Coactivator-associated Arginine Methyltransferase Research Articles

CARM1 Regulates Estrogen-Stimulated Breast Cancer Growth through Up-regulation of E2F1

Estrogen receptor alpha (ER alpha) mediates breast cancer proliferation through transcriptional mechanisms involving the recruitment of specific coregulator complexes to the promoters of cell cycle genes. The coactivator-associated arginine methyltransferase CARM1 is a positive regulator of ER alpha-mediated transcriptional activation. Here, we show that CARM1 is essential for estrogen-induced cell cycle progression in the MCF-7 breast cancer cell line. CARM1 is specifically required for the estrogen-induced expression of the critical cell cycle transcriptional regulator E2F1 whereas estrogen stimulation of cyclin D1 is CARM1 independent. Upon estrogen stimulation, the E2F1 promoter is subject to CARM1-dependent dimethylation on histone H3 arginine 17 (H3R17me2) in a process that parallels the recruitment of ER alpha. Additionally, we find that the recruitment of CARM1 and subsequent histone arginine dimethylation are dependent on the presence of the oncogenic coactivator AIB1. Thus, CARM1 is a critical factor in the pathway of estrogen-stimulated breast cancer growth downstream of ER alpha and AIB1 and upstream of the cell cycle regulatory transcription factor E2F1. These studies identify CARM1 as a potential new target in the treatment of estrogen-dependent breast cancer.

Ligand-dependent interactions of the Ah receptor with coactivators in a mammalian two-hybrid assay

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) is a high affinity ligand for the aryl hydrocarbon receptor (AhR). In this study, we investigated structure-dependent differences in activation of the AhR by a series of halogenated aromatic hydrocarbons. TCDD, 1,2,3,7,8-pentachlorodibenzo- p-dioxin (PeCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), and 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) induced CYP1A1-dependent activities in HEK293 human embryonic kidney, Panc1 pancreatic cancer, and Hepa1c1c7 mouse hepatoma cell lines. There was a structure-dependent difference in the efficacy of TCDF and PCB126 in HEK293 and Panc1 cells since induced CYP1A1 mRNA levels were lower than observed for the other congeners. A mammalian two-hybrid assay in cells transfected with GAL4-coactivator and AhR-VP16 chimeras was used to investigate structure-dependent interactions of these chimeras in Panc1, HEK293, and Hepa1c1c7 cells. The reporter construct pGAL4-luc contains five tandem GAL4 response elements linked to the luciferase gene and the GAL4-coactivator chimeras express several coactivators including steroid receptor coactivator 1 (SRC-1), SRC-2 and SRC-3, the mediator coactivator TRAP220, coactivator associated arginine methyl transferase 1 (CARM-1), and peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1). Results of the mammalian two-hybrid studies clearly demonstrate that activation of pGAL4-luc in cells transfected with VP-AhR and GAL4-coactivator chimeras is dependent on the structure of the HAH congener, cell context, and coactivator, suggesting that the prototypical HAH congeners used in this study exhibit selective AhR modulator activity.

Functional insights from structures of coactivator-associated arginine methyltransferase 1 domains

Coactivator-associated arginine methyltransferase 1 (CARM1), a protein arginine methyltransferase recruited by several transcription factors, methylates a large variety of proteins and plays a critical role in gene expression. We report, in this paper, four crystal structures of isolated modules of CARM1. The 1.7 A crystal structure of the N-terminal domain of CARM1 reveals an unexpected PH domain, a scaffold frequently found to regulate protein-protein interactions in a large variety of biological processes. Three crystal structures of the CARM1 catalytic module, two free and one cofactor-bound forms (refined at 2.55 A, 2.4 A and 2.2 A, respectively) reveal large structural modifications including disorder to order transition, helix to strand transition and active site modifications. The N-terminal and the C-terminal end of CARM1 catalytic module contain molecular switches that may inspire how CARM1 regulates its biological activities by protein-protein interactions.

Insights into histone code syntax from structural and biochemical studies of CARM1 methyltransferase

Coactivator-associated arginine methyltransferase (CARM1) is a transcriptional coactivator that methylates Arg17 and Arg26 in histone H3. CARM1 contains a conserved protein arginine methyltransferase (PRMT) catalytic core flanked by unique pre- and post-core regions. The crystal structures of the CARM1 catalytic core in the apo and holo states reveal cofactor-dependent formation of a substrate-binding groove providing a specific access channel for arginine to the active site. The groove is supported by the first eight residues of the post-core region (C-extension), not present in other PRMTs. In vitro methylation assays show that the C-extension is essential for all histone H3 methylation activity, whereas the pre-core region is required for methylation of Arg26, but not Arg17. Kinetic analysis shows Arg17 methylation is potentiated by pre-acetylation of Lys18, and this is reflected in k(cat) rather than K(m). Together with the absence of specificity subsites in the structure, this suggests an electrostatic sensing mechanism for communicating the modification status of vicinal residues as part of the syntax of the 'histone code.'

The functional role of the CARM1-SNF5 complex and its associated HMT activity in transcriptional activation by thyroid hormone receptor

We have investigated the function and mechanisms of the CARM1-SNF5 complex in T3-dependent transcriptional activation. Using specific small interfering RNAs (siRNA) to knock down coactivators in HeLa alpha2 cells, we found that coactivator associated arginine methyltransferase 1 (CARM1) and SWI/SNF complex component 5 (SNF5) are important for T3-dependent transcriptional activation. The CARM1- SWI/SNF chromatin remodeling complex serves as a mechanism for the rapid reversal of H3-K9 methylation. Importantly, siRNA treatment against CARM1 and/or SNF5 increased the recruitment of HMTase G9a to the type 1 deiodinase (D1) promoter even with T3. Knocking-down either CARM1 or SNF5 also inhibited the down-regulation of histone macroH2A, which is correlated with transcriptional activation. Finally, knocking down CARM1 and SNF5 by siRNA impaired the association of these coactivators to the D1 promoter, suggesting functional importance of CARM1- SNF5 complex in T3-dependent transcriptional activation.

Phosphorylation-mediated inactivation of coactivator-associated arginine methyltransferase 1

Multiple protein arginine methyltransferases are involved in transcriptional activation of nuclear receptors. Coactivator-associated arginine methyltransferase 1 (CARM1)-mediated histone methylation has been shown to activate nuclear receptor-dependent transcription; however, little is known about the regulation of its enzymatic activity. Here, we report that the methyltransferase activity of CARM1 is negatively regulated through phosphorylation at a conserved serine residue. When the serine residue is mutated to glutamic acid, which mimics the phosphorylated serine residue, the mutant CARM1 exhibits diminished ability to bind the methyl donor adenosylmethionine and diminished histone methylation activity. Moreover, such mutation leads to the inhibition of CARM1 transactivation of estrogen receptor-dependent transcription. Our results provide an example for the regulation of protein arginine methyltransferase activity by phosphorylation. As CARM1 is a potent transcriptional coactivator of estrogen receptor, our results suggest that phosphorylation of CARM1 serves as a unique mechanism for inactivating CARM1-regulated estrogen-dependent gene expression.

Expression, purification, crystallization and preliminary crystallographic study of isolated modules of the mouse coactivator-associated arginine methyltransferase 1.

Coactivator-associated arginine methyltransferase 1 (CARM1) plays a crucial role in gene expression as a coactivator of several nuclear hormone receptors and also of non-nuclear receptor systems. Its recruitment by the transcriptional machinery induces protein methylation, leading to chromatin remodelling and gene activation. CARM1(28-507) and two structural states of CARM1(140-480) were expressed, purified and crystallized. Crystals of CARM1(28-507) belong to space group P6(2)22, with unit-cell parameters a = b = 136.0, c = 125.3 A; they diffract to beyond 2.5 A resolution using synchrotron radiation and contain one monomer in the asymmetric unit. The structure of CARM1(28-507) was solved by multiple isomorphous replacement and anomalous scattering methods. Crystals of apo CARM1(140-480) belong to space group I222, with unit-cell parameters a = 74.6, b = 99.0, c = 207.4 A; they diffract to beyond 2.7 A resolution and contain two monomers in the asymmetric unit. Crystals of CARM1(140-480) in complex with S-adenosyl-L-homocysteine belong to space P2(1)2(1)2, with unit-cell parameters a = 74.6, b = 98.65, c = 206.08 A; they diffract to beyond 2.6 A resolution and contain four monomers in the asymmetric unit. The structures of apo and holo CARM1(140-480) were solved by molecular-replacement techniques from the structure of CARM1(28-507).

Contrasting Effects of Two Alternative Splicing Forms of Coactivator-Associated Arginine Methyltransferase 1 on Thyroid Hormone Receptor-Mediated Transcription in Xenopus laevis

Thyroid hormone receptors (TRs) can repress or activate target genes depending on the absence or presence of thyroid hormone (T3), respectively. This hormone-dependent gene regulation is mediated by the recruitment of corepressors in the absence of T3 and coactivators in its presence. Many TR-interacting coactivators have been characterized in vitro. Among them is coactivator-associated arginine methyltransferase 1 (CARM1), which methylates histone H3. We are interested in investigating the role of CARM1 in TR-mediated gene expression in vivo during postembryonic development by using T3-dependent frog metamorphosis as a model. We first cloned the Xenopus laevis CARM1 and obtained two alternative splicing forms, CARM1a and CARM1b. Both isoforms are expressed throughout metamorphosis, supporting a role for these isoforms during the process. To investigate whether Xenopus CARM1s participate in gene regulation by TRs, transcriptional analysis was conducted in Xenopus oocyte, where the effects of cofactors can be studied in the context of chromatin in vivo. Surprisingly, overexpression of CARM1b had little effect on TR-mediated transcription, whereas CARM1a enhanced gene activation by liganded TR. Chromatin immunoprecipitation assays showed that both endogenous CARM1a and overexpressed CARM1a and b were recruited to the promoter by liganded TR. However, the binding of liganded TR to the target promoter was reduced when CARM1b was overexpressed, accompanied by a slight reduction in histone methylation at the promoter. These results suggest that CARM1 may play a role in TR-mediated transcriptional regulation during frog development and that its function is regulated by alternative splicing.

The Arginine Methyltransferase CARM1 Regulates the Coupling of Transcription and mRNA Processing

The coactivator-associated arginine methyltransferase CARM1 is recruited by many different transcription factors as a positive regulator. To understand the mechanism by which CARM1 functions, we sought to isolate its substrates. We developed a small-pool screening approach for this purpose and identified CA150, SAP49, SmB, and U1C as splicing factors that are specifically methylated by CARM1. We further showed that CA150, a molecule that links transcription to splicing, interacts with the Tudor domain of the spinal muscular atrophy protein SMN in a CARM1-dependent fashion. Experiments with an exogenous splicing reporter and the endogenous CD44 gene revealed that CARM1 promotes exon skipping in an enzyme-dependent manner. The identification of splicing factors that are methylated by CARM1, and protein-protein interactions that are regulated by CARM1, strongly implicates this enzyme in the regulation of alternative splicing and points toward its involvement in spinal muscular atrophy pathogenesis.

Signaling within a coactivator complex: methylation of SRC-3/AIB1 is a molecular switch for complex disassembly.

Recent studies indicate that steroid receptor-mediated transcriptional initiation is a cyclical process involving multiple rounds of coactivator assembly and disassembly. Steroid receptor coactivator 3 (SRC-3) coactivator phosphorylation has been shown to regulate coactivator complex assembly, but the mechanisms by which coactivator disassembly is triggered are not well understood. In this study, we provide in vitro and in vivo evidence that members of the SRC coactivator family serve as substrates for the enzymatic coactivator coactivator-associated arginine methyltransferase 1 (CARM1). Methylation of SRC-3 was localized to an arginine in its CARM1 binding region and correlated with decreased estrogen receptor alpha-mediated transcription, as seen with both cell-based and in vitro transcription assays. Consistent with this finding, we demonstrated that methylation promotes dissociation of the SRC-3/CARM1 coactivator complex. Methylation of SRC-3 is regulated by estrogen signaling in MCF7 cells and serves as a molecular switch for disassembly of the SRC-3 transcriptional coactivator complex. We propose that CARM1 is a dual-function coactivator, as it not only activates transcription by modifying core histone tails but also terminates hormone signaling by disassembly of the coactivator complex.

Coactivator-Associated Arginine Methyltransferase 1 Enhances Transcriptional Activity ofthe Human T-Cell Lymphotropic Virus Type 1 Long Terminal Repeat through Direct Interactionwith Tax

In this study, we demonstrate that the coactivator-associated arginine methyltransferase 1 (CARM1), which methylates histone H3 and other proteins such as p300/CBP, is positively involved in the regulation of Tax transactivation. First, transfection studies demonstrated that overexpression of CARM1 wild-type protein resulted in increased Tax transactivation of the human T-cell lymphotropic virus type 1 (HTLV-1) long terminal repeat (LTR). In contrast, transfection of a catalytically inactive CARM1 methyltransferase mutant did not enhance Tax transactivation. CARM1 facilitated Tax transactivation of the CREB-dependent cellular GEM promoter. A direct physical interaction between HTLV-1 Tax and CARM1 was demonstrated using in vitro glutathione S-transferase-Tax binding assays, in vivo coimmunoprecipitation, and confocal microscopy experiments. Finally, chromatin immunoprecipitation analysis of the activated HTLV-1 LTR promoter showed the association of CARM1 and methylated histone H3 with the template DNA. In vitro, Tax facilitates the binding of CARM1 to the transcription complex. Together, our data provide evidence that CARM1 enhances Tax transactivation of the HTLV-1 LTR through a direct interaction between CARM1 and Tax and this binding promotes methylation of histone H3 (R2, R17, and R26).

Bile Salt Excretory Pump

Bile acids are the major determinant and driving force for the generation of bile flow. Bile acid transport across the canalicular membrane is primarily an ATP-dependent process. The predominant transporter is the bile salt excretory pump (BSEP, ABCB11), a member of the adenosine triphosphate-binding cassette (ABC) family of transporters. Regulatory mechanisms that can coordinate the genes encoding bile acid transport proteins are critically important to avoid hepatocyte damage from intracellar accumulation of bile acids. Bile salts are natural ligands for several nuclear hormone receptors expressed in liver and intestine. Nuclear receptors are transcription factors that bind specific ligands such as bile acids and regulate gene expression according to the metabolic requirements of the cell. In cloning of the BSEP gene, we found a binding site in the promoter for the farnesoid X receptor (FXR), a nuclear receptor for bile acids. FXR activity requires heterodimerization with the 9-cis retinoid receptor (RXR alpha), and when bound by bile acids and retinoic acid, the complex effectively activates the transcription of BSEP. There is a growing body of evidence for the activation of nuclear hormone receptors through the remodeling of chromatin by histone modification involving acetylation, in concert with methylation of H3 and H4 histones. We have recently demonstrated a role for the coactivator-associated arginine methyltransferase 1 (CARM1), as a coactivator of the FXR/RXR receptor and regulator of FXR responsive genes such as BSEP. Chromatin immunoprecipitation showed that the bile acid-dependent activation of the human BSEP is associated with a simultaneous increase of FXR and CARM1 occupation of the BSEP promoter. The increased occupation of the BSEP locus by CARM1 also corresponds with the increased deposition of Arg-17 methylation and Lys-9 acetylation of histone H3 within the FXR DNA-binding element of BSEP. Our work on the role of nuclear receptors in regulation of bile acid homeostasis has led to an increased understanding of the pathogenesis of the disorder, progressive familial intrahepatic cholestasis, type 1 (PFIC1) or Byler disease. The gene mutated in PFIC1 is called FIC1 and codes for a type IV P-type ATPase whose function is unknown. Increased ileal apical sodium-dependent bile acid transporter messenger RNA (mRNA) expression was detected in 3 patients with PFIC1. Ileal FXR and short heterodimer partner (an inhibitory nuclear receptor) messenger RNA levels were reduced in the same 3 patients. In studies of cells after antisense-mediated knock-down of endogenous FIC1, the activity of the ileal apical bile acid transporter promoter was enhanced, whereas the activities of the human FXR and BSEP promoters were reduced. Nuclear but not cytoplasmic localization of FXR is markedly decreased in FIC1-negative cells, indicating that FIC1 is necessary for posttranslational modifications necessary for the nuclear translocation of FXR. This defect leads to enhanced ileal bile salt uptake and impaired canalicular bile salt secretion by BSEP. In PFIC1, an increased load of bile acids is retained in the liver leading to cholestasis and progressive liver injury.

Regulation of Nrf2 Transactivation Domain Activity by p160 RAC3/SRC3 and Other Nuclear Co-Regulators

Transcription factor NF-E2-related factor 2 (Nrf2) regulates the induction of Phase II detoxifying enzymes and antioxidant enzymes in response to many cancer chemopreventive compounds. In this study, we investigated the role of receptor associated coactivator (RAC3) or steroid receptor coactivator-3 (SRC3) and other nuclear co-regulators including CBP/p300 (CREB-binding protein), CARM1(Coactivator-associated arginine methyltransferase), PRMT1(Protein arginine methyl-transferase 1), and p/CAF (p300/CBP-associated factor) in the transcriptional activation of a chimeric Gal4-Nrf2-Luciferase system containing the transactivation domain (TAD) of Nrf2 in HepG2 cells. The results indicated that RAC3 up-regulated the transactivation activity of Gal4-Nrf2-(1-370) in a dose-dependent manner. The enhancement of transactivation domain activity of Gal4-Nrf2-(1-370) by RAC3 was dampened in the presence of dominant negative mutants of RAC3. Next we studied the effects of other nuclear co-regulators including CBP/ p300, CARM1, PRMT1 and p/CAF, and the results showed that they had different level of positive effects on this transactivation domain activity of Gal4-Nrf2-(1-370). But importantly, synergistic effects of these co-regulators in the presence of RAC3/SRC3 on the transactivation activity of Gal4-Nrf2-(1-370) were observed. In summary, our present study showed for the first time that the 160 RAC3/SRC3 is involved in the functional transactivation of TAD of Nrf2 and that the other nuclear co-regulators such as CBP/p300, CARM1, PRMT1 and p/CAF can also transcriptionally activate this TAD of Nrf2 and that they could further enhance the transactivation activity mediated by RAC3/SRC3.

Modulation of glucocorticoid receptor‐interacting protein 1 (GRIP1) transactivation and co‐activation activities through its C‐terminal repression and self‐association domains

Glucocorticoid receptor-interacting protein 1 (GRIP1), a p160 family nuclear receptor co-activator, possesses at least two autonomous activation domains (AD1 and AD2) in the C-terminal region. AD1 activity appears to be mediated by CBP/p300, whereas AD2 activity is apparently mediated through co-activator-associated arginine methyltransferase 1 (CARM1). The mechanisms responsible for regulating the activities of AD1 and AD2 are not well understood. We provide evidence that the GRIP1 C-terminal region may be involved in regulating its own transactivation and nuclear receptor co-activation activities through primary self-association and a repression domain. We also compared the effects of the GRIP1 C terminus with those of other factors that functionally interact with the GRIP1 C terminus, such as CARM1. Based on our results, we propose a regulatory mechanism involving conformational changes to GRIP1 mediated through its intramolecular and intermolecular interactions, and through modulation of the effects of co-repressors on its repression domains. These are the first results to indicate that the structural components of GRIP1, especially those of the C terminus, might functionally modulate its putative transactivation activities and nuclear receptor co-activator functions.

CARM1 Regulates Proliferation of PC12 Cells by Methylating HuD

HuD is an RNA-binding protein that has been shown to induce neuronal differentiation by stabilizing labile mRNAs carrying AU-rich instability elements. Here, we show a novel mechanism of arginine methylation of HuD by coactivator-associated arginine methyltransferase 1 (CARM1) that affected mRNA turnover of p21cip1/waf1 mRNA in PC12 cells. CARM1 specifically methylated HuD in vitro and in vivo and colocalized with HuD in the cytoplasm. Inhibition of HuD methylation by CARM1 knockdown elongated the p21cip1/waf1 mRNA half-life and resulted in a slow growth rate and robust neuritogenesis in response to nerve growth factor (NGF). Methylation-resistant HuD bound more p21cip1/waf1 mRNA than did the wild type, and its overexpression upregulated p21cip1/waf1 protein expression. These results suggested that CARM1-methylated HuD maintains PC12 cells in the proliferative state by committing p21cip1/waf1 mRNA to its decay system. Since the methylated population of HuD was reduced in NGF-treated PC12 cells, downregulation of HuD methylation is a possible pathway through which NGF induces differentiation of PC12 cells.

Histone H3 Lysine 9 Methyltransferase G9a Is a Transcriptional Coactivator for Nuclear Receptors

Methylation of Lys-9 of histone H3 has been associated with repression of transcription. G9a is a histone H3 Lys-9 methyltransferase localized in euchromatin and acts as a corepressor for specific transcription factors. Here we demonstrate that G9a also functions as a coactivator for nuclear receptors, cooperating synergistically with nuclear receptor coactivators glucocorticoid receptor interacting protein 1, coactivator-associated arginine methyltransferase 1 (CARM1), and p300 in transient transfection assays. This synergy depends strongly on the arginine-specific protein methyltransferase activity of CARM1 but does not absolutely require the enzymatic activity of G9a and is specific to CARM1 and G9a among various protein methyltransferases. Reduction of endogenous G9a diminished hormonal activation of an endogenous target gene by the androgen receptor, and G9a associated with regulatory regions of this same gene. G9a fused to Gal4 DNA binding domain can repress transcription in a lysine methyltransferase-dependent manner; however, the histone modifications associated with transcriptional activation can inhibit the methyltransferase activity of G9a. These findings suggest a link between histone arginine and lysine methylation and a mechanism for controlling whether G9a functions as a corepressor or coactivator.

Coactivator-Associated Arginine Methyltransferase-1 Enhances Nuclear Factor-κB-Mediated Gene Transcription through Methylation of Histone H3 at Arginine 17

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-kappaB (NF-kappaB) and enhance NF-kappaB 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-kappaB 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-kappaB 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-kappaB and inflammatory genes in monocytes. These results demonstrate that CARM1 participates in NF-kappaB-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.

Ligand-specific allosteric regulation of coactivator functions of androgen receptor in prostate cancer cells

The androgen receptor not only mediates prostate development but also serves as a key regulator of primary prostatic cancer growth. Although initially responsive to selective androgen receptor modulators (SARMs), which cause recruitment of the nuclear receptor-corepressor (N-CoR) complex, resistance invariably occurs, perhaps in response to inflammatory signals. Here we report that dismissal of nuclear receptor-corepressor complexes by specific signals or androgen receptor overexpression results in recruitment of many of the cohorts of coactivator complexes that permits SARMs and natural ligands to function as agonists. SARM-bound androgen receptors appear to exhibit failure to recruit specific components of the coactivators generally bound by liganded nuclear receptors, including cAMP response element-binding protein (CBP)/p300 or coactivator-associated arginine methyltransferase 1 (CARM1) to the SARM-bound androgen receptor, although still causing transcriptional activation of androgen receptor target genes. SARM-bound androgen receptors use distinct LXXLL (L, leucine; X, any amino acid) helices in the p160 nuclear receptor interaction domains that may impose selective allosteric effects, providing a component of the molecular basis of differential responses to different classes of ligands by androgen receptor.

Signal-dependent Control of Gluconeogenic Key Enzyme Genes through Coactivator-associated Arginine Methyltransferase 1

Together with impaired glucose uptake in skeletal muscle, elevated hepatic gluconeogenesis is largely responsible for the hyperglycemic phenotype in type II diabetic patients. Intracellular glucocorticoid and cyclic adenosine monophosphate (cAMP)/protein kinase A-dependent signaling pathways contribute to aberrant hepatic glucose production through the induction of gluconeogenic enzyme gene expression. Here we show that the coactivator-associated arginine methyltransferase 1 (CARM1) is required for cAMP-mediated activation of rate-limiting gluconeogenic phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) and glucose-6-phosphatase genes. Mutational analysis showed that CARM1 mediates its effect via the cAMP-responsive element within the PEPCK promoter, which is identified here as a CARM1 target in vivo. In hepatocytes, endogenous CARM1 physically interacts with cAMP-responsive element binding factor CREB and is recruited to the PEPCK and glucose-6-phosphatase promoters in a cAMP-dependent manner associated with increased promoter methylation. CARM1 might, therefore, represent a critical component of cAMP-dependent glucose metabolism in the liver.

The AT-hook of the Chromatin Architectural Transcription Factor High Mobility Group A1a Is Arginine-methylated by Protein Arginine Methyltransferase 6

The HMGA1a protein belongs to the high mobility group A (HMGA) family of architectural nuclear factors, a group of proteins that plays an important role in chromatin dynamics. HMGA proteins are multifunctional factors that associate both with DNA and nuclear proteins that have been involved in several nuclear processes, such as transcriptional regulation, viral integration, DNA repair, RNA processing, and chromatin remodeling. The activity of HMGA proteins is finely modulated by a variety of post-translational modifications. Arginine methylation was recently demonstrated to occur on HMGA1a protein, and it correlates with the apoptotic process and neoplastic progression. Methyltransferases responsible for these modifications are unknown. Here we show that the protein arginine methyltransferase PRMT6 specifically methylates HMGA1a protein both in vitro and in vivo. By mass spectrometry, the sites of methylation were unambiguously mapped to Arg(57) and Arg(59), two residues which are embedded in the second AT-hook, a region critical for both protein-DNA and protein-protein interactions and whose modification may cause profound alterations in the HMGA network. The in vivo association of HMGA and PRMT6 place this yet functionally uncharacterized methyltransferase in the well established functional context of the chromatin structure organization.