MKL1 mediates TNF-αinduced pro-in flammatory transcription by bridging the crosstalk between BRG1 and WDR5

Wenping Xu,Quanyi Zhao,Min Wu,Mingming Fang,✉,Yong Xu

1 Department of Medicine,Jiangsu Jiankang Vocational College,Nanjing,Jiangsu 211800,China;

2 Department of Biochemistry and Molecular Biology,Collegeof Life Sciences,Wuhan University,Wuhan,Hubei430072,China;

3 Department of Pathophysiology,Key Laboratory of Cardiovascular Disease and Molecular Intervention,Nanjing Medical University,Nanjing,Jiangsu 211166,China.

AbstractTumor necrosis factor alpha(TNF-α)is a cytokine that can potently stimulate the synthesis of a range of proin flammatory mediatorsinmacrophages.Theunderlying epigenetic mechanism,however,isunderexplored.Herewe report that the transcriptional modulator megakaryocytic leukemia 1(MKL1)is associated with a histone H3K4 methyltransferaseactivity.Re-ChIPassay suggests that MKL1 interactswith and recruits WDR5,a componentof the COMPASS complex responsible for H3K4 methylation,to the promoter regions of pro-in flammatory genes in macrophages treated with TNF-α.WDR5 enhances the ability of MKL1 to stimulate the promoter activities of proin flammatory genes.In contrast,silencing of WDR5 attenuates TNF-αinduced production of pro-in flammatory mediators and erases the H3K4 methylation froMthe gene promoters.Ofinterest,the chromatin remodeling protein BRG1 also plays an essential role in maintaining H3K4 methylation on MKL1 target promoters by interacting with WDR5.MKL1 knockdown disrupts the interaction between BRG1 and WDR5.Together,our data illustratea role for MKL1 in moderating the crosstalk between BRG1 and WDR5 to activate TNF-αinduced pro-in flammatory transcription in macrophages.

Keywords:MKL1,WDR5,BRG1,macrophage,transcriptional regulation

Introduction

Macrophages constitute a major source ofin flammation in both health and disease states[1].Macrophageassociated in flammation is generally believed to be a double-edged sword serving both protective and pathogenic roles.When orderly programmed,it helps maintain tissue homeostasis.When aberrantly constructed,it disrupts physiologic integrity and contributes to a host of human diseases such as metabolic syndrome[2],atherosclerosis[3],and cancer[4-5].In macrophages,in flammation-related transcriptional programs are dictated by a handful of conserved transcriptional factors(TF)that include NF-κB,AP-1,and STAT[6].The activities of these sequence-speci fi c TFs are modulated by various co-factors,which may either enhance or suppress the in flammatory response.We have previously identi fi ed megakaryocytic leukeMia 1(MKL1)as a co-factor for NF-κB[7].MKL1 interacts with NF-κB and stimulates NF-κB-dependent transactivation of targetgenes to promote thepathogenesisof pulmonary hypertension and colitis[8-10].The mechanisMwhereby MKL1 mediates NF-κB-dependent proin flammatory transcription,however,is not completely de fi ned.

Engagement of the epigenetic machinery representsa paradigMof transcriptional regulation in eukaryotes[11].Typically,transcriptionally active chromatin is marked by methylated histone H3K4 while transcriptional repression is synonymous with methylated H3K9 and H3K27[12].In eukaryotes,histone H3K4 methylation is catalyzed by the COMPASS complex(short for complex proteinsassociated with SET1)[13].COMPASS operates in amodular mode:whereascatalytic subunits,including MLL1,MLL2,MLL3,MLL4,and SET1 are exchangeable,structural/regulatory subunits,including ASH2,WDR5,WDR82,and RpBP5,are invariable.How individual COMPASS components regulate proin flammatory transcription in macrophages remains to be clearly sorted out.

A range of epigenetic factors have been documented to participate in the regulation of pro-in flammatory transcription in macrophages.For instance,brahmarelated gene 1,or BRG1,was reported to control temporal production of pro-in flammatory mediators induced by lipopolysaccharide(LPS)and free fatty acids[14-15].Several histone methyltransferases,including MLL1[16],MLL4[17],and SET7/9[18],have all been implicated in the regulation of macrophage-derived in flammation by modulating NF-κB activity.Here we report that MKL1 regulates NF-κB-dependent proin flammatory transcription by moderating the crosstalk between WDR5 and BRG1.

Materials and methods

Cell cu lture

Human monocytic/macrophage-like cells(THP-1,ATCC)and human embryonic kidney fi broblast cells(HEK293,ATCC)were maintained in DMEMsupplemented with 10%FBS.Mouse embryonic fi broblast(MEF)cells were isolated froMw ild type or MKL1 de fi cient mice[19].Procedures involving animals were approved by the intramural Ethics ComMittee on Humane Treatment of Experimental Animals.

PlasMids,transfection,and reporter assay

Expression constructs for MKL1,WDR5,as well as promoter constructs have been described before[9].siRNAs for MKL1 and WDR5 were purchased froMDharmacon.Transient transfections were performed with LipofectaMine LTX(Invitrogen).Luciferase activities were assayed 24-48 h after transfection using a luciferase reporter assay system(Promega).All experiments were repeated three times.

Ch IP and Re-Ch IP assay

ChIPassays were performed essentially as described before[20-21].A liquots of lysates containing 200μg of nuclear protein wereused for each immunoprecipitation reaction with anti-MKL1(Santa Cruz),anti-BRG1(Abcam),anti-WDR5(Bethyl Laboratories),antidimethylated H3K4(Millipore/Upstate),and anti-trimethylated H3K4(Millipore/Upstate).For re-ChIP,immune complexes were eluted with the elution buffer(1%SDS,100mmol/L NaCO3),diluted with the re-ChIP buffer(1%Triton X-100,2 mmol/L EDTA,150 mmol/L NaCl,20 mmol/L Tris pH 8.1),and subject to immunoprecipitation with a second antibody ofinterest.All experiments were repeated three times.

RNA extraction and real-time PCR

RNA was extracted using an RNeasy RNA isolation kit(Qiagen).Reverse transcriptase reactions were performed using a SuperScript First-strand synthesis system(Invitrogen).Real-time PCR reactions were performed on an ABISTEPONEPlus(Life Tech)with primers and Taqman probes purchased froMApplied Biosystems.All experimentswere repeated three times.

In vitro HMT assay

In vitro histone methyltransferase assay was performed as described before[22].Brie fly,precipitated immune complex was mixed with histone H3(Millipore),S-adenosyl methionine(SAM,Sigma),BSA,and MAB buffer(50 mmol/L Tris pH 8.5,20 mmol/L KCl,10 mmol/L MgCl2,10 mmol/Lβ-mercaptoethanol,250 mmol/L sucrose).A fter incubation at 37°C overnight,SDS loading buffer wasadded to stop reactions,and the methylation of histone H3 was deterMined by Western blotting.

Statistical analysis

One-way ANOVA with post-hoc Scheffe analyses were performed using an SPSS package.P values smaller than 0.05 were considered statistically signi ficant(*).

Results

MKL1 is associated with a histone H3K 4 methyltransferase activity in macrophages

Wehavepreviously shown that MKL1mediates LPSinduced in flammatory response by in fluencing histone H3K4 methylation[9].We asked whether MKL1 Might be in a complex with an catalytically capable H3K4 methyltransferase in macrophages.To this end,we performed In vitro histone methyltransferase(HMT)assay.MKL1 was isolated froMTHP-1 cells by immunoprecipitation and incubated with recombinant histone H3 in the presence of the methyl donor SAM;RbBP5,a known component of the H3K4 methyltransferase complex[13],was included as a positive control whereas pre-immune IgG was included as a negative control.As shown in Fig.1A,MKL1 was coprecipitated with an activity suf fi cient to catalyze H3K4 methylation.Re-ChIP assay showed that in response to TNF-αstimulation MKL1 was incorporated into a complex with WDR5,a well-de fi ned component of themammalian H3K4methyltransferasecomplex[23],on the promoter regions of several pro-in flammatory mediators(Fig.1B).Indeed,TNF-αtreatmentenhanced the occupancies of WDR5 on gene promoters in macrophages;MKL1 depletion with siRNA,however,signi fi cantly downregulated thebinding of WDR5(Fig.1C),suggesting that MKL1 Might play a role in recruiting WDR5 to the promoter regions of proin flammatory mediators.Ofinterest,we also observed that MKL1 was responsible for recruiting BRG1(Fig.1D),the catalytic subunit of the mammalian chromatin remodeling complex,and,to a lesser extent,BRM(Fig.1E)to thegenepromoters.Taken together,we conclude that MKL1 is associated with a histone H3K4 methyltransferase complex that includes WDR5,BRG1,and possibly BRM.

WDR5 cooperates with MKL1 to activate proin flammatory transcription

Next,weprobed the involvementof WDR5 in TNF-α induced trans-activation of pro-in flammatory mediators in macrophages.As shown in Fig.2A,WDR5 was a relatively weak activator of pro-in flammatory genes in reporter assays.In the presence of MKL1,however,WDR5 markedly enhanced the trans-activation of proin flammatory genes by TNF-α.Additionally,WDR5 cooperated with MKL1 to upregulate the activity of a κB reporter constructed by fusing two tandeMrepeatsof the NF-κB responseelement to the luciferasegene(Fig.2B).On the contrary,WDR5 knockdown by siRNA(Fig.2C for ef fi ciency)attenuated the induction of endogenous pro-in flammatory mediators by TNF-α(Fig.2D).Collectively,these data suggest that WDR5 might play a role in mediating the activation of proin flammatory genes in response to TNF-αtreatment in macrophages.

WDR5 and BRG1 regulate histone H 3K 4 methylation surrounding gene promoters

Next,we tackled the potential mechanisMwhereby WDR5 contributes to the regulation of pro-in flammatory transcription.Knockdown of WDR5 almost completely erased histone H3K4 di-methylation(Fig.3A)and tri-methylation(Fig.3B),two proMinent markers for transcriptional activation[24],surrounding the proximal promoter regions of pro-in flammatory genes.Of note,BRG1 also appeared to play a role in the regulation of histone H3K4 methylation surrounding gene promoters because BRG1 knockdown achieved siMilar effects as WDR5 knockdown(Fig.3C-3D),suggesting of a possible interplay between WDR5 and BRG1 in the regulation of pro-in flammatory transcription.Indeed,Re-ChIP assays showed that WDR5 formed acomplex with BRG1 on theproximal promoter regions of pro-in flammatory genes in macrophages treated with TNF-α(Fig.4A).Therefore,WDR5 might regulate TNF-αinduced pro-in flammatory transcription by interacting with BRG1 to promote histone H3K4 methylation.

MKL1 mediates the crosstalk between WDR5 and BRG1

Finally,we asked whether MKL1 could broker the communication between WDR5 and BRG1 to regulate TNF-αinduced pro-in flammatory transcription.Depletion of MKL1 with siRNA signi fi cantly weakened the interaction between WDR5 an BRG1 on the proximal promoter regions of pro-in flammatory genes(Fig.4B).Similarly,the interaction between WDR5 and BRG1 on the proximal promoter regions of pro-in flammatory genes was much stronger in bone-marrow derived macrophages(BMDM)isolated froMw ild type(WT)mice than froMMKL1 knockout(KO)Mice(Fig.4C).Finally,we found that co-expression of WDR5 and BRG1 enhanced the 2xκB reporter activity in WT mouse embryonic fi broblast(MEF)cells but not in KO MEF cells;re-introduction of ectopic MKL1 restored the cooperation between WDR5 and BRG1(Fig.4D).In summary,we propose that MKL1 could moderate the crosstalk between different epigenetic factors to stimulate TNF-αinduced pro-in flammatory transcription.

Discussion

Fig.1 MKL1 is associated with histone H3K 4 methyltransferase activity in macrophages.A:Whole cell lysates were extracted froMTHP-1 cells.HMTassay wasperformed asdescribed in Methods.Whiteasterisk,non-speci fi c band.B:THP-1 cellswere treated with or without TNF-αfor 12 hours.Re-ChIPassay wasperformed with indicated antibodies.C-D:THP-1 cellswere transfected with siRNA targeting MKL1 or scrambled siRNA(SCR)followed by treatment with TNF-αfor 12 hours.ChIPassay wasperformed with anti-WDR5(C),anti-BRG1(D),or anti-Brm(E).

Epigenetic regulation of gene expression during the pathogenesis of human diseases remains an intriguing and everlasting area of research[25-27].We present evidence here that MKL1 plays an essential role in bridging the dialog between BRG1,a chromatin remodeling protein,and WDR5,a subunit of the H3K4 methyltransferase complex,to stimulate the transcription of NF-κB dependent pro-in flammatory transcription.Several independent investigations have implicated BRG1 in the regulation of H3K4 methylation.For instance,Zager and Johnson have shown that during renal ischeMia-reperfusion injury,enhanced binding of BRG1 to gene promoters is paralleled by progressiveelevationsof H3K4 methylation although it remains undeterMined whether there is a cause-effect relationship[28].Davis et al have reported that treatment of the BRG1 null SW-13 cells with HDAC inhibitors restores BRG1 expression and at the same time stimulates H3K4 hypermethylation[29].We have previously shown that BRG1 is essential in maintaining H3K4 methylation on the endothelin(ET-1)gene promoter,likely through interacting with WDR5,in response to angiotensin II stimulation[22].These data clearly establish a role for BRG1 in regulating locusspeci fi c H3K4 methylation.It isnot known at this point how genomewide H3K4 methylation patterns would be in fluenced by BRG1.In human embryonic steMcells,BRG1 has been found to co-occupy with monomethylated H3K4,catalyzed by the MLL3/4-containing COMPASScomplex[30],on early developmental enhancers[31].In cancer cells,loss-of-function mutation of BRG1 appears to in fluence genomewide distribution of H3K4 trimethylation although the mechanisMis not clear[32].Future studies should make an effort in deciphering the connection between BRG1 and H3K4 methylation in a cell-and context-dependent manner.

Fig.3 WDR5 and BRG1 regulate histone H3K 4 methylation surrounding gene promoters.A-B:THP-1 cells were transfected with siRNA targeting WDR5 or scrambled siRNA(SCR)followed by treatment with TNF-αfor 12 hours.ChIP assay was performed with anti-H3K4Me2(A)or anti-H3K4Me3(B).C-D:THP-1 cellswere transfected with siRNA targeting BRG1 or scrambled siRNA(SCR)followed by treatment with TNF-αfor 12 hours.ChIP assay was performed with anti-H3K4Me2(C)or anti-H3K4Me3(D).

Fig.4 MKL1 mediates the crosstalk between WDR5 and BRG1.A:THP-1 cellswere treated with or without TNF-αfor 12 hours.Re-ChIP assay was performed with indicated antibodies.B:THP-1 cells were transfected with siRNA targeting MKL1 or scrambled siRNA(SCR)followed by treatment with TNF-αfor 12 hours.Re-ChIPassay wasperformed with indicated antibodies.C:MEF cells isolated froMw ild type(WT)or MKL1 knockout(KO)micewere treated with or without TNF-αfor 12 hours.Re-ChIPassay wasperformed with indicated antibodies.D:AκB reporter was transfected into WTor KOMEFcellswith MKL1,BRG1,and/or WDR5 followed by treatment with TNF-αfor 12 hours.Luciferase activities were normalized by both protein concentration and GFP fluorescence.

We show that WDR5 cooperates with BRG1 to stimulate NF-κB dependentpro-in flammatory transcription.Of note,WDR5 has been reported to forMextensive communicationswith other epigenetic factors to regulate transcription.For instance,WDR5 can interact with the acetyltransferase p300 in cancer cells and renal epithelial cells to regulate p53-dependent[33]and SMAD-dependent[34]transcription,respectively.WDR5 can also collaborate with PRMT5,a histone arginine methyltransferase,to activate the transcription of class II major histocompatibility complex(MHC II)genes[35].Ofinterest,the interaction between WDR5 and GClnc1,a long non-coding RNA(lncRNA),ensures its recruitment to a subset of pro-metastasis genes to activate transcription in gastric cancer cells[36],suggesting thatnon-protein factorscould also contribute to the tethering of WDR5 to selective promoters although it is not clear whether these processes are independent of each other or act in concert.The dynamic interplay between WDR5 and other epigenetic factors likely re flect the extricate nature of transcriptional regulation,which deserves further investigation.

Our data suggest that MKL1 contributes to NF-κB dependent pro-in flammatory transcription by moderating the crosstalk between WDR5 and BRG1.Mounting evidence suggests that MKL1 relies heavily on the epigenetic machinery to regulate transcription[8-10,21-22,34,37-41].In keeping with our fi nding,Yu et al.have demonstrated that MKL1 is responsible for the dissemination of H3K4 trimethylation on the promoter regions of genes involved in in flammation across the genome[42].It remains unanswered whether this function of MKL1 relieson WDR5 and/or BRG1.ChIP-seq analysis of MKL1 in macrophages has found that in addition to NF-κB,MKL1 also preferentially binds to the target promoters of SRF,STAT,and IRF1,all of which are key regulators ofin flammatory response[43].It would be interesting to determine whether MKL1 exploits a siMilar strategy to stimulate the transcription of those target genes.

In summary,wepresent evidence to show that MKL1 mediates TNF-αinduced pro-in flammatory transcription by bridging the crosstalk between BRG1 and WDR5.A recent study has found that small peptides that disrupt the interaction between WDR5 and MLL1/SET1 speci fi cally inhibits the activity of MLL1/SET1-containing COMPASS[44].It is possible to employ this strategy to interfere with the MKL1-WDR5-BRG1 interaction in order to control cellular in flammatory response in an attempt to treat in flammation-related diseases.

Acknow ledgements

This work was support,in part,by the National Natural Science Foundation of China(81570420).M.M.F.is supported by the Qinglan Project of the Education Commission of Jiangsu Province.