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*(L)-ARGININE
Molecular Endocrinology 20 (7): 1562-1573
Copyright © 2006 by The Endocrine Society

Coactivator-Associated Arginine Methyltransferase-1 Enhances Nuclear Factor-{kappa}B-Mediated Gene Transcription through Methylation of Histone H3 at Arginine 17

Feng Miao, ShuLian Li, Valerie Chavez, Linda Lanting and Rama Natarajan

Gonda Diabetes Center, Beckman Research Institute of City of Hope, Duarte, California 91010

Address all correspondence and requests for reprints to: Dr. Rama Natarajan, Gonda Diabetes Center, Beckman Research Institute of the City of Hope, 1500 East Duarte Road, Duarte, California 91010. E-mail: RNatarajan{at}coh.org.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Coactivator-associated arginine methyltransferase-1 (CARM1) is known to enhance transcriptional activation by nuclear receptors through interactions with the coactivators p160 and cAMP response element binding protein-binding protein (CBP) and methylation of histone H3 at arginine 17 (H3-R17). Here, we show that CARM1 can act as a coactivator for the transcription factor nuclear factor-{kappa}B (NF-{kappa}B) and enhance NF-{kappa}B activity in a CBP (p300)-dependent manner. This enhancement in 293T cells was abolished by cotransfection with a specific short hairpin RNA targeted to knockdown CARM1. Chromatin immunoprecipitation demonstrated CARM1 recruitment in vivo to the promoters of NF-{kappa}B p65-regulated genes along with CBP and steroid receptor coactivator-1. This was accompanied by an increase in histone H3-R17 methylation as well as H3-K9 and H3-K14 acetylation, and a decrease in H3-citrulline. Immunoprecipitation with anti-p65 antibody revealed that CARM1 physically interacts with NF-{kappa}B p65. Furthermore, we demonstrated the physiological significance by observing that similar events occurred when THP-1 monocytic cells were stimulated with TNF-{alpha} or with S100b, a ligand for the receptor of advanced glycation end products, both of which are associated with diabetic complications and also known inducers of NF-{kappa}B and inflammatory genes in monocytes. These results demonstrate that CARM1 participates in NF-{kappa}B-mediated transcription through H3-R17 methylation and support a nonnuclear receptor-associated function for CARM1. They also demonstrate for the first time that CARM1 occupancy, histone H3-R17 methylation, and citrullination are regulated at the promoters of inflammatory genes in monocytes, thereby suggesting a novel role for histone arginine modifications in inflammatory diseases.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
NUCLEAR FACTOR-{kappa}B (NF-{kappa}B) is an inducible transcription factor that plays a pivotal role in cellular responses by regulating the expression of a variety of genes involved in various cellular processes ranging from proliferation, immune responses, inflammation, and cell survival (1, 2, 3). Although NF-{kappa}B consists of homo- or heterodimers of different subunits such as p50, p52, p65/RelA, RelB, and c-Rel, it is predominantly a heterodimeric complex of p65/RelA and p50. In most cells, p65/RelA and p50 exist in an inactive form in the cytoplasm bound to an inhibitory protein, I{kappa}B-{alpha}. Phosphorylation of the inhibitory I{kappa}B-{alpha} subunit by an I{kappa}B kinase complex results in ubiquitination and degradation of I{kappa}B-{alpha} and translocation of the p50-p65 dimer to the nucleus, where it binds to cognate {kappa}B enhancer elements on gene promoters and modulates their expression. A major negative feedback mechanism to down-regulate the activated NF-{kappa}B is the NF-{kappa}B-dependent expression of the I{kappa}B-{alpha} gene. Newly synthesized I{kappa}B-{alpha} shuttles between the cytoplasm and the nucleus and sequesters NF-{kappa}B from the promoters, thus facilitating the return of the NF-{kappa}B-I{kappa}B{alpha} complex to the cytoplasm and terminating the NF-{kappa}B transcriptional response (4, 5).

NF-{kappa}B-dependent gene expression involves various coactivators that function by facilitating, modifying, recruiting or bridging the sequence-specific activators to the basal transcriptional machinery and altering chromatin structure. The p65 component of NF-{kappa}B can bind to the coactivator, cAMP response element binding protein-binding protein (CBP), and its structural homolog p300 (6, 7, 8). Phosphorylation of p65 by protein kinase A has been shown to stimulate NF-{kappa}B-dependent gene expression by enhancing p65 association with CBP (8, 9). CBP-associated protein p/CAF is also a component of the NF-{kappa}B coactivator complex (10). Additionally, NF-{kappa}B-dependent gene expression involves a second class of transcriptional coactivators, namely steroid receptor coactivator-1 (SRC-1) and nuclear receptor coactivator-1 that interact with p50 and potentiate NF-{kappa}B-mediated transactivation (10, 11).

NF-{kappa}B and nuclear receptors exhibit certain key similarities with respect to their specific requirements for p160 coactivators and their acetyltransferase functions (10). Both NF-{kappa}B activity and nuclear receptor-dependent gene expression require a coactivator complex with the p160 family members. These coactivators contain the consensus LXXLL motif that is important for the interaction of the coactivators with both nuclear receptors and CBP (12, 13, 14). This suggests that LXXLL-mediated interactions between the p160 family members and CBP underlie the assembly of both the NF-{kappa}B and the nuclear receptor coactivator complexes. Furthermore, the histone acetyl transferase (HAT) activity of p/CAF was implicated in the activation of NF-{kappa}B-dependent gene expression and nuclear receptor activation (15), suggesting that both have the same selectivity in the type of HAT activity required for their function. However, it is not clear whether NF-{kappa}B and nuclear receptors use the same HAT-containing complexes. Furthermore, the extent of similarity between these two complexes is also unclear.

Coactivator-associated arginine methyltransferase-1 (CARM1) was originally isolated by a yeast two-hybrid screen while evaluating proteins that interact with the p160 family member glucocorticoid receptor interacting protein 1 (16). It enhances nuclear receptor function in a p160-dependent manner (17), although its activity reached a maximum only in the presence of the p160 and p300/CBP coactivators (17, 18, 19). The catalytic activity of CARM1 methyltransferase, as assessed by methylation of arginine (R) 17 of histone H3 (H3-R17), is required for nuclear receptor transactivation and this has been associated with gene activation (20, 21). The fact that CARM1 and CBP/p300 could cooperate to increase the transcriptional activity of the estrogen receptor (16, 17, 18, 19) indicated a potential cross talk between lysine (K) acetylation and H3-R17 methylation (22). In vitro, the CARM1 methylation activity was most effective on preacetylated histone, suggesting that CARM1 acts downstream of the histone acetylation (19, 22).

CBP/p300 and p160-dependent CARM1 coactivator functions have been mainly investigated among nuclear receptors. However, because CBP/p300 and p160 are also coactivators of NF-{kappa}B-dependent transcription (10), it can be speculated that CARM1 could be involved in NF-{kappa}B mediated transcription. Very recently, Covic et al. (23) reported that the expression of a subset of NF-{kappa}B-dependent genes in response to TNF-{alpha} or lipopolysaccharide stimulation is impaired in CARM1 knockout mouse embryo fibroblasts. By comparing CARM1 knockout cells with wild-type cells, they concluded that CARM1 can function as a promoter-specific regulator of NF-{kappa}B. In our current study, by using expressed short hairpin RNAs (shRNAs) targeted to knockdown CARM1, we provide novel evidence that CARM1 is required for NF-{kappa}B-mediated gene expression and that it enhances these NF-{kappa}B effects in a CBP (p300)-dependent manner. Using chromatin immunoprecipitation (ChIP) assays, we showed that, in response to TNF-{alpha} stimulation, there is increased recruitment of CARM1 to the promoters of NF-{kappa}B-dependent genes along with increased H3-R17 methylation and simultaneously decreased H3-citrulline. These were associated with other events related to the activation of NF-{kappa}B-mediated gene transcription such as recruitment of p65, CBP, SRC-1, and histone H3-K9/14 acetylation at these promoters. We further demonstrated for the first time the physiological relevance and implication of this histone arginine modification by showing that this H3-R17 methylation and citrulline connection occurs during TNF-{alpha} stimulation of human THP-1 monocytic cells. Furthermore, CARM1 occupancy at the TNF-{alpha} promoter was also increased in THP-1 cells by S100B, a ligand for the receptor for advanced glycation end products (RAGE), and a known inducer of NF-{kappa}B-dependent inflammatory genes in monocytes.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CARM1 Enhances p65-Mediated Transcription in the Presence of CBP (p300) and SRC-1 and Acts in a CBP (p300)-Dependent Manner
We first evaluated the optimal concentration of p65 needed for CARM1 coactivation functions, because it has been observed in nuclear receptor studies that the secondary coactivator, CARM1, exhibits more significant effects at low levels of expressed primary activator (19). 293T cells were cotransfected with a luciferase reporter construct containing a minimal TNF-{alpha} promoter (295TNF-{alpha} Luc, which includes the NF-{kappa}B response element), along with CBP, SRC-1, and CARM1 expression plasmids and various amounts of the p65 expression vector. Figure 1AGo shows that CARM1 can significantly enhance p65-mediated transcription of the TNF-{alpha} promoter in the presence of CBP and SRC-1 especially at lower concentrations of p65. The optimal p65 vector input range for maximal CARM1 coactivator effects in our system was between 10 and 20 ng. We then tested potential synergy among various coactivators by cotransfection of 293T cells with various combinations of p65, CBP (p300), SRC-1, and CARM1 expression vectors (Fig. 1BGo). It is seen that CARM1 can enhance NF-{kappa}B-mediated transcription in the presence of CBP (p300) alone (third set of bars), although the maximal luciferase activity was observed when p65, CBP (p300), SRC-1, and CARM1 were all cotransfected together (fourth bar set). Interestingly, it has been shown that, in nuclear receptor-mediated transcription, CARM1 acts as coactivator in which synergistic effects between CARM1 and p300 depends on p160 (17). The current observation reveals that CBP(p300) is the primary coactivator in NF-{kappa}B-mediated transcription. This is in contrast to nuclear receptor-mediated transcription, which uses p160 as the primary coactivator (17).


Figure 1
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  		Fig. 1. CARM1 Enhances p65-Mediated Transcription in a CBP- and SRC-1-Dependent Manner

A total of 3 x 105 293T cells was transiently transfected with the indicated expression plasmids in six-well plates for 24 h. Cell extracts were prepared and assayed for luciferase activity. A, Determination of the optimal p65 concentration needed for synergistic effects among the coactivators. A total of 75 ng of TNF{alpha}Luc, 150 ng of CBP, 150 ng of SRC-1, and 150 ng of CARM1 expression vectors were cotransfected with various amounts (0–80 ng) of p65 expression vector. B, Synergistic effect among various combinations of CARM1, CBP, and SRC-1 on p65-mediated transcription. Transfection assays were performed with 75 ng of TNF{alpha}Luc, 10 ng of p65, 150 ng of CBP, 150 ng of SRC-1, and increasing amounts (0, 50, 100 and 150 ng) of CARM1 expression vectors where indicated. Luciferase activity results are expressed as mean ± SEM of four independent experiments performed in triplicate. *, P < 0.05 vs. without CARM1, paired.

 
ShRNA-Mediated Knockdown of CARM1 Leads to a Decrease in the p65-Dependent Transcription Activity
Gene silencing by RNA interference in mammalian cells using small interfering RNAs (siRNAs) and expressed shRNAs has become a routine tool for the identification of components involved in cellular signaling and gene regulation pathways. We designed shRNAs to specifically knockdown CARM1 expression. We used a PCR-based approach (24, 25) to rapidly screen siRNA accessibility sites on the human CARM1 gene and then cloned the most effective one into the pCR3.1 vector to obtain the CARM1 shRNA expression vector. In cotransfections of 293T cells, this CARM1 shRNA expression vector was used in combination with p65 and other gene expression vectors as indicated in Fig. 2Go. At 24 h after transfection, cells were lysed for luciferase activity assays and Western blotting. Figure 2AGo shows that the luciferase activity in cells cotransfected with the CARM1 shRNA was over 75% lower than those without the shRNA (fifth vs. third lane). The luciferase activity of corresponding control with a scrambled shRNA (Scr; lane 4 in Fig. 2AGo) was similar to cells that did not receive any shRNA (lane 3). The last lane (sixth lane) shows that the inhibitory effect of CARM1 shRNA is evident even in the absence of exogenously transfected CARM1. To confirm that our CARM1 shRNA does indeed knockdown endogenous CARM1 under these experimental conditions, we performed Western blots on the cell lysates from these transfections. These Western blot results (Fig. 2AGo, lower panel of blots) are consistent with the luciferase activity data (Fig. 2AGo). Thus, overexpressed and endogenous CARM1 are specifically suppressed in cells receiving the CARM1 shRNA (fifth and sixth lanes; blots labeled CARM1 in Fig. 2AGo) but not in those transfected with scrambled shRNA (Scr; fourth lane). Additionally, the expression of total p65 and loading control actin remained unchanged under these conditions (Fig. 2AGo, middle and lower panels of blots). These results support the involvement and coactivator role of CARM1 in NF-{kappa}B-mediated gene expression.


Figure 2
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  		Fig. 2. CARM1 shRNA Inhibits p65-Mediated Transcription

293T cell were transiently transfected with the indicated expression plasmids. Cell extracts were prepared and assayed for luciferase activity or used in Western blots. A, TNF-{alpha} transcription as measured by luciferase activity is inhibited in the presence of CARM1 shRNA. A total of 37.5 ng of TNF-{alpha}Luc, 10 ng of p65, 75 ng of CBP, 75 ng of SRC-1, 75 ng of CARM1, and 375 ng of CARM1 shRNA or Scr expression vectors were transfected as indicated and described in Materials and Methods. CARM1 expression levels were monitored by Western blotting (lower panel). For this, half the samples from transfection experiments were lysed and sonicated. A total of 25 ng protein per sample was loaded on 10% SDS-protein gels and analyzed by Western blotting with anti-CARM1, anti-p65, and antiactin antibodies. Luciferase activity data are shown as mean ± SEM from three independent experiments performed in triplicate. *, P < 0.05 vs. without CARM1; **, P < 0.05 vs. without CARM1 shRNA. Western blot results shown are representative of three independent experiments. B, TNF-{alpha} transcription (before and after TNF-{alpha} stimulation) as measured by luciferase activity is inhibited in the presence of CARM1 shRNA. A total of 37.5 ng of TNF-{alpha}Luc, 10 ng of p65, 75 ng of CBP, 75 ng of SRC-1, 75 ng of CARM1, 375 ng of CARM1 shRNA or Scr expression vectors and balance plasmid were transfected into 293T cells as indicated and described in Materials and Methods. Cells were stimulated with or without 20 ng/ml TNF-{alpha} for 6 h. Luciferase activity data are shown as mean ± SEM from three independent experiments performed in triplicate. *, P < 0.01 vs. lanes 1 or 2; **, P < 0.01 vs. lanes 4 or 5.

 
In the next step, we examined whether CARM1 is involved in NF-{kappa}B-mediated gene transcription in response to TNF-{alpha} stimulation. This was performed by testing the effects of CARM1 shRNA in luciferase assays as illustrated in Fig. 2BGo. We observed that CARM1 shRNA again strongly blocked basal and TNF-{alpha}-stimulated luciferase activity not only when p65, p300, SRC-1, and CARM1 were added together (Fig. 2BGo, lane 6) but also in the presence of p65 only (Fig. 2BGo, lane 3). The corresponding Scr controls had no effects (lanes 2 and 5). Thus, when CARM1 is overexpressed, its augmenting effects on NF-{kappa}B-dependent transcription need additional CBP and SRC-1 due to limited endogenous cellular levels of these coactivators. On the other hand, the knockdown of CARM1 directly affects p65-mediated transcription, and this is not dependent on added exogenous CBP and SRC-1. Both these data support an essential role for CARM1 in p65 mediated gene transcription and also provide additional evidence that CARM1 participates in TNF-{alpha}-stimulated NF-{kappa}B-dependent transcription.

To evaluate the effect of CARM1 in p65-mediated gene expression, 293T cells were transfected with Scrambled, CARM1 shRNA, and CARM1 expression vectors as indicated in Fig. 3AGo, and treated with or without TNF-{alpha}. Total RNA was then prepared for relative RT-PCR analyses of several known NF-{kappa}B-regulated genes. CARM1 knockdown by the shRNA led to clearly reduced CARM1 levels, although it was not a complete inhibition. Interestingly, when CARM1 levels were reduced by its shRNA, there was a marked reduction in TNF-{alpha}-induced expression of IL-8, and a partial reduction of TNF-{alpha} and interferon-inducible protein 10 (IP-10). We also confirmed these results by performing quantitative real-time PCR (Fig. 3BGo), which shows that TNF-{alpha}-induced expression of IL-8, IP-10, and TNF-{alpha} were all significantly reduced by the CARM-1 shRNA (P < 0.01) but not by control Scr vector. On the other hand, overexpression of CARM1, however, did not seem to increase the expression of these genes. Among the genes shown in Fig. 3AGo, the expressions of TP53 and IL-6 were not affected by CARM1 or CARM1 shRNA. This was somewhat surprising because evidence shows that CARM1 can strongly enhance TP53 transcription in vitro (26). Overall, these results demonstrate that a reduction in the levels of endogenous CARM1 can impair the responses to TNF-{alpha} on a subset of inflammatory genes that are known to be regulated by NF-{kappa}B.


Figure 3
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  		Fig. 3. CARM1 shRNA Inhibits a Subset of NF-{kappa}B-Regulated Genes

293T cells were transfected with either CARM1, shRNA, or with control Scrambled plasmid for 24 h. A, Total RNA was prepared for relative RT-PCR analysis with 18S internal standards as described in Materials and Methods. The details of primer sequences are provided in Table 1Go. B, Real-time PCR analysis of TNF-{alpha}-induced IL-8, IP-10, and TNF-{alpha} mRNA expression with and without CARM1 shRNA or control scrambled (Scr) vector. Results were analyzed as described in Materials and Methods. Data shown are mean ± SEM from triplicates. *, P < 0.01 vs. without shRNA or Scr, paired.

 
p65-Mediated Transcription Correlates with in Vivo CARM1 Recruitment and Methylation at Arginine 17 of Histone H3
In an effort to further elucidate the coactivator role of CARM1 in p65-mediated transcription in an in vivo context and to determine whether this is associated with corresponding methylation of histone H3 at R17, we carried out a series of ChIP assays with antibodies to p65, CBP, SRC-1, and histone H3-K9 acetyl, H3-K14 acetyl, CARM1, and histone H3-R17Me. The ChIPed DNA samples from each of these ChIPs were then subjected to subsequent PCR analyses with primers (Table 1Go) designed to amplify the human TNF-{alpha} and IL-8 promoter regions around the NF-{kappa}B binding sites. Figure 4Go reveals that p65-dependent transcription of TNF-{alpha} and IL-8 in 293T cells is associated with several key in vivo nuclear chromatin events including the recruitment of p65, CBP, and SRC-1 to their promoters, and acetylation of histone H3 at K9 and K14. When the 293T cells were cotransfected with p65, CBP, and CARM1 (lanes 2, 5), or with p65, CBP, SRC-1, and CARM1 (lanes 3 and 6), the factor occupancy at these promoters and histone modifications representing transcriptional activation events were all clearly augmented compared with those with only p65 (lanes 1 and 4). Cotransfection of p65 along with CBP, SRC-1, and CARM1 showed the maximum effects including that of CARM1 accumulation at the TNF-{alpha} and IL-8 promoters. Importantly, at the same time, the methylation of histone H3 at R17 was also increased simultaneously as assessed by the ChIP assays. Combined with the synergistic coactivator role of CARM1 in p65-mediated TNF-{alpha} promoter transcription (Fig. 1BGo), these in vivo assessments of nuclear chromatin events by the ChIP assays demonstrate that CARM1 participates (along with CBP and SRC-1) in p65-dependent transcription and exercises its effects through the methylation of histone H3 at R17.


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  		Table 1. Sequences of the Primers Used for PCR and ChIP

 

Figure 4
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  		Fig. 4. ChIP Analyses of Coactivator Recruitment and Histone Modifications under Conditions of Coactivator Overexpression

A total of 106 293T cells was transiently transfected with 12 ng of p65, 450 ng of p300, 450 ng of SRC-1, and 450 ng of CARM1 expression vectors in 60-mm plates as indicated for 24 h. ChIP assays with anti-p65, anti-CBP, anti-SRC-1, anti-acetyl-histone H3-K9, anti-acetyl-histone H3-K14, anti-CARM1, and anti-dimethyl-histone H3-R17 antibodies were performed as described in Materials and Methods. Subsequent PCR analyses with the ChIPed DNA were performed to amplify the selected regions on the TNF-{alpha} and IL-8 promoters.

 
We further performed a time course evaluation of the effects of TNF-{alpha} in 293T cells and performed ChIPs with the abovementioned antibodies as well as an antibody that recognizes H3-citrulline followed by ChIP-PCR analyses with primers amplifying the TNF-{alpha}, IL-8, and IP-10 promoter regions containing NF-{kappa}B binding sites. These three genes were selected due to their reduction by CARM1 shRNA in response to TNF-{alpha} stimulation (Fig. 3Go). Here, the effects were examined in the absence of exogenously transfected expression vectors. Thus, Fig. 5Go shows that the factor recruitment and histone modification events around these promoters in response to TNF-{alpha} stimulation have very similar patterns. TNF-{alpha} stimulation clearly increased the recruitment of p65, CBP, SRC-1, and CARM1 at all three promoters, and these events also coincided with marked increases in lysine (K9 and K14) acetylation and arginine 17 methylation of HH3. Overall, histone H3 K9/14 acetylation and R17 methylation are increased in frame with CBP, SRC-1, and CARM1 recruitment, respectively. Although H3-R17 methylation might appear to slightly precede CARM1 recruitment at the IL-8 promoter, it could be due to the fact that some CARM1 prebound at the promoter may induce some methylation. Furthermore, the antibody that we used (anti-dimethyl H3-R17, Upstate 07-214) can also recognize mono-methyl H3-R17, albeit to a much lower degree, and H3-R17 may be mono- or dimethylated to some extent even before TNF-{alpha} stimulation. The lowest panel shows amplification of control input DNA.


Figure 5
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  		Fig. 5. ChIP Analysis of Coactivator Recruitment and Histone Modifications in Response to TNF-{alpha} Stimulation in Human 293T Cells

Formaldehyde cross-linked DNA samples from 293T cells were prepared at 0, 60, 120, and 240 min after stimulation with 20 ng/ml TNF-{alpha}. ChIP assays were carried out with anti-p65, anti-CBP, anti-SRC-1, anti-CARM1, anti-acetyl histone H3K9 or K14, anti-dimethyl-H3R19, and anti H3-citrulline. ChIPed DNA samples were analyzed by PCR with TNF-{alpha}, IL-8, or IP-10 promoter primer sets (sequences listed in Table 1Go).

 
During the same time frame of recruitment of p65, CBP, SRC-1, and CARM1, histone H3-R17Me levels were clearly increased, and, interestingly, H3-citrulline levels were decreased. The peptidyl arginine deiminase (PADI4) has been recently identified as a histone H3-specific arginine deiminase (27, 28) that blocks arginine methylation by converting arginine to citrulline and also functions as a repressor in PS2 transcription (27). Thus, the deimination of H3-R17Me is considered as a novel mechanism for antagonizing the transcription activation functions of CARM1. Here, for the first time, we provide evidence that histone H3-citrulline levels are decreased during the activation of p65-dependent transcription and that the recently proposed histone arginine/CARM1/PADI4 interaction also plays a novel regulatory role in NF-{kappa}B-mediated gene expression.

CARM1 Interacts with p65 during Transcription
CARM1 was originally isolated as a protein interacting with p160 (16), and evidence shows that it also directly interacts with CBP/p300 (17, 18, 19). The results in Figs. 1Go and 2Go suggest that there could be an interaction between CARM1 and p65 through either CBP or SRC-1 or both. To evaluate this further, we carried out immunoprecipitation with an anti-p65 antibody in 293T cells in which we overexpressed relevant expression vectors and then examined by Western blotting whether the p65 immunoprecipitates contain CARM1. Figure 6AGo shows that CARM1 is not detectable in the p65 immunoprecipitates in the basal state (lane 3). However, when the 293T cells were cotransfected with p65 along with CARM1, CARM1 was present in the p65 immunoprecipitates (lane 4), which provides evidence that CARM1 physically associates with p65 during transcription. Lane 5 shows that CARM1 is also present in the p65 immunoprecipitates in cells that were also cotransfected with CBP and SRC-1. The positive control (input) is seen in the first lane (lane 1), whereas negative control and control for specificity with IgG alone is seen in lane 2.


Figure 6
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  		Fig. 6. CARM1 Coimmunoprecipitates with p65 in Vivo

A total of 106 293T cells were transiently transfected with 12 ng of p65, 450 ng of CBP, 450 ng of SRC-1, and 450 ng of CARM1 expression vectors as indicated in 60-mm plates for 24 h. A, Coimmunoprecipitation of CARM1 with p65. Transfected cells were lysed with radioimmunoprecipitation assay buffer and immunoprecipitated with anti-p65 antibody or IgG control. After three washes, the precipitates were loaded onto 10% SDS-protein gel and analyzed by Western blotting with anti-CARM1 antibody. B, The p65 immunoprecipitates were incubated with 1 µg of histone H3 substrate and [3H-Me]S-adenosyl-methionine to assay CARM1 methyltransferase activity as described in Materials and Methods. Immunoprecipitates with CARM1 antibody were used as positive control for methylation activity (first solid bar). Methylation activity in p65 immunoprecipitates are shown as hatched bars. Methylation activity assay results (in counts per minute) are presented as mean ± SEM from three independent experiments. *, P < 0.05 vs. without CARM1.

 
We further verified the interaction of CARM1 with p65 by examining CARM1 methylation activity in the p65 immunoprecipitates (Fig. 6BGo). Immunoprecipitates obtained with antibody to CARM1 were used as positive control for methylation activity assay. These CARM1 immunoprecipitates, as expected, were found to contain significant methylation activity (solid bar) thereby demonstrating that the methylation activity is CARM1-specific. The hatched bars show that the methylation activity in the p65 immunoprecipitates was low when 293T cells were transfected with p65 alone, but it increased sequentially when the cells were cotransfected with p65 along with CARM1, SRC-1 plus CARM1, CBP plus CARM1, with maximum activity seen with a combination of all four, namely p65, SRC-1, CBP, and CARM1 (last bar). This is consistent with the transcription and ChIP data (Figs. 1Go and 4Go), and provides further direct evidence that CARM1 can interact with p65 through CBP and SRC-1 and therefore plays a coactivator role in p65-mediated transcription through methylation of histone arginine residues.

Histone H3-R17 Methylation Plays a Role in NF-{kappa}B-Regulated Inflammatory Gene Expression in Human Monocytes
NF-{kappa}B-mediated gene expression in human monocytes has been extensively investigated due to the important physiological and pathological roles of inflammatory genes in these cells and relevance to several human diseases (29, 30). Our experiments in Figs. 1–6GoGoGoGoGoGo clearly demonstrate that CARM1 and arginine modifications (methylation and citrullination) play important roles in inflammatory gene expression and regulation in 293T cells that can be transfected with high efficiency. To demonstrate the physiological relevance, we investigated these events in THP-1 monocytes. We recently demonstrated the role of histone lysine acetylation in NF-{kappa}B-dependent inflammatory gene expression in response to TNF-{alpha} and diabetic conditions in these cells (31). We now investigated for the first time the potential involvement of histone R17 methylation in NF-{kappa}B-dependent effects in these cells. We performed ChIP analysis to examine events at TNF-{alpha} and IL-8 promoters in human monocytic THP-1 cells. Figure 7AGo shows that, after TNF-{alpha} treatment, CARM1 is recruited and H3-R17 methylation is increased in a time-dependent fashion at the TNF-{alpha} and IL-8 promoters in THP-1 cells treated with TNF-{alpha}. In parallel, there was a concomitant decrease in histone H3 arginine citrullination at these promoter regions. These results are similar to those obtained with the 293T cell line (Fig. 5Go).


Figure 7
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  		Fig. 7. ChIP Analysis of CARM1 Participation in p65-Mediated Gene Transcription in Response to TNF-{alpha} and S100b Stimulation in Human THP-1 Cells

A, Formaldehyde cross-linked DNA from THP-1 cells were prepared 0, 60, 120, and 240 min after stimulation with 20 ng/ml TNF-{alpha}. ChIP assays with anti-CARM1, anti-dimethyl-histone H3-R17 antibodies, and anti H3-citrulline were performed as described in Materials and Methods. ChIPed DNA samples were analyzed by PCR with TNF-{alpha} and IL-8 promoter primers. B, Formaldehyde cross-linked DNA samples from THP-1 cells were prepared at 0, 10, 30, and 60 min after stimulation with 10 µg/ml S100b. ChIP assays with anti-p65, anti-CBP, anti-acetyl-histone H3-K9 or H3K14, anti-CARM1, and anti-dimethyl-histone H3-R17 antibodies were performed as described in Materials and Methods. ChIPed DNA samples were analyzed by PCR with TNF-{alpha} promoter primers.

 
Because diabetic conditions such as high glucose and advanced glycation end products (AGEs) can induce TNF-{alpha} expression in monocytes, including THP-1 cells (32, 33), we further evaluated the relevance of our findings in THP-1 cells treated with S100b. S100b, a specific ligand for RAGE and a member of a multigenic family of Ca2+-regulated proteins of the EF-hand type, is associated with inflammatory properties (34, 35). We used S100b because it can mimic diabetic conditions and also induce a variety of proinflammatory events through the RAGE receptor such as the activation of NF-{kappa}B and its target genes including vascular cell adhesion molecule-1, TNF-{alpha}, IL-6, and cyclooxygenase-2 (32, 33, 34, 35, 36). The ChIP assays in Fig. 7BGo show that S100b treatment of THP-1 cells could trigger nuclear events related to gene expression at the TNF-{alpha} promoter similar to those occurring when THP-1 cells or 293T cells were stimulated with TNF-{alpha} as shown in Figs. 4Go, 5Go, and 7AGo. CARM1 occupancy and histone H3-R17 methylation were increased concomitantly with p65 and CBP recruitment and increased acetylation of histone H3-K9 and H3-K14. Taken together, our results demonstrate for the first time that CARM1, histone H3-R17 methylation, and citrullination are part of a transcriptional network that is regulated by inflammatory stimuli in THP-1 monocytes. This also suggests a potential relevance of histone arginine modifications in inflammatory human diseases such as diabetes.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Although the mechanism of NF-{kappa}B-mediated gene regulation has been extensively studied, several new discoveries (37, 38) have revealed that NF-{kappa}B-mediated transactivation is far more complicated than anticipated. Beyond the fact that new coactivators and mediators have been implicated, the role of factors that covalently modify tails of core histone in chromatin is now becoming evident. The recent rapid advances in deciphering the histone code and functional roles of histone modifications have now changed the classic concept of chromatin structure from a static carrier to an active platform that mediates dynamic changes in gene function and expression. In particular, histone methylation events at key lysine residues by various specific methylases, and at arginine by protein arginine methyltransferases and CARM1 have elicited enormous interest recently due to increasing evidence that they provide an additional layer in the control of chromatin remodeling and gene expression (39, 40, 41, 42, 43).

In the current study, we have shown that CARM1 functions as a coactivator in NF-{kappa}B-mediated gene transcriptional activity in a p300 (CBP)-dependent manner instead of the p160-dependent manner reported in nuclear receptor-mediated transcription. The combination of CBP (p300), SRC-1, and CARM1 showed the highest activity in the luciferase assays. The specific involvement of CARM1 was confirmed by demonstrating that shRNA-mediated knockdown of CARM1 could down-regulate NF-{kappa}B transcriptional activity. Notably, we used ChIP assays to demonstrate that CARM1 was recruited to the TNF-{alpha} and IL-8 promoters along with p65, CBP, and SRC-1, and these were associated with increased H3-R17 methylation and H3K9 and H3K14 acetylation as well as decreased H3-citrullination. We performed coimmunoprecipitation studies to confirm that p65 directly associates with CARM1 and also showed that the p65 coimmunoprecipitates were associated with CARM1 histone methylation activity. Finally, we extended our study to show the physiological relevance in human monocytic THP-1 cells by demonstrating that TNF-{alpha} stimulation also increased the recruitment of CARM1 and H3-R17 methylation while decreasing H3-citrullination around the TNF-{alpha} and IL-8 promoters in a similar fashion as in 293T cells. We also showed that similar nuclear chromatin events were induced by treatment of THP-1 cell with S100b, an inflammatory diabetogenic stimulus that binds to the RAGE receptor, suggesting potential relevance to the actions of AGEs and the pathology of diabetes and its complications. Effects of CARM1 shRNA on inflammatory genes in THP-1 cells could not be demonstrated clearly due the low efficiency of transfection of THP-1 cells. Overall, our results demonstrate for the first time that CARM1 recruitment, histone H3-R17 methylation, and citrullination are regulated by inflammatory stimuli in monocytes and could be part of a transcriptional network mediating inflammatory gene expression. Because NF-{kappa}B-regulated genes are key modulators of immune, inflammatory, and acute phase responses and are also implicated in the control of cell proliferation and apoptosis (44, 45), our studies suggest that CARM1 actions could confer an additional level of regulation in these important cellular functions, and furthermore, changes in histone H3-R17 modification status may play a role in human inflammatory diseases such as diabetes.

A basic question that arises is what is the role of arginine methylation in this context? One possibility is that CARM1-mediated arginine methylation makes the histone a better substrate for recognition and modification by other cofactors. Another possibility is that the arginine-methylated histones can help in the recruitment of other transcriptional coactivators or antagonize the association of corepressors. Previously, it has been shown that acetylation of histone H3-K18 can augment methylation of H3-R17 in vitro, thus indicating that acetylated H3 is a better substrate for CARM1 (22). The mechanistic implication is that the HAT coactivator CBP (p300) may bind to the promoter first, acetylate histone H3, and then enhance the recruitment of CARM1, which in turn methylates H3-R17. However, it is quite possible that the methylation of H3-R17 can also accelerate specific histone acetylation. Such stepwise histone modifications and cooperative events implicated by the histone code are not yet completely understood and could be essential steps in the initiation of transcription.

Although it is well known that CARM1 functions as a coactivator in nuclear receptor-mediated transcription, recent reports provide evidence that CARM1 can also function as a secondary coactivator in a nonnuclear receptor system. One example is a recent study showing that CARM1 is necessary for muscle differentiation by acting as a secondary coactivator for monocyte enhancer factor-2C-mediated transcription in a SRC-2/glucocorticoid receptor interacting protein 1-dependent manner (46). ChIP assays demonstrated the in vivo recruitment of CARM1 to the endogenous muscle creatine kinase promoter in a differentiation-dependent manner (46). Another example is the very recent study by An et al. which provides compelling evidence that CARM1 and protein arginine methyltransferase 1, another arginine methyltransferase, act as coactivators for p53-mediated transcriptional activation via direct interactions with p53 and CBP (26, 47). Covic et al. (23) and, now, our current study show that CARM1 plays a novel coactivator role in NF-{kappa}B-mediated gene expression. In all of these transcription systems, CBP (p300) or SRC-1 plays primary coactivator roles and CARM1 actions occur in a CBP (p300)- or p160-dependent manner. This strongly links CARM1 to CBP (p300)- and p160-mediated gene transcription, and provides clear evidence that CARM1 participates in these transactivation events. Furthermore, apart from histones, evidence shows that CARM1 can also methylate arginines on CBP and p300 coactivators and thereby modulate coactivator transcriptional activity (48, 49). Poly(A) binding protein 1 was identified as another nonhistone protein target of CARM1 (50). A methylation-mediator complex was isolated containing at least eight components of human SWI/SNF complex, and CARM1, which revealed the link between chromatin remodeling and histone arginine methylation (51). In addition, CARM1 knockout embryos are small in size and die during late embryonic development or immediately after birth (52). Taken together, these data indicate that CARM1 has multiple and potentially critical roles in various cellular processes.

Additionally, two very recent papers reveal that PADI4 can act as a histone demethylase and regulate histone arginine methylation levels by converting methyl-arginine to citrulline and releasing methylamine (27, 28). These data have created a new dimension to our understanding of histone arginine methylation. Because evidence indicates that CARM1 has several critical functions, the regulation of arginine demethylation at core histone tails by PADI4 actions may provide a novel cellular mechanism for regulating CARM1 target genes as well as chromatin status. This is supported by data showing that core histone citrullination can block the transcription initiation of pS2 gene (27). It should also be pointed out that PADI4 has recently been identified as a gene associated with susceptibility to rheumatoid arthritis (53), an inflammatory disease. Hence our new data showing the effects of TNF-{alpha} and S100b on H3R-citrulline at inflammatory gene promoters may provide additional clues regarding the disease relevance of PADI4 and provide novel new links between CARM1, NF-{kappa}B, PADI4, and inflammation, especially in diseases such as diabetes. Additional work is needed to confirm this connection.

Similar to histone lysine methylation, the interest in histone arginine methylation is rapidly increasing (41, 42, 43). Based on current knowledge, the histone modification status of H3-R17 in cells can exist in at least three modes: arginine, methylated arginine, and citrullinated arginine. A mechanism has been proposed to illustrate conversions between these states (27). This is further complicated by the fact that arginine methylation can have mono- and di- levels and that dimethylation of arginine in vitro results in both symmetric and asymmetric NG,NG-dimethylarginine (54). The biological meaning of the three modes or the "H3-R17 code" is slowly emerging but needs more investigation. We have just begun to understand that H3-R17 methylation is associated with gene activation and that H3 arginine citrullination might be associated with repression of nuclear receptor and NF-{kappa}B-regulated genes. The well-known connections of nuclear receptors and NF-{kappa}B to several human endocrine and inflammatory diseases suggest that the methylation status of H3-R17 could be an important signal that reflects the transcriptional status of key genes or even cellular states. This could provide new information on the connections between the epigenetic characteristics of arginine modifications and human diseases.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
Anti-p65, anti-p50, anti-SRC-1, and anti-CBP antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-histone H3-citrulline was obtained from Abcam (Cambridge, MA). All other antibodies were purchased from Upstate Biotechnology (Lake Placid, NY). TNF-{alpha} promoter-luciferase construct (295-TNF-{alpha} Luc) was from Dr. J. S. Economou (University of California, Los Angeles, CA). p65 expression vector (HA tagged) was from Dr. E. Zandi (University of Southern California, Los Angeles, CA). CBP/p300 and SRC-1 expression vectors were from Dr. B. Forman (Beckman Research Institute, Duarte, CA). CARM1 expression construct was from Dr. M. Stallcup (University of Southern California, Los Angeles, CA). S100b was obtained from Calbiochem (La Jolla, CA). THP-1 cells were from American Type Culture Collection (Manassas, VA).

Transfections and Luciferase Assays
HEK 293T cells were grown in DMEM (Irvine Scientific, Santa Ana, CA) supplemented with 10% fetal calf serum (FCS; Irvine Scientific), 1 mM L-glutamine, 100 U/ml penicillin/ streptomycin, and 25 mM glucose. A total of 2 x 105 cells was placed in six-well culture dishes in RPMI 1640 medium and transfected with combinations of the TNF{alpha} Luc construct, p65 and CBP or p300, SRC-1, and CARM1 expression vectors as needed, using Effectene transfection reagent (QIAGEN, Valencia, CA) in 2% FCS-containing medium overnight. Samples were balanced for total DNA content with control plasmid. Cells were then washed once and allowed to recover in DMEM containing 10% FCS for 24 h. The cells were then lysed and luciferase assays performed according to the Promega (Madison, WI) Luciferase Assay System protocol. All assays were performed in triplicate in each experiment.

RT-PCR Assays and Real-Time Quantitative PCRs
293T cells were transfected with CARM1 shRNA or scramble control and then treated with or without 20 mM TNF-{alpha} for 6 h. Total RNA was isolated by RNA-STAT-60 method (Tel-Test, Friendswood, TX). cDNA was generated with 0.8 µg of RNA using random hexamers and Moloney murine leukemia virus reverse transcriptase using a Gene Amp RNA PCR kit (Applied Biosystems, Foster City, CA). An aliquot was used in multiplex relative RT-PCRs with cytokine-specific primers paired with Quantum RNA 18S internal standards (Ambion, Austin, TX) as described previously (31). PCRs were carried out at 95 C for 2 min followed by 30 cycles at 95 C for 30 sec, 58 C for 30 sec, and 72 C for 45 sec, and a final extension at 72 C for 7 min. PCR primer sequences are shown in Table 1Go. Real-time quantitative PCR (50 C for 2 min, 95 C for 10 min, followed by 40 cycles of 95 C for 15 sec, 58 C for 1 min) were performed with an ABI 7300 real-time PCR thermal cycler (Applied Biosystems). SYBR Green PCR Master Mix kit was from Applied Biosystems. All reactions were performed in a 25-µl reaction volume in triplicate. Standard curves were generated using glyceraldehyde-3-phosphate dehydrogenase. Dissociation curves were run to detect nonspecific amplification and we confirmed that single products were amplified in each reaction. Quantitations of the expression of each test gene and 18S RNA were then determined from the standard curve using the Applied Biosystems software. Relative amounts of mRNA of test genes were normalized to 18S mRNA internal control. Primers for real-time PCR are provided in Table 1Go.

Construction of CARM1 shRNA and Cell Transfections
The shRNA against human CARM1 was constructed according to a recently described method for making shRNAs (24, 25), which uses a rapid PCR method to initially identify the best siRNA accessibility sites on the target gene. The CARM1-specific siRNA sequence under the control of the U6 promoter was prepared by PCR amplification using pTZ/U6+1 containing U6 promoter as a template. The forward primer (5'-CGCGGATCCAAGGTCGGGCAGG-3') was located upstream of the U6 promoter and the reverse primer contained the 21-bp sense sequence (5'-GTCTTTAAGTGCTCAGTGTCC-3'), 9-bp hairpin (5'-TTTGTGTAG-3'), and 21-bp antisense sequence. The PCR products included the U6 promoter sequence, the sense and the antisense sequence with a 9-nucleotide loop in between them, a terminator sequence, and a stuffer tag sequence at the 3' end of the product. We chose one of the PCR products containing the U6+shRNA sequence for the knockdown experiments based on sequence-specific gene silencing with greatest efficiency and this was cloned into the pCR3.1 vector (Invitrogen, San Diego, CA) to obtain the CARM1 shRNA expression vector. As an important control, the sequence 5'-GGATATATCCCGAACTAGACA-3' was used to make a control scrambled nonspecific shRNA. For Western blot analysis of CARM1 levels after shRNA treatments, 293T cells were seeded into 12-well plates at 3–5 x 105 per well 1 d before transfection. Transfections were carried out using Lipofectamine 2000 reagent according to manufacturer’s instructions (Invitrogen) and our reported methods (31, 55). Briefly, 48 h after transfection, the cells were washed twice with 1x PBS buffer [137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4 (pH 7.3)], lysed with 1% sodium dodecyl sulfate (SDS) in 20 mM Tris-HCl buffer (pH 6.8), and about 25 µg of protein was fractionated on a 10% SDS-PAGE gel (56). Immunoblotting was performed with a CARM1-specific antibody (1:2000 dilution) according to our reported procedures (57).

Coimmunoprecipitations
A total of 2 x 106 293T cells were grown in DMEM with 25 mM high glucose and transfected with various combinations of p65, CARM1, CBP, and SRC-1 expression plasmids as described above in Transfections and Luciferase Assays. After 24 h, extracts were prepared by lysing the cells with radioimmunoprecipitation assay buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Nonidet P-40 (NP-40), 0.5% sodium deoxycholate, 0.1% SDS] and centrifuging for 10 min at 12,000 rpm. The supernatants were incubated with 2 µg of anti-p65 antibody and 30 µl of a 50% suspension of protein A-agarose (Upstate Biotechnology) overnight at 4 C, followed by washing four times with PBS containing 150 mM NaCl. Immunoblotting was performed with an antibody against CARM1. Control for specificity was done with IgG.

ChIP
ChIPs were performed according to Farnham and coworker (58) with some modifications (31). Briefly, 2–3 x 106 cells were cross-linked with 1% formaldehyde for 30–60 min, washed twice with cold PBS, resuspended in lysis buffer [1% SDS, 10 mM EDTA, 50 mM Tris-HCl (pH 8.1), 1x protease inhibitor cocktail (Roche Molecular Biochemicals, Basel, Switzerland)], and sonicated one to three times for 30 sec each at 40% maximum setting (Branson Sonifier; model 250) followed by centrifugation for 10 min. One-tenth of total lysate was used for total genomic DNA as "Input DNA" control. Supernatants were collected and diluted in buffer [1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl (pH 8.1), and 1x protease inhibitor mixture] followed by immunoclearing with 1 µg of sheared salmon sperm DNA, 10 µl of rabbit IgG, and 20 µl of protein A-agarose (Upstate Biotechnology) for 1 h at 4 C. Immunoprecipitation was performed for 15 h at 4 C with 2–5 µg each of specific relevant antibodies. Precipitates were washed sequentially for 10 min each in TSE I buffer [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl (pH 8.1)], TSE II buffer (TSE I with 500 mM NaCl), and TSE III [0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl (pH 8.1)]. Precipitates were then washed twice with TE buffer [10 mM Tris-HCl (pH 7.5), 1 mM EDTA] and extracted twice with 1% SDS containing 0.1 M NaHCO3. Eluates were pooled and heated at 65 C for at least 6 h to reverse the formaldehyde cross-linking. DNA fragments were purified with QIAGEN Qiaquick spin kits. One microliter of a 40-µl DNA extract was used for PCR. ChIP PCR primers corresponding to sequences within the promoter regions of various genes are listed in Table 1Go.

In Vitro Histone Methylation Activity Assay (48)
One microgram of histone H3 (Upstate Biotechnology) was incubated with p65 immunoprecipitates and [3H-Me]S-adenosyl methionine (New England Nuclear, Boston, MA; 80 Ci·mmol–1) in buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 5 mM EDTA, and 0.5% NP-40 (48) at 30 C for 2 h. Antihistone H3 and protein A agarose beads were added and incubated for 1 h. The samples were spun for 1 min at 2000 rpm and washed four times with the same buffer and then resuspended in 50 µl of water. Five microliters was then taken for liquid scintillation counting.

Data Analyses
Results are expressed as means ± SEM of multiple experiments. Student’s t tests were used to compare two groups, or ANOVA with Dunnet’s posttests for multiple groups using Prism software (GraphPad, San Diego, CA). Statistical significance was detected at the 0.05 level.


   ACKNOWLEDGMENTS
 
We are very grateful to Drs. M. Stallcup, B. Forman, E. Zandi, and J. Economou for the generous gifts of plasmids. We thank Saurabh Sahar for his critical comments and helpful discussions and LingXiao Zhang for her technical assistance.


   FOOTNOTES
 
This work was supported by grants from the National Institutes of Health (RO1 DK65073, PO1 HL55798) and the Juvenile Diabetes Research Foundation.

F.M., S.L.L., V.C., L.L., and R.N. have nothing to declare.

First Published Online February 23, 2006

Abbreviations: AGE, Advanced glycation end product; CARM1, coactivator-associated arginine methyltransferase-1; ChIP, chromatin immunoprecipitation; CBP, cAMP response element binding protein-binding protein; FCS, fetal calf serum; HAT, histone acetyl transferase; IP-10, interferon-inducible protein 10; NF-{kappa}B, nuclear factor-{kappa}B; NP-40, Nonidet P-40; PADI4, peptidyl arginine deiminase; RAGE, receptor for advanced glycation end products; SDS, sodium dodecyl sulfate; shRNA, short hairpin RNA; siRNA, small interfering RNA; SRC-1, steroid receptor coactivator-1.

Received for publication September 7, 2005. Accepted for publication February 8, 2006.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
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 DISCUSSION
 MATERIALS AND METHODS
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