution buffer (50 mM Tris, pH 8.0, 300 mM NaCl, 4 mM DTT, 10% glycerol, and 10 mM reduced L-glutathione). The eluted protein was mixed with Precision Protease (GE Healthcare) and exhaustively dialyzed against binding buffer, without ZnCl 2 , over the course of 20 h at 4°C. The cleaved protein sample was applied to a pre-equilibrated 5-ml glutathione-agarose gravity flow column at a flow rate of ϳ0.5 ml/min at 4°C, and the flow-through was collected and concentrated using an Amicon-Ultra 15 concentrator (Millipore). The Bradford assay and SDS-PAGE analysis were used to determine the quantity and purity of the protein samples, respectively. The SETD2 R1625C mutant was generated by site-directed mutagenesis using the QuikChange kit (Agilent) and expressed and purified as described above. Note that a small amount of GST-SETD2 WT and R1625C was not treated with Precision Protease but was extensively dialyzed against binding buffer and then used for peptide pull-down experiments (see below).
Histone Methyltransferase Assays-Histone methyltransferase assays were performed by incubating wild type SETD2 or the R1625C variant at a final concentration of 500 nM with 1 g of chicken oligonucleosomes (EpiCypher) and 1 Ci of [ 3 H]AdoMet (PerkinElmer Life Sciences) in a buffer containing 50 mM HEPES, pH 8.0, 150 mM NaCl, 2.5 mM MgCl 2, 1 M ZnCl 2 , and 2.5% glycerol for 16 h at room temperature (total reaction volume was 20 l). The reactions were quenched with 0.5% TFA and then spotted onto Whatman filter paper, airdried, and washed four times with ϳ200 ml of a sodium bicarbonate (pH 9.0) solution. Then they were air-dried again and added to liquid scintillation vials containing 5 ml of Ultima Gold F (PerkinElmer Life Sciences). Samples were counted for 1 min each using an all-purpose Beckman Coulter liquid scintillation counter in 3 H mode. A reaction without enzyme was used as the negative control and to determine background counts.
CD Spectroscopy-For CD experiments, proteins were exhaustively dialyzed into a buffer containing 20 mM sodium phosphate (pH 7.0), 150 mM sodium fluoride (NaF), and 0.2 mM tris(2-carboxyethyl)phosphine at 4°C. CD spectra were collected using a 0.1-cm quartz cuvette and a Chirascan Plus instrument (Applied Photophysics Inc.) at 20°C over the wavelength range 185-260 nm with a step size of 0.5 nm. A sample of the buffer was collected over the same wavelength scan, and absorbance values were subtracted from the final data sets. Each sample was scanned three times, and the final plots represent the average scan with the CD signal (in millidegrees) converted to molar ellipticity. For thermal melt curves, the CD absorbance at 207 nm was collected over the temperature range from 20 to 95°C with 1°C temperature ramping and a temperature tolerance range set to 0.2°C. Proteins were diluted to 0.25 mg/ml for all CD data collection (protein stock concentrations determined by A 280 ).
Peptide Pull-downs-A total of 50 pmol of GST-tagged wild type SETD2 or the R1625C variant was incubated with 500 pmol of each biotinylated histone peptide for 1 h at 4°C in peptide binding buffer (50 mM Tris, pH 8.0, 300 mM NaCl, 0.1% Nonidet P-40) supplemented with 2 mM DTT and 1 M ZnCl 2 . Following incubation, the protein/peptide mixture was incubated with streptavidin-coated magnetic beads (Pierce), preequilibrated with peptide binding buffer, for an additional 1 h at 4°C. The beads were washed three times with peptide binding buffer, and bound complexes were eluted with 1ϫ SDS loading buffer, resolved via SDS-PAGE, and transferred to a PVDF membrane. The membrane was probed with anti-GST antibody diluted to 1:4000 in 5% BSA in PBS-T. The peptides con-

SETD2/Set2 Domain Mutations and H3K36 Methylation
tained the budding yeast histone H3 residues 27-45 and were mono-, di-, or trimethylated at Lys-36. In this region, the human and budding yeast H3 sequences differ by an Ala to Ser substitution at residue 31 and by an Arg to Lys substitution at residue 42.
Yeast Growth Assays-Parental yeast strains were transformed with the indicated plasmids and were grown to saturation in appropriate selection medium. Saturated cultures were diluted to an A 600 of 0.5 and 5-fold serially diluted and plated with or without 6-AU or plated with or without phleomycin; pictures were taken 2-3 days after spotting. Similarly, strains with an integrated cryptic initiation cassette (as shown in Fig. 4) were serially diluted and plated on ϪURA-HIS plates with or without galactose for 3 days to detect growth. Growth on ϪURA acted as a control for equivalent growth for all of the strains.
Immunofluorescence Staining for ␥H2A.X-HKC were cultured for 16 -18 h followed by 2-Gy radiation (RS 2000 Biological Research Irradiator), fixed for 15 min in 4% paraformaldehyde, washed with cold PBS, and permeabilized (0.25% Triton X-100 in PBS) for 10 min. After blocking with 1% hydrogen peroxide and then 5% BSA, cells were incubated with anti-␥H2A.X (1:500) at 4°C overnight. Cells were washed using PBST and incubated with goat anti-rabbit IgG (1:500) Cy5 for 1 h at room temperature. After washing, cells were counterstained using DAPI. Fluorescence signals were visualized using confocal microscopy (LSM 700, Zeiss), and the number of foci per cell was analyzed using Zen (LSM 700, Zeiss). 5 images/ coverslip (total of 15 images) were collected in three independent experiments. For the radiation colony formation assay, cells were diluted to a single cell suspension, and 300 cells were plated on a 10-cm plate. Plates were irradiated at 0, 37, 75, 150, and 300 rads; allowed to grow for 10 days; and then stained with crystal violet. Colonies were counted manually.