Guangdong Sun,*† Marpadga A. Reddy,* Hang Yuan,*† Linda Lanting,* Mitsuo Kato,*and Rama Natarajan*
*Gonda Diabetes Center, Beckman Research Institute of the City of Hope, Duarte, California; and †Division ofNephrology, 2nd Hospital of Jilin University, Changchun, China
ABSTRACTTGF-�1–induced expression of extracellular matrix (ECM) genes plays a major role in the developmentof chronic renal diseases such as diabetic nephropathy. Although many key transcription factors areknown, mechanisms involving the nuclear chromatin that modulate ECM gene expression remainunclear. Here, we examined the role of epigenetic chromatin marks such as histone H3 lysine methylation(H3Kme) in TGF-�1–induced gene expression in rat mesangial cells under normal and high-glucose (HG)conditions. TGF-�1 increased the expression of the ECM-associated genes connective tissue growthfactor, collagen-�1[�], and plasminogen activator inhibitor-1. Increased levels of chromatin marks asso-ciated with active genes (H3K4me1, H3K4me2, and H3K4me3), and decreased levels of repressive marks(H3K9me2 and H3K9me3) at these gene promoters accompanied these changes in expression. TGF-�1also increased expression of the H3K4 methyltransferase SET7/9 and recruitment to these promoters.SET7/9 gene silencing with siRNAs significantly attenuated TGF-�1–induced ECM gene expression.Furthermore, a TGF-�1 antibody not only blocked HG-induced ECM gene expression but alsoreversed HG-induced changes in promoter H3Kme levels and SET7/9 occupancy. Taken together,these results show the functional role of epigenetic chromatin histone H3Kme in TGF-�1–mediatedECM gene expression in mesangial cells under normal and HG conditions. Pharmacologic and othertherapies that reverse these modifications could have potential renoprotective effects for diabeticnephropathy.
J Am Soc Nephrol 21: 2069 –2080, 2010. doi: 10.1681/ASN.2010060633
TGF-�1 has been implicated in various human dis-orders including vascular and renal diseases.1–3 Di-abetic nephropathy (DN) is a chronic renal com-plication characterized by the thickening ofglomerular and tubular basement membranesand progressive accumulation of extracellularmatrix (ECM) proteins such as type I and type IVcollagens, fibronectin, and laminin in the tubularinterstitium and mesangium.3–5 Induction ofprofibrotic TGF-�1 by diverse mediators such ashigh glucose (HG), advanced glycation endprod-ucts (AGEs), and angiotensin II in glomerularmesangial cells (MCs) and other renal cells hasbeen implicated in these events.2–9 TGF-�1 alsoincreases ECM accumulation through inductionof its downstream effector, connective tissue
growth factor (CTGF),10,11 and by decreasing ma-trix degradation through inhibition of proteasesor activation of protease inhibitors such as plas-minogen activator inhibitor-1 (PAI-1).12 A TGF-�1–specific antibody had significant anti-fibroticeffects in animal models of DN, including db/dbtype 213,14 and streptozotocin-induced type 1 di-
Received June 15, 2010. Accepted July 28, 2010.
Published online ahead of print. Publication date available atwww.jasn.org.
Correspondence: Dr. Rama Natarajan, Department of Diabetes,Beckman Research Institute of the City of Hope, 1500 East DuarteRoad, Duarte, CA 91010. Phone: 626-256-4673, ext. 62289; Fax:626-301-8136; E-mail: [email protected]
abetic mice15 and prevented HG-induced increase in matrixprotein synthesis in renal cells.4,16 TGF-�1 can regulate geneexpression through Smad transcription factors and E-box–dependent mechanisms.1,17–20 However, the subtle nuclear chro-matin mechanisms involved in TGF-�1–induced expression ofkey ECM genes in MCs are not clear.
Gene regulation by extracellular stimuli involves not only tran-scription factors binding to their cognate DNA binding sites butalso epigenetic changes in chromatin without alterations in DNAsequence. Post-translational modifications on amino-terminaltails of nucleosomal histones such as histone H3 and H4, includ-ing acetylation, methylation, and ubiquitination at key lysines,play key roles in modulating chromatin structure and gene tran-scription.21,22 They form a “histone code” that can dictate tran-scriptional outcomes of gene activation or repression.23 In gen-eral, acetylation of histone H3 lysines (H3KAc) is associated withactive gene transcription, whereas methylation (H3Kme) can beassociated with either active or inactive gene promoters depend-ing on the position of lysine modified. H3KAc is mediated byhistone acetyl transferases and H3Kme by histone methyltrans-ferases (HMTs). HMTs can mono-, di-, or tri-methylate (H3K-me1, -me2, -me3) specific lysine residues, thereby adding anotherepigenetic regulatory layer.22
Histone H3K4me is usually associated with gene activationand transcriptional elongation and is mediated by HMTs such asSET1, MLL1–4, and SET7/9.22,24–26 H3K9me, on the other hand,is generally associated with gene repression and is mediated byHMTs such as SUV39H1, G9a, and SETDB1/ESET.24 Other ly-sines, including H3K27, H3K36, and H3K79, can also be methyl-ated to various degrees.24 In addition, the discovery of histonelysine demethylases has added another dimension to gene regula-tion.27 Together, these factors create a fine balance of gene regu-lation, a disruption of which could result in abnormal gene ex-pression and disease phenotypes.
To date, it is not known whether promoter histone H3 lysinemethylation plays a role in TGF-�1–induced transcription ofECM-associated genes in MCs or whether the effects of HG onsuch epigenetic events can be mediated through TGF-�1. Here weshow that TGF-�1 leads to the enrichment of H3K4me1/2/3 anddepletion of H3K9me2/3 marks at ECM-associated gene promot-ers in rat MCs. A TGF-�1 antibody could reverse HG-inducedchanges in H3Kme at these fibrotic gene promoters along withreductions in their expression. Furthermore, the H3K4 HMTSET7/9 seemed to play a role in TGF-�1–induced ECM gene expres-sion. These data show novel epigenetic chromatin mechanisms inTGF-�1 actions in MCs related to ECM deposition and DN.
RESULTS
ECM-Associated Genes Are Increased, Whereas,Reciprocally, Repressive H3K9me Levels Are Decreased atTheir Promoters in TGF-�1–Treated Rat Mesangial CellsWe first examined whether TGF-�1–induced expression ofkey ECM-related genes was associated with changes in the
repressive epigenetic marks H3K9me2 and H3K9me3 at their pro-moters. Serum-depleted rat mesangial cells (RMCs) werestimulated with TGF-�1 (10 ng/ml) for various time peri-ods, and gene expression levels were analyzed by RT-QPCR.Collagen-�1(I) chain (Col1a1), CTGF, and PAI-1 mRNAlevels were significantly increased by TGF-�1 from 2 to 24hours compared with control, whereas the housekeepinggene CypA showed no difference under these conditions(Figure 1A). Immunoblotting showed that protein levels ofcollagen I, CTGF, and PAI-1 (Figure 1B) were also similarlyincreased by TGF-�1. These results confirmed that TGF-�1can upregulate ECM-associated genes in RMCs.
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Figure 1. TGF-�1 increases the expression of ECM-associatedgenes in RMCs. (A) Serum depleted RMCs were stimulated withTGF-�1 (10 ng/ml) for various time periods (0.5 to 24 hours), andmRNA levels of ECM-associated genes (Col1a1, CTGF, and PAI-1)and housekeeping gene cyclophilin A (CypA) were analyzed byRT-QPCR. Gene expression was normalized to internal control�-actin gene, and results are expressed as fold stimulation overcontrol (ctrl) (mean � SEM; *P � 0.05; **P � 0.01 versus ctrl, n �3). (B) Western blot analysis of RMC cell lysates from control andTGF-�1–treated RMCs using collagen I, CTGF, PAI-1, and �-actinantibodies. Results shown are representative of two separateexperiments.
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We next performed chromatin immunoprecipitation(ChIP) assays with H3K9me2- and H3K9me3-specific anti-bodies. ChIP-enriched DNA samples were analyzed by quan-titative PCR (QPCR) using primers spanning Smad bindingsites and nearby cis-elements at these promoters (Figure 2A).Levels of both H3K9me2 (Figure 2B) and H3K9me3 (Figure2C) at the Col1a1, CTGF, and PAI-1 promoters were signifi-cantly reduced in RMCs treated with TGF-�1 from 2 to 24hours compared with control. In contrast, there were no sig-nificant differences at the CypA promoter. These results sug-gest that TGF-�1–induced expression of these genes may becaused, at least in part, by a loss of repressive epigenetic histonemodifications at their promoters.
TGF-�1 Enhances H3K4me Levels at the Promotersof ECM-Associated GenesWe next examined whether TGF-�1 could alter promoter lev-els of H3K4me, an epigenetic “active” mark, using ChIP assayswith H3K4me1, H3K4me2, or H3K4me3 antibodies. As shownin Figure 3A, TGF-�1 increased H3K4me1 levels at the Col1a1and CTGF promoters in RMCs at 2 hours, and this was sus-tained up to 24 hours. At the PAI-1 promoter, H3K4me1 levelswere significantly increased only at 24 hours. TGF-�1 enhancedH3K4me2 levels from 6 to 24 hours at Col1a1 and PAI-1 promot-ers, with no significant changes at the CTGF promoter (Figure3B). TGF-�1 significantly increased H3K4me3 levels at theCol1a1, CTGF, and PAI-1 promoters (Figure 3C). These increases
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Figure 2. TGF-�1 decreases H3K9me2/3 levels at ECM-associated gene promoters in RMCs. (A) Map showing locations of Col1a1,CTGF, and PAI-1 promoter primers used for ChIP-QPCRs. TSS, transcription start site; SBE, Smad binding elements. (B and C) Bargraphs showing H3K9me2 (B) and H3K9me3 (C) levels at the indicated gene promoters in control and TGF-�1 (10 ng/ml)-stimulatedRMCs. ChIP assays were performed with H3K9me2 and H3K9me3 antibodies as described in Concise Methods. ImmunoprecipitatedDNA and input DNA were subjected to QPCRs with primers specific for the indicated gene promoters to measure enrichment levels.QPCR data were analyzed using the 2���Ct method, and results normalized to input DNA were expressed as fold over respectiveuntreated control (ctrl) cells (mean � SEM; *P � 0.05; **P � 0.01 versus ctrl, n � 3).
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in promoter H3K4me1/2/3 levels correlated with the increasedexpression of these genes by TGF-�1. On the other hand, theCypA promoter showed no significant changes in these marks,confirming specificity. These results suggest that increases in pro-moter H3K4me may be involved in TGF-�1–induced upregula-tion of ECM-associated genes in RMCs.
TGF-�1–Specific Antibody Reverses HG-InducedInhibition of Repressive H3K9me in RMCsSerum-depleted RMCs were pretreated with TGF-�1–specificantibody (25 �g/ml) or control mouse IgG (25 �g/ml) andthen treated with either normal glucose (5 mM) plus 25 mMmannitol (NG), or HG (30 mM) for 48 hours. Gene expressionwas evaluated by RT-QPCR, whereas promoter enrichments ofH3K9me2 or H3K9me3 were assessed by ChIP-QPCRs. Asshown in Figure 4A, HG significantly increased Col1a1, CTGF,and PAI-1 mRNA levels compared with NG. These changeswere significantly inhibited by TGF-�1 antibody but not con-trol IgG. More interestingly, ChIP assays showed that HG sig-nificantly decreased H3K9me2 (Figure 4B) and H3K9me3 lev-els (Figure 4C) at the Col1a1, CTGF, and PAI-1 promoterscompared with NG, and this inhibitory effect of HG was sig-
nificantly reversed by TGF-�1 antibody but not IgG control.There were no significant differences at the CypA promoter.These results suggest that HG-induced ECM gene expressionin RMCs is associated with decreased repressive marksH3K9me2 and H3K9me3, which can be reversed, at least inpart, by the TGF-�1 antibody.
TGF-�1–Specific Antibody Reverses HG-InducedIncreases in Promoter H3K4me in RMCsWe next tested whether HG can increase active H3K4memarks at the promoters of ECM-associated genes andwhether this can be reversed by the TGF-�1 antibody. Se-rum-depleted RMCs were pretreated with TGF-�1 antibodyor IgG for 1 hour and then treated with NG or HG for 48hours, followed by ChIP assays with specific antibodies. Re-sults showed that HG significantly increased H3K4me1(Figure 5A), H3K4me2 (Figure 5B), and H3K4me3 (Figure5C) levels at the Col1a1, CTGF, and PAI-1 promoters com-pared with NG. TGF-�1 antibody, but not IgG, significantlyattenuated these HG-induced increases (except forH3K4me3 at the Col1a1 promoter; Figure 5, A–C). In con-trast, the CypA promoter showed no significant differences
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Figure 3. TGF-�1 upregulates H3K4me1/2/3 levels at ECM-associated gene promoters. H3K4me1 (A), H3K4me2 (B), and H3K4me3(C) levels at indicated promoters in RMCs treated without (control, ctrl) or with TGF-�1 (10 ng/ml) for various time periods. ChIP assayswere performed as described in Figure 2 with specific antibodies, and results normalized to input DNA were expressed as foldenrichment over respective untreated ctrl (mean � SEM; *P � 0.05; **P � 0.01 versus ctrl, n � 3).
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2072 Journal of the American Society of Nephrology J Am Soc Nephrol 21: 2069–2080, 2010
among all of the groups. These findings, coupled with theeffects seen on H3K9me2/3, show the mediatory role ofTGF-�1 in HG-induced epigenetic events at promoters ofECM genes and their subsequent expression and that block-ade of these events may be a key mechanism for the anti-fibrotic and renoprotective effects of the TGF-�1 antibody.
SET7/9 Expression Is Increased by TGF-�1 in RMCsWe next examined the role of SET7/9, a H3K4 mono-methyl-transferase, in ECM gene expression. We first observed thatSET7/9 mRNA levels were increased by TGF-�1 (10 ng/ml) in
a time-dependent fashion in RMCs (Figure 6A). SET7/9 pro-tein levels were also increased (Figure 6, B and C). Pretreat-ment with actinomycin D (2 �g/ml) abolished TGF-�1 in-duced SET7/9 mRNA (Figure 6D), showing that TGF-�1 mayregulate SET7/9 at the level of transcription.
TGF-�1 Increases SET7/9 Recruitment toECM-Associated Gene PromotersNext, we examined whether TGF-�1 alters SET7/9 occupancyusing ChIP assays with SET7/9 antibodies. Results showed thatSET7/9 recruitment was significantly increased at the Col1a1,
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Figure 4. TGF-�1–specific antibody reverses HG-induced expression of ECM-associated genes and HG-induced changes inH3K9me2/3 at their promoters in RMCs. (A) mRNA levels of ECM-associated genes in RMCs. Serum-depleted RMCs were pretreatedwith TGF-�1–specific antibody (25 �g/ml) or mouse IgG (25 �g/ml) for 1 hour and then treated with NG (5 mM glucose � 25 mMmannitol) or HG (30 mM glucose) for 48 hours. Gene expression was analyzed by RT-QPCR, and results are expressed as fold stimulationover NG cells (mean � SEM; **P � 0.01 versus NG; ##P � 0.01 versus HG, n � 3). (B) H3K9me2 and (C) H3K9me3 enrichment levelsat ECM and CypA gene promoters in RMCs treated with NG or HG pretreated with IgG or TGF-�1 antibodies. ChIP assays wereperformed as described in Figure 2, and results are expressed as fold enrichment relative to NG (mean � SEM; *P � 0.05 versus NG;#P � 0.05 versus HG, n � 3).
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CTGF, and PAI-1 promoters from 2 to 24 hours after TGF-�1stimulation compared with control, with no change at the CypApromoter (Figure 7A). The SET7/9 recruitment pattern was quitesimilar to the increased H3K4me1 (Figure 3A), except at thePAI-1 promoter, suggesting a key role for SET7/9 in TGF-�1–mediated increase in H3K4me1 in the induction of ECM genes.
TGF-�1–Specific Antibody Can Block HG-InducedSET7/9 RecruitmentWe next observed that HG can also enhance SET7/9 recruitmentto the Col1a1, CTGF, and PAI-1 promoters compared with NG(Figure 7B). Furthermore, the TGF-�1 antibody (but not IgGcontrol) could significantly block these HG-induced increases inSET7/9 recruitment (Figure 7B). SET7/9 occupancy at the CypApromoter showed no differences under these conditions. These
results suggest a mediatory role for TGF-�1 in HG-induced SET7/9recruitment and increased H3K4me1 at ECM gene promoters.
SET7/9 Knockdown Can Attenuate TGF-�1–InducedExpression of ECM-Associated GenesWe further studied the functional role of SET7/9 in TGF-�1–induced gene expression. RMCs were first transfectedwith various concentrations of siRNA oligonucleotides tar-geting SET7/9 (siSET7/9) or control siRNAs (siNeg). After48 hours, total RNA from transfected cells was analyzed byRT-QPCR. As shown in Figure 8A, SET7/9 mRNA levelswere significantly reduced in RMCs transfected with si-SET7/9, with maximum inhibition at 300 ng and no furtherdecrease at 450 ng. Next, RMCs were transfected with 300ng of siSET7/9 or siNeg, and 72 hours later, lysates were
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Figure 5. TGF-�1–specific antibody reverses HG-induced H3K4me at ECM gene promoters in RMCs. (A–C) Bar graphs showing theH3K4me1 (A), H3K4me2 (B), and H3K4me3 (C) levels at ECM and CypA gene promoters in RMCs pretreated with TGF-�1–specific antibody(25 �g/ml) or mouse IgG (25 �g/ml) for 1 hour, followed by treatment with NG or HG for 48 hours. ChIP assays were performed as describedin Figure 2, and results are expressed as fold enrichment relative to NG (mean � SEM; *P � 0.05 versus NG; #P � 0.05 versus HG, n � 3).
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2074 Journal of the American Society of Nephrology J Am Soc Nephrol 21: 2069–2080, 2010
analyzed by immunoblotting with SET7/9 antibody. Asshown in Figure 8B, siSET7/9 also significantly reducedSET7/9 protein levels compared with siNeg.
We next examined whether SET7/9 siRNA can affect
H3K4me in RMCs because previous studies showed thatSET7/9 can regulate H3K4me1 in endothelial cells and mono-cytes28 –30 and induce H3K4me2 in pancreatic islet cells,31
whereas others reported that SET7/9 may not mediateH3K4me132 or that it can activate transcription by also meth-ylating nonhistone protein substrates.33–36 We transfectedRMCs with siSET7/9 and examined global H3K4me levels byimmunoblotting. Total H3K4me1 levels were clearly decreasedin SET7/9 knockdown RMCs compared with siNeg, whereasH3K4me2 and H3K4me3 levels were not affected (Figure 8C).Thus, SET7/9 most likely mediates H3K4me1, but notH3K4me2 or H3K4me3, in RMCs. Furthermore, TGF-�1–in-duced Col1a1, CTGF, and PAI-1 mRNA levels were signifi-cantly attenuated by siSET7/9 compared with siNeg (Figure8D). In contrast, CypA mRNA levels were not affected (Figure8D). These results further support a key role for SET7/9 inmodulating TGF-�1 responses in RMCs.
DISCUSSION
In this report, we first confirmed that TGF-�1 can upregulateECM-associated genes Col1a1, CTGF, and PAI-1 in RMCs. Wethen examined changes in key epigenetic chromatin marks, in-cluding histone H3K9me2/3 and H3K4me1/2/3 levels, at thesegene promoters. Our results showed that these marks and theH3K4 HMT and SET7/9 are involved in TGF-�1– and HG-in-duced up-regulation of ECM-associated genes in RMCs. Further-more, a TGF-�1 antibody could reverse HG-induced changes inthe levels of these promoter marks, and this correlated with theantibody-induced inhibition of their expression in RMCs.
Increasing evidence shows that H3K9me2 and H3K9me3marks are recognized by heterochromatin protein 1 and gen-erally correlate with gene silencing and transcriptional repres-sion.24,37,38 Our previous study showed that, in vascularsmooth muscle cells (VSMCs) derived from diabetic db/dbmice, H3K9me3 levels were decreased at key inflammatorygenes promoters and inversely correlated with the increasedexpression of these genes under basal and TNF-�–treated con-ditions.39 Furthermore, human VSMCs and endothelial cellscultured under HG conditions also exhibited decreased levelsof H3K9me3,30,39 suggesting that a loss of the repressiveH3K9me3 mark can increase the expression of pathologicgenes under diabetic conditions. Our current results showedfor the first time that TGF-�1 decreased H3K9me2 andH3K9me3 levels on Col1a1, CTGF, and PAI-1 promoters, andthis inversely correlated with increased expression of thesegenes, thereby further supporting the notion that a relief oftranscriptional repression caused by decreases in repressivechromatin histone modifications may contribute to increasedexpression of fibrotic genes by TGF-�1.
We also showed that up-regulation of H3K4me1/2/3 marksusually associated with active chromatin occurs in parallel withthe downregulation of H3K9me2/3 by TGF-�1, suggestingthat this can further contribute to the increased gene expres-
ctrl 0.5h 2h 6h 24h0
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Figure 6. TGF-�1 induces the expression of SET7/9 mRNA and pro-tein in RMCs. (A) SET7/9 mRNA expression in RMCs after TGF-�1treatment for various time periods was analyzed by RT-QPCR, normal-ized to internal control �-actin gene, and expressed as fold stimulationover control (ctrl) (mean � SEM; *P � 0.05; **P � 0.01 versus ctrl, n �3). (B) Cell lysates from control (ctrl) RMCs and cells treated with TGF-�1for indicated time periods were analyzed by immunoblotting withSET7/9 and �-actin antibodies. (C) SET7/9 protein levels were quanti-fied by scanning densitometry, and results are expressed as fold overctrl (mean � SEM; *P � 0.05; **P � 0.01 versus ctrl, n � 3). (D) SET7/9mRNA expression in RMCs pretreated with or without actinomycin Dfollowed by stimulation with TGF-�1. Gene expression was analyzed byRT-QPCR and normalized to internal control �-actin gene, and resultswere expressed as fold stimulation over no actinomycin D and noTGF-�1 treatment (mean � SEM; *P � 0.05; **P � 0.01 versus noactinomycin D and no TGF-�1; $P � 0.01 versus no actinomycin D andTGF-�1, n � 3).
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J Am Soc Nephrol 21: 2069–2080, 2010 TGF-� and Histone Methylation 2075
sion. Recent studies showed that methylated histone H3K4correlates with transcriptionally competent chromatin and isassociated with active genes.22–26 This supports our observationthat TGF-�1 increased the expression of Col1a1, CTGF, andPAI-1 in RMCs, and this positively correlated with increasedH3K4me1, H3K4me2, and H3K4me3 at their promoters.
Evidence showed that HG treatment caused dynamicchanges in H3K4me2 and H3K9me2 in human monocytes40
and H3K4me1 in endothelial cells,29 whereas H3K9me3 lev-els were decreased in VSMCs from diabetic db/db mice rel-ative to control db/� and in HG-treated VSMCs39 and en-dothelial cells.30 This is in line with our results showingincreases in H3K4me1–3 and decreases in H3K9me2–3 atthe Col1a1, CTGF, and PAI-1 promoters under HG condi-tions in RMCs, which correlated with HG-induced up-regulation of these genes. Furthermore, the TGF-�1 anti-body could reverse HG-induced epigenetic alterations.
This study also showed an increase, not only in the recruit-ment of H4K4 HMT SET7/9 at the Col1a1, CTGF, and PAI-1promoters, but also in the expression of SET7/9 in RMCs stim-ulated by TGF-�1, suggesting that this HMT is involved inTGF-�1–induced up-regulation of ECM genes in RMCs. Thiswas supported by our observations that SET7/9 gene silencingpartially, but significantly, blocked TGF-�1–induced ECM-as-
sociated genes. Knockdown of SET7/9 with siRNAs could de-crease global H3K4me1, but not H3K4me2 or H3K4me3, lev-els, suggesting that SET7/9-mediated H3K4me1 plays a keyrole, at least in part, in ECM-associated gene expression andthat SET7/9 may be a potential therapeutic target for fibroticdisorders such as DN. The observed increases in H3K4me1might synergize with other complementary events occurring atthese promoters, such as increases in H3K4me2–3 and de-creases in H3K9me2/3, to enhance ECM gene expression inresponse to TGF-�1 (Figure 9). Furthermore, the HMTs andhistone demethylases (HDMs), as well as histone acetyl trans-ferases (HATs) and histone deacetylases (HDACs) regulatingthese other chromatin marks, may also play cooperative roles.Additional studies are needed to assess these factors, includingthe role of HMTs that mediate H3K4me2/3 and H3K9me2/3.
It was suggested that SET7/9-mediated H3K4 methylationfunctions in transcriptional activation by competing withHDACs to enhance H3-K9 acetylation and prevent H3-K9methylation.41,42 SET7/9 can also methylate nonhistone pro-teins including p53,33,43 DNMT1,44 TAF10,34 and p65.35,36 Fur-thermore, SET7/9 could regulate a subset of TNF-�–inducedNF-�B– dependent inflammatory genes in monocytes28 andHG-induced expression of NF-�B p65 and inflammatorygenes in endothelial cells.29 These data show the diverse phys-
ctrl 2h 6h 24h0
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Figure 7. TGF-�1 enhances SET7/9 recruitment at ECM-associated gene promoters, and the TGF-�1–specific antibody reversesSET7/9 occupancy under HG conditions in RMCs. (A) SET7/9 recruitment at the indicated gene promoters in RMCs stimulated withTGF-�1 (10 ng/ml) for various time periods. (B) Inhibition of HG-induced SET7/9 enrichment at ECM gene promoters by TGF-�1 Ab.Serum-depleted RMCs were pretreated with TGF-�1–specific antibody (Ab) (25 �g/ml) or mouse IgG (25 �g/ml) for 1 hour and thentreated with normal glucose (5 mM) plus mannitol (25 mM) (NG) or high glucose (30 mM) (HG) for 48 hours. SET7/9 recruitment wasdetermined by ChIP assays with SET7/9 Ab as described in Figure 2. Results were normalized to input, and SET7/9 occupancy wasexpressed as fold enrichment over respective control samples (mean � SEM; *P � 0.05; **P � 0.01 versus ctrl, n � 3) (A) or over NGsamples (mean � SEM; *P � 0.05 versus NG; #P � 0.05 versus HG, n � 3) (B).
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2076 Journal of the American Society of Nephrology J Am Soc Nephrol 21: 2069–2080, 2010
iologic roles of SET7/9 in gene transactivation. It is possiblethat, in this study, SET7/9 may also act through methylatingother nonhistone proteins and possibly even Smads. Furtherstudies are needed to evaluate these aspects.
Evidence showed that TGF-�1 signaling through acetyla-tion of Smad3 itself can up-regulate gene transcription,45 andacetylation of histones in the TGF-�RII promoter regions canup-regulate its transcription.46 A few studies in diabetic kid-neys have shown the involvement of HDACs in TGF-�1–me-diated ECM production and kidney fibrosis.47,48 These studiessuggest a role for HDACs in the pathogenesis of renal fibrosisand TGF-�1 actions by possibly silencing key protective genes.In unpublished observations, we showed that TGF-�1 can alsoincrease levels of the active chromatin histone acetylation markH3K9Ac at the PAI-1 promoter, and this correlated with in-creased PAI-1 expression in MCs, suggesting that a balancebetween active and repressive marks at the K9 position may
control TGF-�1–induced gene expression. Further studies areneeded to determine such functional interplay between thesetwo antagonistic histone modifications. It is also well knownthat tubulointerstitial disease contributes substantially to DN.In the future, it would be interesting to explore analogous ap-proaches that examine epigenetic mechanisms in matrix geneexpression in tubular epithelial and/or interstitial cells exposedto TGF-�1. Evidence showed that HDAC inhibitors attenuatefibrotic changes in tubulointerstitial injury.49 Epigeneticmechanisms may affect ECM expression by also regulating theexpression of metalloproteases and other proteases, becausehistone acetylation has been shown to regulate metallopro-teases in some cells.50
Taken together, our studies showed that TGF-�1 and HGcan promote significant changes in promoter histone H3K4and H3K9 methylation in MCs that correlate with parallel in-creases in the expression of genes related to ECM accumula-
siNeg
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Figure 8. SET7/9 is involved in TGF-�1–induced regulation of ECM-associated genes in RMCs. (A) RMCs were transfected with variousconcentrations of SET7/9 siRNA (siSET7/9) or control (siNeg) oligonucleotidies. Forty-eight hours after transfection, SET7/9 mRNAlevels were analyzed by RT-QPCR. Results were normalized to internal control �-actin gene, and SET7/9 levels were expressed as foldover siNeg 150 ng (mean � SEM; **P � 0.01 versus siNeg 150 ng). (B) SET7/9 protein levels in RMCs transfected with 300 ng ofsiSET7/9 or siNeg oligonucleotidies. Total cell lysates were prepared 72 hours after transfection and immunoblotted (IB) with SET7/9or �-actin antibodies. Bar graph below represents the quantification of SET7/9 protein levels determined by densitometry andexpressed as fold over siNeg (mean � SEM; **P � 0.01 versus siNeg, n � 3). (C) Global levels of H3K4me1, H3K4me2, and H3K4me3in RMCs transfected with siNeg or siSET7/9 were determined by immunoblotting with indicated antibodies (mean � SEM; **P � 0.01versus siNeg, n � 3). (D) RMCs were transfected with 150 ng of siSET7/9 or siNeg oligonucleotidies, serum depleted for 24 hours, andstimulated with or without TGF-�1 (10 ng/ml) for 6 hours. ECM-associated genes (Col1a1, CTGF, and PAI-1) and CypA mRNAexpression were analyzed by RT-QPCR and normalized to internal control �-actin gene. Results were expressed as fold over siNeg(mean � SEM; **P � 0.01 versus ctrl siNeg; †P � 0.05 versus siNeg � TGF-�1, n � 3).
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J Am Soc Nephrol 21: 2069–2080, 2010 TGF-� and Histone Methylation 2077
tion and the pathogenesis of DN. Because histone lysine meth-ylation has been associated with metabolic memory in othercells,29,30,39 it is possible that these epigenetic changes may alsocontribute to sustained diabetic renal complications that per-sist despite glycemic control.
CONCISE METHODS
MaterialsRecombinant human TGF-�1 (240-B) and pan-specific TGF-� anti-
body (MAB1835) were from R&D systems (Minneapolis, MN); nor-
mal mouse IgG(12–371), normal rabbit IgG (PP64B), and protein A
agarose/salmon sperm DNA(16 –157) were from Upstate, (Billerica,
MA). The following antibodies were used in the Western blotting
analyses and ChIP assays: anti-CTGF (ab6992), anti-collagen I
Figure 9. Schematic representation of histone H3 lysine methyl-ation in TGF-�1–mediated fibrotic gene expression in mesangialcells. Diabetic conditions and TGF-�1–induced expression ofECM-associated genes Col1a1, CTGF, and PAI-1 in MC result infibrosis in the pathogenesis of DN through increases in promoterlevels of active chromatin marks (H3K4me1, H3K4me2, andH3K4me3) and promoter occupancy or expression of K4-HMTssuch as SET7/9, and in parallel, through decreases in promoterlevels of repressive chromatin marks H3K9me2 and H3K9me3.Under diabetic (HG) conditions, a TGF-�1 antibody can reversethese chromatin modifications to exert renoprotective effects.
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2078 Journal of the American Society of Nephrology J Am Soc Nephrol 21: 2069–2080, 2010
RNA Isolation and Real-Time QPCRTotal RNA was isolated from RMCs using RNA-STAT60 reagent ac-
cording to the manufacturer’s instructions. Total RNA (2 �g) was
used to synthesize cDNA using Moloney murine leukemia virus
(MuLV) reverse transcriptase and random hexamers in a final volume
of 20 �l as described by the manufacturer. QPCRs using SYBR green
reagent with gene-specific primers (listed in Table 1) and �-actin gene
primers (internal control) were performed in triplicate in a final vol-
ume of 20 �l in an ABI 7300 real-time PCR thermal cycler (Applied
Biosystems). Dissociation curves were run to detect nonspecific am-
plification, and we confirmed that single products were amplified in
each reaction. Relative gene expression levels were calculated after
normalization with internal control �-actin gene using the 2���Ct
method, where Ct is the threshold value.39 Results were expressed as
fold over control.
Western BlottingRMCs were lysed in 1.5� SDS sample buffer, fractionated on 4 to 15%
SDS-PAGE gels (Bio-Rad, Hercules, CA), and immunoblotted with
antibodies to PAI-1 (1:10,000), CTGF (1:5000), collagen I (1:2000),
and SET7/9 (1:2000) as reported earlier.11 The blots were stripped and
reprobed with an antibody to �-actin (1:200,000). Immunoblots were
developed using a chemiluminescence method and scanned with a
GS-800 densitometer to determine the intensity of protein bands with
Quantity One software (Bio-Rad).
ChIP AssaysChIPs were performed, and ChIP-enriched DNA was analyzed by
real-time QPCR as described earlier.39 Briefly, cells were fixed with
1% formaldehyde at 37°C for 10 minutes, washed with cold PBS con-
taining protease inhibitors, and lysed in Tris, pH 8.1, containing 1%
SDS, 1 mM PMSF, and complete protease inhibitor cocktail. Cell
lysates were sonicated to fragment chromatin to 500-bp size, diluted
in ChIP dilution buffer, and immunoprecipitated overnight at 4°C
with indicated specific antibodies, with IgG control, or without anti-
body (no antibody control). Next day, immune complexes were col-
lected on protein-A agarose beads, and the beads were washed to
remove nonspecific binding. DNA was eluted from the beads, cross-
links were reversed, and DNA was extracted. ChIP-enriched DNA
samples and input DNA samples were analyzed by QPCR with SYBR
reagent in a real-time PCR machine (ABI 7300; Applied Biosystems)
using primers specific for Col1a1, CTGF, or PAI-1 promoters span-
ning Smad binding elements or the control cyclophilin A promoter.
All reactions were performed in triplicate in a final volume of 20 �l.
Dissociation curves were run to detect nonspecific amplification, and
we confirmed that single products were amplified in each reaction.
QPCR data were analyzed using the 2���Ct method as described ear-
lier39 and normalized with input samples. Results were expressed as
fold over control. In all of the experiments, we verified that ChIP
samples obtained with specific antibodies exhibited significant en-
richment relative to IgG or no antibody controls.
Statistical AnalysisData are expressed as mean � SEM of multiple experiments.
Paired t tests were used to compare two groups or ANOVA with
Dunnet post tests for multiple groups, using PRISM software
(GraphPad, San Diego, CA). Statistical significance was deter-
mined at the 0.05 level.
ACKNOWLEDGMENTS
We thank Lingxiao Zhang and Jehyun Park for all their help and those
who generously provided reagents. This work was supported by Na-
tional Institutes of Health NIDDK Grants R01 DK 058191 and R01
DK081705 to R.N.
DISCLOSURESNone.
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