dna double - stranded breaks ( dsbs ) are potentially lethal lesions if unrepaired , and their misrepair can give rise to genome instability , a hallmark of cancer ( jeggo and lavin , 2009 ) . to maintain genome stability in response to such lesions , cells employ either homologous recombination ( hr ) or nonhomologous end - joining ( nhej ) pathways to repair dsbs . hr is initiated by 5 end resection to generate a 3 single - stranded dna ( ssdna ) overhang . resection is a two - step process initiated by removing a short oligonucleotide through the activities of the mre11-rad50-nbs1 ( mrn ) complex and ctip . extensive resection is performed by exo1 or dna2-blm in conjunction with replication protein a ( rpa ) , which binds ssdna and removes secondary structures ( sugiyama et  al . , 1998 , symington and gautier , 2011 ) . this , in turn , leads to rad51 nucleofilament formation , which promotes strand invasion of a homologous chromatid , leading to accurate repair ( heyer et  al . , the broken ends are rapidly bound and protected by the ku70/ku80 heterodimer , which acts as a platform to recruit the dna - pk catalytic subunit ( dna - pkcs ) . damaged ends are then processed and subsequently joined by the ligase 4 ( lig4 ) , xrcc4 , xlf complex in a template - independent manner , which can lead to inaccurate repair ( lieber , 2010 ) . dsbs may also be repaired through alternative end - joining pathways , such as microhomology - mediated end - joining ( mmej ) , which do not require ku or lig4 . like hr , mmej is initiated by resection , and end - joining is mediated through annealing of short direct repeats of microhomology . mmej leads to deletions and is frequently associated with chromosomal rearrangements ( mcvey and lee , 2008 ) . dsb repair is further facilitated through chromatin modification by chromatin remodeling complexes , by incorporation of histone variants , and by histone modification ( smeenk and van attikum , 2013 ) . histone h3k36 dimethylation has recently been proposed to facilitate nhej , where metnase ( setmar ) directly mediates h3k36 dimethylation near the break site , leading to recruitment and stabilization of nbs1 and ku70 ( fnu et  al . histone h3k36 is also trimethylated , which in mammalian cells is performed uniquely by the setd2/hypb methyltransferase ( edmunds et  al . , 2008 ) . h3k36me3 is associated with transcriptional elongation and is found in gene coding regions , peaking at 3 ends ( edmunds et  al . , 2008 ) . setd2-dependent h3k36 trimethylation facilitates a number of processes within the cell , including splicing , repression of intragenic transcripts , and chromatin accessibility ( li et  al . setd2 is also mutated in a number of cancer types , including breast , lung , acute lymphoblastic leukemia , clear cell renal cell carcinoma , and glioma , supporting its role as a tumor suppressor ( al sarakbi et  al . , 2009 , dalgliesh et  al . , 2010 , fontebasso et  al . , 2013 , newbold and mokbel , 2010 , zhang et  al . , 2012 ) . setd2-dependent h3k36 trimethylation has recently been shown to regulate dna mismatch repair ( li et  al . , 2013 ) . however , setd2-deficient cancers exhibit a wide range of mutations , including insertions , deletions ( indels ) , and chromosomal aberrations ( sato et  al . , 2013 , zhu et  al . , 2014 ) , suggesting an additional role for setd2 in genome stability . these contain jmjn - jmjc and tandem - tudor domains that specifically remove the tri- and dimethyl forms of both h3k9 and h3k36 , with preference for the trimethyl form being observed in the case of kdm4a / jmjd2a ( couture et  al . kdm4 family proteins are frequently overexpressed in cancers and are associated with poor patient survival ( berry and janknecht , 2013 , black et  al . , 2013 ) . here in addition , we define a role for setd2 and rad51 in maintaining genome stability through suppressing mmej at break sites . tumor suppressors can suppress carcinogenesis by preventing  genome instability , a hallmark of cancer . to investigate the impact of loss of setd2 , a tumor suppressor , on genome stability following dsb induction , we developed an i - scei - induced loss of function assay ( hprt : i - scei ) . intron - encoded endonuclease 1 from saccharomyces cerevisiae ( i - scei ) is a rare - cutting endonuclease that has no predicted recognition site in the mammalian genome ( jasin , 1996 ) . an i - scei recognition sequence was inserted into the endogenous hprt exon 6 in ht1080 ( fibrosarcoma ) cells . the i - scei site maintains the hprt reading frame and makes only a single amino acid change that does not impair the function of hprt ( figure  1a ) . following i - scei induction , inaccurate dsb repair generates hprt - negative mutants , which can be selected for using 6-thioguanine ( 6-tg ) , and indels can be detected by pcr amplification using primers flanking the break site ( figure  1a ) . following small interfering rna ( sirna ) knockdown and i - scei induction , the frequency of hprt loss ( mutation frequency ) in setd2-depleted cells was 1.65% , significantly higher ( p  = 0.0014 ) than that of nontargeting controls ( 1.1% ) . these findings resembled those in rad51-depleted cells in which the frequency of hprt loss was also significantly increased to 1.88% ( p  = 0.0022 ) compared to nontargeting controls ( figure  1b ) . setd2 and rad51 codepletion ( sis+sir ) also significantly increased the mutation frequency to 1.52% ( p  = 0.037 ) , which was similar to the frequency observed following either setd2 or rad51 depletion ( figure  1b ) . we confirmed that i - scei protein expression and cleavage efficiency were the same in cells transfected with either control sirna or setd2 sirna ( figures s1a  s1c ) . in addition , hprt gene transcription was not affected by setd2 knockdown , as shown by  quantitative rt - pcr ( qrt - pcr ) of spliced and unspliced hprt mrna ( figures s1d and s1e ) . therefore , the mutation frequency in this system is likely to be directly associated with misrepair of the dsb at the i - scei site . further , we studied the mutation patterns across the break site  of 30 individually isolated hprt - negative clones from each background by pcr amplification and sequencing . hprt - negative clones from cells treated with nontargeting control sirna indicated the presence of microdeletions of 25  bp , consistent with dsb repair by c - nhej . larger microdeletions were also observed that were associated with regions of microhomology located either side of the break site , consistent with dsb repair through mmej ( figures 1c and s1f ) . sequence analysis indicated that setd2-depleted cells exhibited a significant increase in average deletion length ( 11  bp , p  < 0.0001 ) compared to control cells ( 5  bp ) ( figures 1c and 1d ) . rad51-depleted cells exhibited a further significant increase in the average deletion length ( 22  bp , p  = 0.014 ) ( figures 1c and 1d ) . rad51 and setd2 codepletion generated deletion lengths ( an average of 8  bp ) comparable to those observed following setd2 knockdown , which in turn was significantly higher than the nontargeting control ( p  = 0.026 ) ( figures 1c and 1d ) . however , the frequency and size of the insertions was not significantly altered between setd2-depleted cells , setd2/rad51 codepleted and nontargeting controls . we found that the frequency of microhomology - mediated end - joining ( mmej ) was significantly increased in setd2 and rad51-depleted cells ( p  < 0.05 ) ( figures 1e and s1 g ) . this suggested that hr - deficient cells use mmej as an alternative repair mechanism , which gives rise to a mutation signature of sequence loss between regions of microhomology . these results support a role for setd2 in tumor suppression by maintaining genome stability through preventing deleterious mutations arising in response to dsbs . these findings further identify a similar break - induced mutation signature between setd2- and rad51-depleted cells , suggesting an early role for setd2 in hr repair . to test the role for setd2 in dsb repair , we depleted setd2 in u2os and hela cells using two independent sirnas and measured the clonogenic survival following exposure to dna - damaging agents . setd2 knockdown significantly reduced survival after treatment with mitomycin c ( mmc ) , camptothecin ( cpt ) , or ionizing radiation ( ir ) in u2os cells and hela cells compared to nontargeting controls ( figures 2a and s2a ) . these results identify a role for setd2 in the cellular response to dna damage and are consistent with a role for setd2 in dsb repair . western blotting also showed that setd2 knockdown significantly reduced global levels of h3k36me3 , confirming setd2 as the major h3k36me3 methyltransferase in human cells ( figure  2b ) . because rad51 is more likely to be affected among dna repair genes by sirna off - target effects ( adamson et  al . , 2012 ) , we also confirmed that setd2 knockdown by sirna did not affect the rad51 protein levels ( figure  2b ) . we  confirmed that setd2 knockdown had no effect on the expression of major hr proteins , including brca2 , rad50 , ctip , mre11 , and rpa and the nhej protein ku80 ( figure  s2b ) . we also observed no changes to the cell cycle after setd2 knockdown ( figure  s2c ) . both mmc and cpt induce s phase - specific dsbs , which are predominantly repaired by hr ( arnaudeau et  al . , 2001 , , we used a well - characterized gfp - based reporter for hr ( dr - gfp ) . the hr reporter contains an i - scei recognition sequence , which upon i - scei expression is cleaved to generate a dsb . dsb repair by hr using a direct repeat within the reporter cassette as  a template results in an intact gfp gene ( figure  2c)(pierce et  al . , 1999 ) . setd2 knockdown by two independent sirnas significantly reduced hr repair of an i - scei induced dsb by 67%77% compared to nontargeting control cells ( p  <  0.0003 ) ( figure  2d ) . as a control for the hr reporter , 0.0001 ) , whereas dna - pkcs inhibitor nu7441 had no effect on hr ( figure  2d ) . in contrast , setd2 knockdown had no effect on repair of an i - scei induced dsb in an nhej reporter ( ires - tk - egfp ) in which joining of two i - scei sites separated by a thymidine kinase gene ( tk ) results in gfp expression ( figures 2e and 2f)(ogiwara et  al . , 2011 ) . as a control , rad51 had no effect on  the same nhej reporter , whereas the dna - pk inhibitor nu7441 reduced nhej activity by 98% ( p to further investigate the role of setd2 in dsb repair , we examined the rate of repair following ir . we used h2ax foci as a marker of dna damage and found that following exposure to 5  gy ir , setd2 knockdown significantly delayed removal of h2ax foci at 48  hr ( p  < 0.003 ) compared to nontargeting controls , supporting a role for setd2 in efficient dsb repair ( figure  3a ) . we next investigated the effect of setd2 depletion on the recruitment of hr repair proteins following dna damage . setd2 knockdown resulted in significantly reduced rad51 foci formation 4 and 8  hr after exposure to ir compared to nontargeting controls ( p  < setd2 knockdown also significantly reduced rad51 foci formation after exposure to cpt ( p  < we also examined the effect of setd2 depletion on rpa recruitment , where we induced dsbs in s phase cells using cpt or mmc . a significant reduction in rpa foci formation was observed after cpt or mmc treatment following setd2 knockdown compared to nontargeting controls ( p  < 0.023 ) ( figures 3c and s2e ) . setd2 knockdown leads to reduced h3k36me3 in the nuclei as confirmed by costaining the cells with antibodies against h3k36me3 and rpa ( figure  s2f ) . together , these findings support a role for setd2 in promoting hr through facilitating recruitment of rpa and rad51 to dna damage sites . to further confirm the impact of setd2 loss on the recruitment of hr factors to the site of dsbs , we performed chromatin immunoprecipitation ( chip ) near the i - scei site in the dr - gfp cassette ( figure  s3a ) . the i - scei site is located within the dr - gfp cassette under the control of the highly active cmv promoter , and thus h3k36me3 , which is associated with active transcription , is predicted to be present throughout the cassette . after induction of dsbs by i - scei , no change in h3k36me3 or  setd2 levels was detected proximal or distal to the break site after 8 , 24 , or 48  hr following i - scei transfection ( figure  s3b ) . the results were also quantified by chip - qpcr ( figure  s3c ) . although other groups also observed no changes to h3k36me3 level after dsb , globally ( fnu et  al . , 2011 ) or locally at the break site ( pei et  al . , 2011 , 2014 ) , we were surprised by the lack of reduction in h3k36me3 or setd2 knowing that regional transcriptional silencing has been observed at sites of dsbs ( shanbhag et  al . , 2010 ) . we speculate that the unexpected maintenance of h3k36me3 at the dsb site by setd2 may serve as an anchor to mediate hr repair . we monitored recruitment of major hr factors to the dsb site  in the chip system . rpa and rad51 were recruited to the dsb site , resulting in increased binding to the  up  site 18  hr after i - scei transfection , compared to uncut controls ( figure  3d ) . setd2 depletion substantially reduced the recruitment of rpa  and rad51 after the i - scei cut ( figure  3d ) . setd2 protein depletion was validated by western blotting ( figure  s3d ) , and was further confirmed by the absence of setd2 or h3k36me3 at the break site ( figure  3d ) . these findings are consistent with the rpa and rad51 foci data , indicating that setd2 is required to recruit rpa and rad51 to dsb sites . together , these findings  confirm a presynaptic role for setd2 in facilitating hr repair of dsbs . to investigate if the methyltransferase activity of setd2 is required for its role in hr , we generated two doxycycline - inducible u2os cell lines , where either a wild - type setd2 cdna or a methyltransferase - dead setd2 cdna was inserted behind a tet operator in the genome of u2os cells . upon addition of doxycycline to the cell culture medium , the tet operator was derepressed and the wild - type or mutant setd2 was expressed . the methyltransferase - dead setd2 was mutated at two amino acid residues located within the catalytic site of the set domain , which are conserved with yeast set2 . the corresponding mutations in yeast were shown to abolish the methyltransferase activity ( rea et  al . , 2000 , strahl et  al . , both arginine ( r ) and cysteine ( c ) residues were mutated in the methyltransferase - dead ( md ) cdna by site - directed mutagenesis ( see supplemental experimental procedures ) . western blot analysis confirmed that doxycycline - induced expression of  wild - type setd2 could restore the level of h3k36me3 after sisetd2 treatment , whereas expression of methyltransferase - dead setd2 could not restore the level of h3k36me3 ( figure  s4a ) . we depleted endogenous setd2 with sirna#3 and examined the rescue of the phenotype by the two constructs . although the wild - type construct increased foci formation of rpa and rad51 after dna damage in sisetd2 cells , the methyltransferase - dead mutant did not ( figures 4b and 4c ) . this is consistent with the observation that the wild - type construct partially restored hr efficiency whereas the methyltransferase - dead ( md ) mutant did not ( figure  4d ) . all wt clones were able to restore hr efficiency , but none of the md clones showed rescue ( figures s4b and s4c ) . the observation that the methyltransferase - dead setd2 mutant could not rescue hr efficiency suggested that h3k36me3 is required for efficient hr . to test this first , we overexpressed kdm4a , an oncogene and a demethylase specific for tri- and dimethylated histone h3k36 and h3k9 , predicting that overexpression of kdm4a would reduce the level of h3k36me3 and thus impair hr . upon addition of doxycycline , kdm4a was overexpressed , which led to a global reduction in h3k36me3 levels ( figure  4e ) . we then compared the recruitment of the hr factors in this cell  line with or without kdm4a overexpression . we found that  kdm4a overexpression inhibited the formation of rpa and rad51  foci after dna damage ( figures 4f and 4 g ) . accordingly , kdm4a overexpression also reduced hr efficiency as assessed by the hr reporter ( p  = 0.0078 ) ( figure  4h ) . using the same cell line without kdm4a cdna integration , we found that doxycycline itself had no effect on hr efficiency of u2os cells ( figure  s4d ) . these findings indicate that overexpression of the oncogene kdm4a significantly reduces levels of h3k36me3 and hr . second , we utilized a dominant - negative mutation in the histone h3.3 gene ( h3.3k36 m ) that was recently shown to reduce h3k36me3 specifically without affecting other histone methylations ( lewis et  al . , 2013 ) . accordingly , in cells expressing the  mutant h3.3k36 m transgene , global levels of h3k36me3 were depleted compared to the wild - type h3.3 control ( figure  4i ) . cells expressing h3.3k36 m also exhibited delayed rpa and rad51 foci formation following dna damage ( figures 4j and 4k ) , consistent with similar delays seen in kdm4a overexpressing cells or setd2-depleted cells . neither kdm4a overexpression nor h3.3k36 m expression affected cell cycle progression as assessed by brdu incorporation ( figures s4e  and s4f ) , thus excluding the possibility that the reduction  in  rpa , rad51 foci formation and hr efficacy was due to  cell  cycle effects . the common feature of all three different  systems ( setd2 knockdown and rescue , kdm4a overexpression , and  h3.3k36 m expression ) was the reduction in the recruitment of hr proteins and hr efficiency associated with reduced  h3k36me3 . lens epithelium - derived growth factor p75 ( ledgf ) binds to h3k36me3 constitutively through its pwwp domain . upon dna damage ledgf recruits ctip , which facilitates the resection step during hr repair ( daugaard et  al . , 2012 ) . accordingly , we were able to coprecipitate ledgf and h3k36me3 , independently of dna damage in u2os cells ( figure  5a ) . ledgf binding to chromatin was reduced upon setd2 depletion , consistent with preferential binding of ledgf to h3k36me3 ( eidahl et  al . , 2013 ) , and was dsb independent ( figure  5b ) . we predicted that the reduced ledgf binding following setd2 depletion would reduce recruitment of ctip upon damage . we therefore used micro - irradiation to induce localized dsbs and studied the recruitment of ctip using a u2os cell line expressing gfp - tagged ctip ( sartori et  al . , 2007 ) . we found ctip to be localized to sites of dna damage marked by h2ax ( figure  5c ) . in contrast , setd2 knockdown resulted in reduced recruitment of ctip to the dna damage site as marked by h2ax ( figure  5c ) . if setd2 facilitates hr through ledgf binding and ctip recruitment , we would expect setd2 to be epistatic with ctip . indeed , codepletion of setd2 and ctip ( sic+sis ) showed the same level of reduction in hr as ctip - depletion alone ( figures 5d and s5a ) . these findings are consistent with the hypothesis that setd2-dependent h3k36me3 promotes recruitment of ctip by ledgf to break sites . reduced ctip recruitment , together with the reduced rpa foci formation following setd2 depletion , suggested that setd2-dependent h3k36 trimethylation might promote dsb resection . to directly measure resection , we used an in  vivo resection assay ( zhou et  al . , 2014 ) in which levels of ssdna were assessed at increasing distances from a specific dsb induced by the asisi restriction enzyme , which was found to recruit rad51 ( iacovoni et  al . following dsb induction ( + 4oht ) , ssdna was readily detected 231  bp from the asisi break site ( dsb - ii in aymard et  al . , 2014 ) and at reduced levels further from the break site . following ctip depletion , ssdna levels were significantly reduced at 231  bp ( p  = 2.2   10 ) and 918  bp ( p  = 0.057 ) from the break site compared to control sirnas ( figure  5e ) . following setd2 depletion , ssdna levels were also significantly reduced at 231  bp ( p  = 0.021 ) and 918  bp ( p  = 0.037 ) from the break site compared to control sirnas ( figure  5f ) , and setd2 depletion did not affect dsb induction at this site as assayed by xrcc4 chip ( figure  s5b ) , thus confirming a role for setd2 in facilitating dsb resection . the setd2 gene encoding the h3k36 trimethyltransferase is among the most mutated in human cancers ( lawrence et  al . , 2014 ) . yet , how setd2 functions to suppress the genome instability frequently observed in setd2-deficient cancer cells is unknown ( sato et  al . , 2013 , zhu et  al . , 2014 ) . here , we describe a  role for setd2-dependent h3k36 trimethylation in facilitating error - free dsb repair and genome stability through efficient hr . our data support a model in which ledgf is constitutively  anchored to h3k36me3-decorated chromatin through its pwwp domain . upon dna damage , chromatin - bound ledgf recruits ctip , which promotes resection  an essential step in hr repair ( figure  6a ) . in the absence of setd2-dependent h3k36me3 , the chromatin association of ledgf is compromised , and dna damage - induced ctip recruitment is impaired , leading to reduced resection and hr and an increase in error - prone mmej and subsequent genome instability ( figure  6b ) . our data do not , however , exclude the  possibility that other h3k36me3 binding factors may also contribute to efficient hr . the involvement of h3k36me3 in hr raises the question as to whether this histone mark is dsb inducible or preset to determine the choice of repair pathways . we did not detect any appreciable increase in h3k36me3 levels at the break site following i - scei induction . these findings are consistent with previous observations indicating that h3k36me3 levels do not change following dna damage , either globally ( fnu et  al . , 2011 ) or locally ( aymard et  al . , 2014 , pei et  al . , 2011 ) given that setd2-dependent h3k36 trimethylation strongly correlates with actively transcribed regions of the genome ( edmunds et  al . , 2008 , yoh et  al . , 2008 ) , our findings thus support a spatial link between hr and transcriptionally active , h3k36me3 decorated regions of the genome . indeed , because hr repair is usually error free , this would be expected to protect genome integrity following dsb induction or replication fork collapse at such sites , which would be important in maintaining cell homeostasis . genome - wide sequencing has suggested certain mutation signatures are associated with specific cancer types ( alexandrov et  al . , 2013 ) but lacks the power to identify driver mutations that give rise to that mutation signature . here , we use a system to determine the impact of individual gene dysfunction on mutation patterns arising during the repair of a single dsb . we show that , by depleting setd2 or rad51 hr proteins , dsb induction leads to significantly increased mutation frequencies associated with deletions within six cell cycles ( 5  days ) . these findings identify a role for setd2 , like rad51 , in suppressing break - induced mutations . further mechanistic analysis indicated that deletions arising from setd2 or rad51 depletion resulted from a significant increase in error - prone mmej . setd2 loss results in partial resection , short ssdna 3 ends and reduced rpa foci . these partially resected ends are insufficient to promote efficient hr and are no longer substrates for c - nhej . instead , these partially resected ends become substrates for mmej . similarly , following rad51 depletion , whereas resection proceeds normally , hr is blocked presynaptically , resulting in the accumulation of longer unrepaired resected ends . such ends are no longer substrates for c - nhej , and , because hr can not proceed , the ends are instead rejoined by mmej . extensive resection following rad51 loss facilitates annealing between regions of microhomology further from the break site , thus leading to larger deletions than those observed for setd2 . the observations that hr was significantly reduced whereas c - nhej was unchanged following setd2 or  rad51 depletion are consistent with this model . together , these  findings suggest a role for hr in maintaining genome stability through suppression of mmej . mutation study can therefore provide important insights into how loss of these  genes contributes to somatic mutation and intratumor heterogeneity , and it highlights the importance of hr repair genes in maintaining genome stability . setd2 is a tumor suppressor and its loss is an important step in the progression of a number of cancer types ( dalgliesh et  al . , 2010 , fontebasso et  al . , 2013 , gerlinger et  al . , 2012 ) . moreover , all three tudor domain - containing  kdm4/jmjd2 proteins ( a , b , and c ) are frequently overexpressed in various cancers , including breast , colorectal , lung , and prostate ( berry and janknecht , 2013 ) . our findings predict that overexpression of these kdm4 family members causes  genome instability and tumorigenesis through loss of h3k36me3-mediated hr repair . the identification of a role for h3k36me3 in facilitating hr repair , in addition to its recently described role in mismatch repair ( li et  al . , 2013 ) , establishes the h3k36me3 chromatin mark as an important functional node for maintaining genome stability . the reporter ht1080 cell line with a functional but i - scei - cleavable hprt gene  was generated by inserting an 18  bp i - scei recognition site into exon 6 of the hprt gene using gene targeting . reporter cells were transfected with the i - scei plasmid 48  hr after sirna knockdowns . the cells were allowed to repair in a nonselective medium for 5  days before seeding and selecting for hprt - negative cells using 6-tg ( 15  g / ml ) . the mutation frequency was calculated as the number of hprt - negative colonies divided by number of cells plated , after correcting for plating efficiency . to determine the mutation sequence , individual single - cell colonies were isolated , and genomic dna spanning the dsb junction was pcr - amplified using the primers flanking the i - scei site . sequence alignment of the pcr products was conducted using the dna data bank of japan ( ddbj ) clustalw program . 2014 ] ) were transfected with sirna using the cell line nucleofactor kit v ( amaxa ) according to the manufacturer s instructions . forty - eight hours after sirna transfection , cells were treated or not with 300  nm of 4-hydroxytamoxifen ( 4oht ) ( sigma ; h7904 ) for 4  hr . single - stranded dna generated at an asisi - induced dsb was analyzed using the procedure described in  zhou et  al . the percentage of ssdna was calculated with the following equation : ssdna%  = 1/(2  + 0.5 ) 100 , where ct is calculated by subtracting the ct obtained from mock - digested sample from the bani - digested sample . u2os - flp - in / t - rex cells express the tet repressor and contain a single flp - in site ( gordon et  al . , 2009 ) . u2os - flp - in / t - rex / drgfp cells were generated by integrating the pdr - gfp expression plasmid ( pierce et  al . , 1999 ) into the u2os - flp - in / t - rex cells . the kdm4a cdna ( genecopoeia ) was cloned into the pdestfrtto destination vector by a gateway lr reaction ( life technologies ) . u2os - flp - in / t - rex or u2os - flp - in / t - rex / drgfp cells were cotransfected with the resulting kdm4a destination plasmid and pog44 ( invitrogen ) . stable clones that express the kdm4a protein under the control of the t - rex system were subjected to the treatments indicated . p  < 0.05 , p  < 0.01 , p  < 0.001 , and n.s . represents p  0.05 . l .- p.z . , s.s . , and f.a . designed and performed experiments . experiments in figure  1 were performed by s.a . , with technology developed by a.c.g.p . ; figure  2 was performed by s.x.p . and s.a . l .- p.z . ; figure  4 was performed by s.s . and s.x.p . , ; figure  s3 was performed by l .- p.z . ; figure  s4 was performed by s.x.p . and s.s .