Negative regulation of NF-κB action by Set9-mediated lysine methylation of the RelA subunit
2009; Springer Nature; Volume: 28; Issue: 8 Linguagem: Inglês
10.1038/emboj.2009.55
ISSN1460-2075
AutoresXiaodong Yang, Bo Huang, Mingxi Li, Acacia Lamb, Neil L. Kelleher, Lin‐Feng Chen,
Tópico(s)interferon and immune responses
ResumoArticle5 March 2009free access Negative regulation of NF-κB action by Set9-mediated lysine methylation of the RelA subunit Xiao-Dong Yang Xiao-Dong Yang Department of Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, USA Search for more papers by this author Bo Huang Bo Huang Department of Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, USA Search for more papers by this author Mingxi Li Mingxi Li Department of Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, USA Search for more papers by this author Acacia Lamb Acacia Lamb Department of Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, USA Search for more papers by this author Neil L Kelleher Neil L Kelleher Department of Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, USA Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA Search for more papers by this author Lin-Feng Chen Corresponding Author Lin-Feng Chen Department of Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, USA Search for more papers by this author Xiao-Dong Yang Xiao-Dong Yang Department of Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, USA Search for more papers by this author Bo Huang Bo Huang Department of Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, USA Search for more papers by this author Mingxi Li Mingxi Li Department of Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, USA Search for more papers by this author Acacia Lamb Acacia Lamb Department of Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, USA Search for more papers by this author Neil L Kelleher Neil L Kelleher Department of Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, USA Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA Search for more papers by this author Lin-Feng Chen Corresponding Author Lin-Feng Chen Department of Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, USA Search for more papers by this author Author Information Xiao-Dong Yang1, Bo Huang1, Mingxi Li1, Acacia Lamb1, Neil L Kelleher1,2 and Lin-Feng Chen 1 1Department of Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign, Urbana, IL, USA 2Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA *Corresponding author. Department of Biochemistry, University of Illinois at Urbana-Champaign, 600 S Mathews Avenue, Urbana, IL 61801, USA. Tel.: +1 217 333 7764; Fax: +1 217 244 5858; E-mail: [email protected] The EMBO Journal (2009)28:1055-1066https://doi.org/10.1038/emboj.2009.55 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Proper regulation of NF-κB activity is critical to maintain and balance the inflammatory response. Inactivation of the NF-κB complex relies in part on the proteasome-mediated degradation of promoter-bound NF-κB, but the detailed molecular mechanism initiating this process remains elusive. Here, we show that the methylation of the RelA subunit of NF-κB has an important function in this process. Lysine methyltransferase Set9 physically associates with RelA in vitro and in vivo in response to TNF-α stimulation. Mutational and mass spectrometric analyses reveal that RelA is monomethylated by Set9 at lysine residues 314 and 315 in vitro and in vivo. Methylation of RelA inhibits NF-κB action by inducing the proteasome-mediated degradation of promoter-associated RelA. Depletion of Set9 by siRNA or mutation of the RelA methylation sites prolongs DNA binding of NF-κB and enhances TNF-α-induced expression of NF-κB target genes. Together, these findings unveil a novel mechanism by which methylation of RelA dictates the turnover of NF-κB and controls the NF-κB-mediated inflammatory response. Introduction The transcription factor NF-κB has an important function in regulating immune and inflammatory responses, apoptosis, cell proliferation and differentiation and tumorigenesis (Baldwin, 1996; Ghosh et al, 1998). The prototypical NF-κB complex, consisting of a heterodimer of p50 and RelA, is sequestered in the cytoplasm by its association with its inhibitor IκBα (Baldwin, 1996; Ghosh et al, 1998; Karin, 1999). NF-κB is activated by a variety of stimuli, including various proinflammatory cytokines, T-cell receptor signals and viral and bacterial products. Stimulation by these agonists leads to the activation of the IκB kinase complex (IKK), which then phosphorylates IκBα, triggering its rapid ubiquitination and proteolytic degradation in the 26S proteasome (Karin and Ben-Neriah, 2000). The liberated NF-κB heterodimer rapidly translocates to the nucleus, in which it engages its cognate κB enhancer and stimulates gene expression through the transcriptional activation domain of RelA. NF-κB activates a variety of genes involved in different biological processes including inflammation, proliferation and cell survival (Ghosh and Karin, 2002). Because of its pleiotropic effect and its important role in the inflammatory response, activation of NF-κB is tightly controlled at multiple levels. Activated NF-κB needs to be terminated after induction to limit the inflammation, as sustained NF-κB activity and inflammation lead to a variety of diseases including asthma, arthritis and septic shock (Ghosh and Karin, 2002). In fact, constitutively active NF-κB is frequently encountered in a wide variety of tumours and in some rheumatic conditions such as rheumatoid arthritis and systemic lupus erythematosus (Sethi et al, 2008). Cells use multiple mechanisms for the termination of NF-κB, including NF-κB-dependent resynthesis of IκBα and the deubiquitination of upstream signalling molecules by deubiquitinating enzymes A20 and CYLD (Hayden and Ghosh, 2008; Sun, 2008). A20 and CYLD are also target genes of NF-κB, which together with resynthesized IκBα form a negative feedback regulation of NF-κB (Hayden and Ghosh, 2008). In addition, recent studies also indicate that proteasome-mediated degradation of nuclear DNA-bound NF-κB provides another layer of termination independent of negative feedback regulation (Saccani et al, 2004; Natoli and Chiocca, 2008). These different levels of regulation define the tight control of NF-κB action and the inflammatory response. The highly controlled degradation of proteins through the proteasome-dependent pathway represents a key mechanism for the regulation of a variety of signalling molecules, including transcription factors such as p53 and c-Jun (Muratani and Tansey, 2003; Dornan et al, 2004; Wertz et al, 2004). Termination of NF-κB also requires the polyubiquitination and degradation of the nuclear NF-κB (Natoli and Chiocca, 2008). Saccani et al reported that NF-κB response was terminated even in the absence of the inhibitor IκBα, and the inhibition of the proteasome function in this condition increased the expression of NF-κB target genes. These findings suggest that nuclear degradation of NF-κB contributes to the termination of NF-κB activity and prevents excessive induction of genes involved in inflammation (Saccani et al, 2004; Natoli and Chiocca, 2008). Once NF-κB binds to target gene promoters and activates gene transcription, a significant amount of RelA, estimated to be 25%, is ubiquitinated and degraded by the 26S proteasome in the nucleus (Saccani et al, 2004). In support of this, several E3 ligases, including SOCS1 and PDLIM2, have been identified to mediate the ubiquitination of NF-κB (Ryo et al, 2003; Maine et al, 2007; Tanaka et al, 2007). However, what signal triggers the ubiquitination and degradation of NF-κB is not clear. Set9 (also known as Set7)-mediated lysine methylation has emerged recently as a key posttranslational modification that regulates the function of histone and non-histone proteins (Wang et al, 2001; Nishioka et al, 2002; Chuikov et al, 2004; Kouskouti et al, 2004; Huang et al, 2006; Huang and Berger, 2008). Set9 was originally identified as a methyltransferase that methylates lysine 4 of histone H3 (Wang et al, 2001; Nishioka et al, 2002); more recent studies indicate that Set9 is a methyltransferase preferentially for non-histone proteins, as studies from Set9 knock-out cells indicate no changes at the level of lysine methylation on histones (Ivanov et al, 2007; Kurash et al, 2008). In this regard, more non-histone proteins, especially transcription factors, have been identified as targets for Set9. p53 is the first transcription factor that was identified to be regulated by Set9-mediated methylation (Chuikov et al, 2004). Methylation of p53 by Set9 at lysine 372 stabilizes p53 and enhances its transcriptional activity, probably by enhancing the acetylation of p53, a modification that competes for the same lysine responsible for ubiquitination and subsequent degradation of p53 (Chuikov et al, 2004; Huang et al, 2006; Ivanov et al, 2007; Kurash et al, 2008). In an effort to investigate the potential regulation of NF-κB signalling by lysine methylation, we find that the RelA subunit of NF-κB is monomethylated by Set9 at lysines 314 and 315. Methylation of RelA triggers the degradation of the activated form of the NF-κB complex and down-regulates NF-κB target gene expression. Results RelA is methylated by Set9 in vitro and in vivo in response to TNF-α stimulation To investigate whether NF-κB is subjected to lysine methylation and the potential functional consequence of this modification, we first examined whether NF-κB could be a potential target of Set9 (Wang et al, 2001; Nishioka et al, 2002). In an in vitro methylation assay, when recombinant RelA was incubated with recombinant Set9 in the presence of 3H-S-adenosine-methionine (SAM), we found that recombinant RelA was methylated (Figure 1A, lane 4). Without either SAM or Set9, RelA was not methylated (Figure 1A, lanes 2 and 3), indicating that RelA is indeed methylated by Set9. As a positive control, histone H3 was also methylated by Set9 under the same condition as RelA, validating the in vitro methylation assay (Figure 1A, lane 5). However, when p50 and IκBα, two other key components in the NF-κB signalling pathway, were subjected to Set9-mediated methylation, no methylation was observed (Figure 1B). Figure 1.Set9 methylates RelA in vitro and in vivo. (A) Set9 methylates RelA in vitro. In vitro methylation was performed with 3H-S-adenosine-methionine (3H-SAM), recombinant Set9 and RelA or Histone H3 as indicated. Autoradiography and Coommassie Brilliant Blue staining (CBB) were used to show methylation and protein levels, respectively. (B) Set9 does not methylate p50 or IκBα. In vitro methylation of recombinant RelA, p50 or IκBα by Set9 were performed and detected as described in (A). (C) Enzymatic activity of Set9 is required for the methylation of RelA. In vitro methylation with RelA and recombinant wild-type (WT) Set9 or enzymatically inactive Set9-H297A mutant (Mut) of Set9 was performed as described in (A). Immunoblot (IB) analysis with anti-pan-methyl-lysine antibodies (α-pan-me-K) was used to detect the methylation of RelA. (D) TNF-α stimulates the methylation of RelA in vivo. U2OS cells were transfected with control or Set9 siRNA and stimulated with TNF-α as indicated. Immunoblot analysis of RelA immunoprecipitates from whole cell lysates was conducted with α-pan-me-K to detect methylation of RelA. (E) Only nuclear RelA is methylated upon TNF-α stimulation. U2OS cells were stimulated with TNF-α as indicated and fractionated. Methylation of RelA immunoprecipitates from cytoplasmic or nuclear extracts were assessed as described in (D). Download figure Download PowerPoint An anti-pan-methyl-lysine antibody, which has been successfully used to detect the methylation of TAF10 and histone H3 (Su et al, 2003; Kouskouti et al, 2004), was able to detect the in vitro methylated RelA (Figure 1C). Methylation of RelA was recognized by this antibody when RelA was methylated by wild-type (WT) Set9 (Figure 1C, lane 2). However, when an enzymatically inactive form of Set9 (Set9-H297A) (Wang et al, 2001; Nishioka et al, 2002) was used for the same assay, no signal was detected, further confirming the methylation of RelA by Set9 (Figure 1C, lane 3). We next examined whether endogenous RelA could be methylated in response to stimulation. Compared with unstimulated cells, TNF-α treatment of osteosarcoma U2OS cells led to increased methylation of RelA (Figure 1D). The methylated RelA appeared at 30 min and reached its maximal level around 60 min, then decreased at 90 min (Figure 1D, lanes 2–4), indicating a dynamic methylation status of RelA upon TNF-α stimulation. The signal recognized by the anti-pan-methyl-lysine antibodies in unstimulated cells probably represents a non-specific cross-reaction of the immunoprecipitates with the antibodies (Figure 1D, lane 1). To further examine whether TNF-α-induced methylation of RelA was mediated by Set9, we knocked down the expression of Set9 by siRNA. Depletion of Set9 by siRNA abolished TNF-α-induced methylation of RelA (Figure 1D). Fractionation experiments further demonstrate that only nuclear RelA undergoes signal-dependent methylation (Figure 1E). These results suggest that the activation-dependent RelA methylation requires endogenous Set9 and that nuclear RelA is likely an endogenous substrate for Set9. RelA is methylated at lysines 314 and 315 in vitro and in vivo Given that RelA is methylated in vitro and in vivo by Set9, we next aimed to identify the methylation site(s) of RelA. We purified bacterially expressed GST fusion proteins containing five different segments of RelA (Figure 2A) and used them as substrates for the in vitro methylation assay. Of these five fragments, only GST–RelA (271–364) was methylated by Set9 (Figure 2B, lane 5). There are six lysine residues on this fragment 271–364, so we further divided it into two different fragments: RelA (271–313) and RelA (311–347), with each fragment containing three lysine residues (Figure 2A). An in vitro methylation assay with these two fragments showed that the methylated lysine(s) were within the fragment RelA (311–347) (Figure 2C). To map the methylation site(s) and status, the in vitro methylated RelA (311–347) fragment was subjected to mass spectrometry analysis. Three different types of methylation were detected: single lysine monomethylation either at lysine 314 (K314me1, 30%) or lysine 315 (K315me1, 25%) or double lysine monomethylation at both lysines 314 and 315 (K314me1/K315me1, 35%) (Figure 2D). These data are consistent with the SET domain structure of Set9, which suggests that Set9 only monomethylates its substrate (Wilson et al, 2002; Kwon et al, 2003; Xiao et al, 2003). Figure 2.Set9 monomethylates RelA at lysines 314 and 315. (A) Schematic depiction of WT and deletion mutants of RelA. (B, C) In vitro methylation assay of deletion mutants of RelA shown in (A). (D) Mass spectrometry analysis of RelA fragment (a.a. 311–347) methylated by Set9. Top left: partial Fourier-Transform mass spectrum and monoisotopic masses. Both experimental (Exp) and theoretical (The) masses are shown. Bottom: fragmentation map depicting methylated lysines (circled) in all ECD fragment ions. Top right: percentage of fragments with different methylation states. (E) In vitro methylation of WT or point mutants of GST–RelA –(271–364) fragment. (F) In vitro methylation assay of full-length WT RelA and RelA-K314/315R mutant. (G) TNF-α stimulates the methylation of RelA at lysines 314 and 315. RelA or RelA-K314/315R reconstituted MEFs were stimulated with or without TNF-α (20 ng/ml), methylation of RelA was assessed as described in Figure 1D. ns represents a non-specific band. Download figure Download PowerPoint To further confirm the methylation sites, we mutated lysines 314 and 315, individually or in combination, to arginine in fragment RelA (271–364), and compared the methylation of these mutants with WT RelA. Mutation of either lysine 314 or 315 impaired the methylation of RelA (Figure 2E, lanes 2 and 3). However, when both lysines were mutated, the methylation was completely abolished (Figure 2E, lane 4), Conversely, mutation of other lysines within this fragment, lysines 301, 303, 310 or 343, barely affected the methylation level (Figure 2E, lanes 5–8), confirming that lysines 314 and 315 but not the other lysines are methylated by Set9 in vitro. Further supporting this notion, mutation of both lysines 314 and 315 to arginine in full-length RelA (designated as RelA-K314/315R) also abolished the methylation of RelA (Figure 2F). The abolished methylation of RelA-K314/315R derives directly from the mutated lysines rather than from the possible binding defect of this mutant to Set9, as RelA-K314/315R binds to Set9 at a similar level as the WT RelA (Supplementary Figure S1). To further investigate whether lysines 314 and 315 are methylated in vivo in response to stimuli, we reconstituted RelA-deficient mouse embryonic fibroblasts (MEFs) with WT RelA and RelA-K314/315R and examined the TNF-α-induced methylation of RelA. In the reconstituted MEFs, RelA and RelA-K314/315R express at a similar level as the endogenous RelA in WT MEFs (data not shown). In WT RelA reconstituted MEFs, TNF-α stimulation induced the methylation of RelA (Figure 2G, lane 2). However, TNF-α-induced methylation of RelA was abolished in RelA-K314/315R reconstituted MEFs (Figure 2G, lane 4). The background signal, which was also detected in RelA-deficient MEFs (data not shown), probably represents a non-specific protein captured in the co-immunoprecipitation (Figure 2G). All together, these results show that Set9 methylates RelA at the two lysine residues 314 and 315 in vivo in response to TNF-α. Hereafter, we refer to RelA-K314/315R mutant as the methylation-deficient mutant of RelA. Set9 associates with RelA in vitro and in vivo We next assessed the physical interaction between RelA and Set9 by in vivo co-immunoprecipitation assay. Immunoprecipitation of Flag-Set9 from transfected HEK293T cells co-purified T7-RelA (Figure 3A), indicating a physical interaction between RelA and Set9 in vivo. Reversed immunoprecipitation of T7-RelA also shows such an interaction (Figure 3B). In an in vitro GST pull-down assay, GST–Set9 pulled down a significant amount of RelA (Figure 3C), showing a direct interaction between Set9 and RelA. We next investigated whether there was a physical association between endogenous RelA and Set9. When HEK293T cells were stimulated with TNF-α, and endogenous Set9 was immunoprecipitated and examined for the associated RelA, we found that TNF-α stimulated the interaction between RelA and Set9 (Figure 3D), although levels of association varied at different time points. These data suggest that Set9 associates with RelA and induces its methylation in response to TNF-α stimulation. Figure 3.Set9 associates with RelA in vitro and in vivo. (A, B) Set9 interacts with RelA in vivo. HEK293T cells were transfected with T7-tagged RelA and Flag-tagged Set9 as indicated. At 40 h after transfection, T7-RelA or Flag-Set9 immunoprecipitates were prepared from whole-cell lysates and immunoblotted for Flag-Set9 or T7-RelA, respectively. (C) Set9 interacts with RelA in vitro. GST or GST–Set9 was incubated with recombinant RelA and precipitated with glutathione beads. The recovered materials were immunoblotted for RelA. (D) TNF-α stimulates the interaction between endogenous RelA and Set9. HEK293T cells were left untreated or stimulated with TNF-α (20 ng/ml) for indicated time periods. Set9 immunoprecipitates were prepared from whole-cell lysates and immunoblotted for RelA. Download figure Download PowerPoint Set9-mediated methylation of RelA negatively regulates the transcriptional activation of NF-κB We next investigated the potential effects of Set9 on the RelA-dependent transcriptional activation of NF-κB. We used siRNA to knock-down the expression of Set9 in U2OS cells and evaluated TNF-α-induced activation of NF-κB target genes by quantitative real-time PCR. Depletion of Set9 (Supplementary Figure S2) enhanced TNF-α-induced transcription of both IL-8 and IL-6 genes compared with cells treated with control siRNA (Figure 4A, left and middle panels). Interestingly, depletion of Set9 barely affected TNF-α-induced expression of another NF-κB target gene, A20 (Figure 4A, right panel), indicating that Set9 is involved in the down-regulation of a subset of NF-κB targets and that the negative regulation of NF-κB by Set9 might be promoter specific. Similar results were obtained when Set9 was down-regulated in lung epithelial A549 cells using another siRNA targeting a different region of Set9 (Supplementary Figure S3), excluding a possible off-target effect of the siRNA in U2OS cells. Collectively, these results indicate that Set9 negatively regulates the expression of a subset of NF-κB target genes in different cell types. Figure 4.Set9 negatively regulates NF-κB activity. (A) Depletion of Set9 up-regulates transcription of a subset of NF-κB target genes. U2OS cells were transfected with control siRNA or Set9 siRNA. At 60 h after transfection, cells were left untreated or stimulated with TNF-α (20 ng/ml) for 1, 3 or 5 h. Total RNAs were isolated and relative levels of mRNA for IL-8, IL-6 and A20 were measured by quantitative real-time PCR. (B) Depletion of Set9 enhances TNF-α-induced activation of NF-κB. HEK293T cells were transfected with control siRNA or Set9 siRNA. After 36 h, cells were re-transfected with 5XκB luciferase reporter and Set9 siRNA resistant mutant and stimulated with TNF-α (2 ng/ml) for 5 h. Luciferase activity was measured and expressed as fold induction after normalization with Renilla luciferase. (C) Set9 inhibits TNF-α-induced activation of NF-κB. HEK293T cells were transfected with 5XκB luciferase reporter plasmid DNA and increasing amounts of plasmid DNA encoding WT Set9 or Set9-H279A. TNF-α-stimulated luciferase activity was measured as described in (B). (D) RelA-K314/315R displays more resistance than RelA WT to Set9-induced inhibition of RelA-mediated NF-κB activation. HEK293T cells were transfected with the expression vectors for RelA WT or RelA-K314/315R. After 24 h, cells were re-transfected with κB luciferase reporter plasmids together with increasing amounts of Set9. Luciferase activity was measured and reported as a percentage. (E) RelA-K314/315R displays enhanced and prolonged TNF-α-induced expression of NF-κB target genes in cells. RelA or RelA-K314/315R reconstituted MEFs were treated with TNF-α as indicated and the expression of IL-6, Nos-2 and A20 genes was assessed by real-time PCR as described in (A). (F) Set9 is differentially recruited to promoters of NF-κB target genes. U2OS cells were left untreated or stimulated with TNF-α (20 ng/ml) for 30, 60 or 90 min. ChIP assays were used to assess the binding of RelA and Set9 to the promoters of NF-κB targets IL-8 and A20. A representative PCR result from three independent experiments is shown. Download figure Download PowerPoint To show that the enhanced expression of NF-κB target genes is due to the specific depletion of Set9 by the siRNA but not to a non-specific effect of the siRNA, we generated an siRNA resistant Set9 construct and examined its ability to rescue the effect of Set9 depletion. Consistent with the real-time PCR data (Figure 4A), depletion of Set9 by siRNA enhanced TNF-α-induced activation of NF-κB in a κB-luciferase assay (Figure 4B). However, when the siRNA resistant Set9 was reintroduced into the knock-down cells at a similar level to the endogenous Set9 (Supplementary Figure S4), TNF-α-induced NF-κB activation was reduced to a level comparable to that without siRNA treatment (Figure 4B). These data further support the conclusion that specific depletion of Set9 enhances the TNF-α-induced activation of NF-κB and that Set9-mediated methylation of RelA negatively regulates the transcriptional activation of NF-κB. Additionally, when Set9 was exogenously expressed in cells, TNF-α-induced NF-κB activity was reduced in a dose-dependent manner (Figure 4C). This Set9-mediated inhibition depends on the enzymatic activity of Set9, as Set9-H297A failed to inhibit the κB-luciferase activity (Figure 4C). Similar to the TNF-α-activated NF-κB, WT Set9, but not Set9-H297A, also inhibited RelA-mediated activation of κB-luciferase activity (Supplementary Figure S5). These data are consistent with the Set9 knock-down experiment, indicating that Set9 functions as a negative regulator for TNF-α-mediated activation of NF-κB. To test whether the inhibitory effect of Set9 derives from the methylation of RelA at lysines 314 and 315, we examined the inhibitory effect of Set9 on WT RelA and RelA-K314/315R in a κB-luciferase assay. Transcriptional activation of WT RelA was significantly inhibited by Set9 (Figure 4D). However, transcriptional activity of RelA-K314/315R was only slightly affected by Set9 (Figure 4D). Furthermore, when we examined the TNF-α-induced expression of NF-κB target genes in MEFs reconstituted with WT RelA or RelA-K314/315R, we found that TNF-α-induced expression of IL-6 and Nos-2 was enhanced and prolonged in RelA-K314/315R reconstituted MEFs compared with the expression of the same genes in WT RelA reconstituted MEFs. In contrast, A20, whose expression was not regulated by Set9 (Figure 4A; Supplementary Figure S3), showed no significant expression difference between reconstituted WT and RelA-K314/315R MEFs. Collectively, these data, together with the real-time PCR results from Set9 knock-down cells (Figure 4A; Supplementary Figure S3), strongly indicate that methylation of RelA at lysines 314 and 315 by Set9 negatively regulates the expression of a subset of NF-κB target genes in response to TNF-α. Set9 is differentially recruited to the promoters of NF-κB target genes Having identified that Set9 negatively regulates the activation of NF-κB in a promoter-specific manner, we next used chromatin immunoprecipitation (ChIP) assays to examine the recruitment of Set9 to the promoters of NF-κB target genes. We stimulated U2OS cells with TNF-α for various time points and examined the binding of RelA and Set9 to the promoters of IL-8 and A20, whose expression is differentially regulated by Set9 (Figure 4A). As expected, RelA was recruited to the promoters of IL-8 after TNF-α stimulation. Interestingly, Set9 was found to reside on the promoter even before the stimulation (Figure 4F, left panel). After stimulation, promoter-bound Set9 decreased rapidly (∼30 min) and then increased gradually (Figure 4F), representing a dynamic binding of Set9 on the promoter in response to TNF-α stimulation. However, for the promoter of A20, Set9 was not recruited to the promoter before or after TNF-α stimulation even though RelA was still recruited to the promoter after TNF-α stimulation (Figure 4F, right panel). These ChIP data suggest that Set9 selectively occupies the promoters of NF-κB target genes and that preclearance of Set9 from the occupied promoters might be important for the initial recruitment and activation of NF-κB. Methylation of RelA by Set9 down-regulates the stability of RelA A role for lysine methylation in regulating the stability of transcription factors, including p53 and ERα, was reported recently (Chuikov et al, 2004; Huang et al, 2006; Kurash et al, 2008). Therefore, we examined whether Set9 also affected the stability of RelA. Co-expression of WT Set9 but not Set9-H297A in U2OS cells down-regulated the expression of RelA (Figure 5A), suggesting that Set9-mediated methylation of RelA might be involved in the down-regulation. Treatment with MG-132 gradually reversed the expression level of RelA and stimulated the accumulation of RelA ubiquiitnation in a time-dependent fashion (Figure 5B and C), suggesting that down-regulation of RelA is likely due to the Set9-induced ubiquitination and proteasome-mediated degradation of RelA. Further supporting these findings, we found that the co-expression of Set9 did not alter the RelA mRNA levels (Figure 5D). Figure 5.Set9 induces the degradation of RelA. (A) Set9, but not Set9-H297A, down-regulates the expression of RelA. U2OS cells were transfected with the expression vectors for T7-RelA and increasing amounts of WT Set9 or Set9-H279A. Whole cell lysates were immunoblotted as indicated. (B) Proteasome inhibitor MG-132 blocks Set9-induced down-regulation of RelA. U2OS cells were transfected with the expression vectors for T7-RelA and Flag-Set9. At 24 h after transfection, cells were treated with MG-132 (20 μM) for various time points, and whole cell lysates were immunoblotted as indicated. (C) Set9 induces polyubiquitination of RelA. U2OS cells were transfected and treated as described in (B). T7-RelA immunoprecipitates were prepared and immunoblotted for ubiquitin. (D) U2OS cells were transfected with the expression vectors for T7-RelA and WT Set9 or Set9-H279A. Quantitative real-time PCR with primers spanning the T7 tag and the N-terminus of RelA was performed to measure mRNA levels of T7-RelA. Data represent the average of three independent experiments. (E) Set9 shortens the half-life of RelA. U2OS cells were transfected with the expression vectors for T7-RelA or T7-RelA-K314/315R alone or together with Set9. At 24 h after transfection
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