Coactivator-associated Arginine Methyltransferase 1 Expression Research Articles

Downregulated RBM5 Enhances CARM1 Expression and Activates the PRKACA/GSK3β Signaling Pathway through Alternative Splicing-Coupled Nonsense-Mediated Decay.

Downregulated RNA-binding motif protein 5 (RBM5) promotes the development and progression of various tumors, including bladder cancer (BC). Alternative splicing (AS) plays a crucial role in the progression of cancer by producing protein isomers with different functions or by promoting nonsense-mediated mRNA decay (NMD). However, whether RBM5 modulates the progression of BC through AS-NMD remains unexplored. In this study, we revealed that the downregulation of RBM5 expression promoted the expression of coactivator-associated arginine methyltransferase 1 (CARM1) in BC cells and tissues. Increased expression of CARM1 facilitated the activation of the Wnt/β-catenin axis and cell proliferation, which then contributed to the poor prognosis of patients with BC. Interestingly, RBM5 bound directly to CARM1 mRNA and participated in AS-NMD, downregulating the expression of CARM1. In addition, we revealed that protein kinase catalytic subunit alpha (PRKACA) functioned as a phosphorylated kinase of GSK3β, was regulated by CARM1 at the transcription level, and promoted the growth and progression of BC cells. Furthermore, in this study, we demonstrated a regulatory mechanism of Wnt/β-catenin activation through the RBM5/CARM1/PRKACA axis and identified a novel potential target for treating BC.

Inhibition of CARM1 suppresses proliferation of multiple myeloma cells through activation of p53 signaling pathway

BackgroundMultiple myeloma (MM) is a malignant proliferative disease of plasma cells, the incidence of which is increasing every year and remains incurable. The enzyme co-activator-associated arginine methyltransferase 1 (CARM1) is highly expressed in a variety of cancers, such as Hodgkin's lymphoma and acute myeloid leukemia, and CARM1 is closely associated with tumor cell proliferation. However, the role of CARM1 in MM has not been elucidated.Methods and resultsIn this study, we found that CARM1 is overexpressed in MM and closely associated with poor prognosis in MM. CCK-8 and colony formation assays showed that the proliferation of MM cell lines was downregulated when CARM1 expression was knockdown by specific shRNA. Knockdown of CARM1 reduced the proportion of MM cell lines in the S phase and increased the proportion in G0/G1 phase. RNA-seq analysis of the CARM1-KD cell line revealed that it was closely associated with apoptosis and activated the p53 pathway. CCK-8 and apoptosis results showed that CARM1 knockdown made MM cells more sensitive to standard-of-care drugs.ConclusionThis study provides an experimental basis for elucidating the pathogenesis of multiple myeloma and searching for potential therapeutic targets.

Histone arginine methyltransferase CARM1 selective inhibitor TP-064 induces apoptosis in endometrial cancer

Histone modification is the key epigenetic mechanism that regulates gene expression. Coactivator-associated arginine methyltransferase 1 (CARM1) is an arginine methyltransferase that catalyzes dimethylation of histone H3 (H3R17) at arginine 17. Lately, it has been suggested that CARM1 is associated with human carcinogenesis, and the CARM1-selective inhibitor, TP-064, has been shown to be a potential therapeutic agent for multiple myeloma. However, the physiological significance of CARM1 in endometrial cancer remains unclear. Therefore, we aimed to explore the role of CARM1 and the effect of TP-064 in endometrial cancer. To this end, we analyzed CARM1 expression in endometrial cancer using quantitative real-time polymerase chain reaction and examined the antitumor mechanism with CARM1 knockdown endometrial cancer cells. Moreover, we evaluated the therapeutic capability of TP-064 in endometrial cancer cells. CARM1 was remarkably overexpressed in 52 endometrial cancer tissues compared to normal endometrial tissues. The growth of CARM1 knockdown endometrial cancer cells was suppressed and CARM1 knockdown induced apoptosis. TP-064 also inhibited endometrial cancer cell growth and declined the number of endometrial cancer cell colonies. These data suggest that CARM1 may be a powerful therapeutic target for endometrial cancer.

Systematic pan-cancer landscape identifies CARM1 as a potential prognostic and immunological biomarker

BackgroundBelonging to the protein arginine methyltransferase (PRMT) family, the enzyme encoded by coactivator associated arginine methyltransferase 1 (CARM1) catalyzes the methylation of protein arginine residues, especially acts on histones and other chromatin related proteins, which is essential in regulating gene expression. Beyond its well-established involvement in the regulation of transcription, recent studies have revealed a novel role of CARM1 in tumorigenesis and development, but there is still a lack of systematic understanding of CARM1 in human cancers. An integrated analysis of CARM1 in pan-cancer may contribute to further explore its prognostic value and potential immunological function in tumor therapy.ResultsBased on systematic analysis of data in multiple databases, we firstly verified that CARM1 is highly expressed in most tumors compared with corresponding normal tissues, and is bound up with poor prognosis in some tumors. Subsequently, relevance between CARM1 expression level and tumor immune microenvironment is analyzed from the perspectives of tumor mutation burden, microsatellite instability, mismatch repair genes, methyltransferases genes, immune checkpoint genes and immune cells infiltration, indicating a potential relationship between CARM1 expression and tumor microenvironment. A gene enrichment analysis followed shortly, which implied that the role of CARM1 in tumor pathogenesis may be related to transcriptional imbalance and viral carcinogenesis.ConclusionsOur first comprehensive bioinformatics analysis provides a broad molecular perspective on the role of CARM1 in various tumors, highlights its value in clinical prognosis and potential association with tumor immune microenvironment, which may furnish an immune based antitumor strategy to provide a reference for more accurate and personalized immunotherapy in the future.

Blocking the Spinal Fbxo3/CARM1/K+ Channel Epigenetic Silencing Pathway as a Strategy for Neuropathic Pain Relief.

Many epigenetic regulators are involved in pain-associated spinal plasticity. Coactivator-associated arginine methyltransferase 1 (CARM1), an epigenetic regulator of histone arginine methylation, is a highly interesting target in neuroplasticity. However, its potential contribution to spinal plasticity-associated neuropathic pain development remains poorly explored. Here, we report that nerve injury decreased the expression of spinal CARM1 and induced allodynia. Moreover, decreasing spinal CARM1 expression by Fbxo3-mediated CARM1 ubiquitination promoted H3R17me2 decrement at the K+ channel promoter, thereby causing K+ channel epigenetic silencing and the development of neuropathic pain. Remarkably, in naïve rats, decreasing spinal CARM1 using CARM1 siRNA or a CARM1 inhibitor resulted in similar epigenetic signaling and allodynia. Furthermore, intrathecal administration of BC-1215 (a novel Fbxo3 inhibitor) prevented CARM1 ubiquitination to block K+ channel gene silencing and ameliorate allodynia after nerve injury. Collectively, the results reveal that this newly identified spinal Fbxo3-CARM1-K+ channel gene functional axis promotes neuropathic pain. These findings provide essential insights that will aid in the development of more efficient and specific therapies against neuropathic pain.

CARM1 Regulates AMPK Signaling in Skeletal Muscle.

SummaryCoactivator-associated arginine methyltransferase 1 (CARM1) is an emerging mediator of skeletal muscle plasticity. We employed genetic, physiologic, and pharmacologic approaches to determine whether CARM1 regulates the master neuromuscular phenotypic modifier AMP-activated protein kinase (AMPK). CARM1 skeletal muscle-specific knockout (mKO) mice displayed reduced muscle mass and dysregulated autophagic and atrophic processes downstream of AMPK. We observed altered interactions between CARM1 and AMPK and its network, including forkhead box protein O1, during muscle disuse. CARM1 methylated AMPK during the early stages of muscle inactivity, whereas CARM1 mKO mitigated progression of denervation-induced atrophy and was accompanied by attenuated phosphorylation of AMPK targets such as unc-51 like autophagy-activating kinase 1Ser555. Lower acetyl-coenzyme A corboxylaseSer79 phosphorylation, as well as reduced peroxisome proliferator-activated receptor-γ coactivator-1α, was also observed in mKO animals following acute administration of the direct AMPK activator MK-8722. Our study suggests that targeting CARM1-AMPK interplay may have broad impacts on neuromuscular health and disease.

Investigating the Function of Co‐activator‐associated Arginine Methyltransferase 1 (CARM1) During Denervation‐induced Skeletal Muscle Atrophy

CARM1 is a type I protein arginine methyltransferase (PRMT) that forms asymmetric dimethylarginines (ADMA) by transferring monomethyl groups to arginine residues on target proteins. We previously observed elevated CARM1 expression and methyltransferase activity in response to neurogenic muscle disuse. Transient knockdown of CARM1 in muscle also represses the progression of muscle wasting and the expression of atrophy‐related genes following denervation. Notably, CARM1 regulates intracellular signaling molecules that are important for atrophy such as AMP‐activated protein kinase (AMPK) and downstream unc‐51 like autophagy activating kinase 1 (ULK1), forkhead box class O (FOXO), and transcription factor EB (TFEB) in non‐muscle tissues. The purpose of this study was to more comprehensively elucidate the function of CARM1 during neurogenic muscle disuse. We employed the Cre/loxP system to generate skeletal muscle‐specific CARM1 knockout (mKO) mice. Following unilateral sectioning of the sciatic nerve, mKO and wild type (WT) mice were subjected to 3 and 7 days of denervation. The contralateral limb served as an internal control. Hematoxylin and eosin staining analyses were performed to assess cross‐sectional area (CSA) in the extensor digitorum longus (EDL) muscle. We also employed Western blot and immunoprecipitation analyses to evaluate protein expression and protein interaction levels, respectively, in the non‐denervated and denervated tibialis anterior muscles. Our CSA analyses revealed that the absence of CARM1 mitigated denervation‐induced atrophy in the EDL muscle after 7 days of disuse (p < 0.05). Compared to WT animals, phosphorylation levels of AMPK and ULK1 were lower (p < 0.05) in mKO mice following 3 days of neurogenic muscle disuse. We also report that CARM1 interacts with AMPK, FOXO1, and TFEB in skeletal muscle. Relative to the contralateral, non‐denervated control (CON) limb, the interactions between CARM1‐AMPK and CARM1‐FOXO1 increased (p < 0.05) after 3 days of denervation in WT mice. When compared to the CON limb, the CARM1‐TFEB interaction was lower (p < 0.05) in WT mice following 7 days of neurogenic muscle disuse. In comparison to WT animals, the methylation of AMPK was significantly blunted in mKO mice after 3 days of denervation, suggesting that CARM1 methyltransferase activity regulates AMPK signaling. Collectively, these results suggest that CARM1 interacts with intracellular signaling molecules that are integral for muscle wasting. Importantly, targeting the interplay between CARM1‐AMPK, CARM1‐FOXO1, and CARM1‐TFEB may provide a novel therapeutic strategy for mitigating neurogenic skeletal muscle atrophy.Support or Funding InformationNatural Sciences and Engineering Research Council of CanadaCanada Research Chairs

TRIM28 protects CARM1 from proteasome-mediated degradation to prevent colorectal cancer metastasis

TRIM28 (Tripartite motif-containing protein 28), a member of TRIM family, is aberrantly expressed and reportedly has different functions in many types of human cancer. However, the biological roles of TRIM28 and related mechanism in colorectal cancer (CRC) remain unclear. Here, we showed that TRIM28 was downregulated in colorectal cancer compared with normal mucosa, especially at advanced stages, and acted as an independent prognostic factor of favorable outcome. Functional studies demonstrated that TRIM28 restrained CRC migration and invasion in vitro and in vivo. Mechanistically, we reported that CARM1 (co-activator-associated arginine methyltransferase1) was a critical player downstream of TRIM28. TRIM28 interacted with CARM1, and protected CARM1 from proteasome-mediated degradation through physical protein-protein interaction to suppress CRC metastasis. Further, TRIM28 suppressed the migration and invasion of CRC cells through inhibiting WNT/β-catenin signaling in a CARM1-dependent manner, but independent of CARM1′s methyltransferase activity. The protein expression of CARM1 was positively correlated with TRIM28 in CRC tissues. Patients with high levels of TRIM28 and CARM1 had improved prognosis, whereas patients with low TRIM28 and CARM1 expression had the poor outcomes. Thus, our study reveals an inhibitory role of TRIM28 in CRC metastasis, which was achieved through a TRIM28-CARM1-WNT/β-catenin axis. This work provides potential prognostic and therapeutic targets for CRC treatment.

The co-activator-associated arginine methyltransferase 1 (CARM1) gene is overexpressed in type 2 diabetes.

We examined the expression of a panel of epigenetic enzymes catalyzing histone tails post-transcriptional modifications, together with effectors of metabolic and inflammatory alterations, in type 2 diabetes. Cross-sectional, case-control study of 21 people with type 2 diabetes and 21 matched controls. Total RNA was extracted from white cells and reverse transcribed. PCR primer assays for 84 key genes encoding enzymes known to modify genomic DNA and histones were performed. Western blot was performed on lysates using primary antibodies for abnormally expressed enzymes. Hormones and cytokines were measured by multiplex kits. A Bayesian network was built to investigate the relationships between epigenetic, cytokine, and endocrine variables. Co-activator-associated aRginine Methyltransferase 1 (CARM1) expression showed a five-fold higher median value, matched by higher protein levels, among patients who also had increased GIP, IL-4, IL-7, IL-13, IL-17, FGF basic, G-CSF, IFN-γ, and TNFα and decreased IP-10. In a Bayesian network approach, CARM1 expression showed a conditional dependence on diabetes, but was independent of all other variables nor appeared to influence any. Increased CARM1 expression in type 2 diabetes suggests that epigenetic mechanisms are altered in human diabetes. The impact of lifestyle and pharmacological treatment on regulation of this enzyme should be further investigated.

P53 mediated regulation of coactivator associated arginine methyltransferase 1 (CARM1) expression is critical for suppression of adipogenesis.

Coactivator-associated arginine methyltransferase 1 (CARM1/PRMT4) is a type I arginine methyltransferase that mediates transcriptional activation via methylation of histone H3 on R17, R26, and R42. CARM1 is also a coactivator of transcription of various transcription factors such as NF-kB, MEF2C, β-catenin, p53, PPAR-gamma etc. CARM1 has been functionally implicated in maintenance of pluripotency, cellular differentiation, and tumorigenesis; where its expression status plays an important role. Although its expression has been shown to be regulated by a few miRNAs in different contexts at post-transcriptional level, transcriptional regulation of CARM1 gene is still unexplored. In this report we demonstrate that CARM1 is a p53 responsive gene, where p53 could suppress CARM1 promoter-driven luciferase expression. CARM1 gene expression was found to be repressed by p53 in 3T3L1 preadipocytes when activated with Nutlin-3a treatment. Ectopic overexpression of CARM1 could rescue inhibitory effect of p53 on adipogenesis, suggesting a role of p53-CARM1 axis of regulation operational in the context of adipocyte differentiation. p53 and CARM1 showed antagonistic regulatory influence on PPAR-gamma expression; which suggests that p53-mediated suppression of adipogenesis could be partly via repression of CARM1 expression. Taken together these observations provide convincing mechanistic explanation for p53 function in the context of adipocyte differentiation process.

P53 mediated regulation of Coactivator associated arginine methyltransferase 1 (CARM1) expression is critical for suppression of adipogenesis

Coactivator associated arginine methyltransferase 1 (CARM1/PRMT4) is a type I arginine methyltransferase that mediates transcriptional activation via methylation of histone H3 on R17, R26 and R42. CARM1 acts as a coactivator of transcription of various transcription factors such as NF‐kB, MEF2C, β‐catenin, p53, PPAR‐gamma etc. CARM1 has been functionally implicated in maintenance of pluripotency, cellular differentiation and tumorigenesis; where its expression status plays an important role. Although its expression has been shown to be regulated by a few miRNAs in different contexts at post‐transcriptional level, transcriptional regulation of CARM1 gene is still unexplored. In the present study we demonstrate that CARM1 is a p53 responsive gene, where p53 could suppress CARM1 promoter driven luciferase expression. CARM1 gene expression was found to be repressed by p53 in 3T3L1 preadipocytes when activated with Nutlin‐3a treatment. Ectopic overexpression of CARM1 could rescue inhibitory effect of p53 on adipogenesis, suggesting a role of p53‐CARM1 axis of regulation operational in the context of adipocyte differentiation. p53 and CARM1 showed antagonistic regulatory influence on PPAR‐gamma expression; which suggests that p53 mediated suppression of adipogenesis could be partly via repression of CARM1 expression. Taken together these observations provide convincing mechanistic explanation for p53 function in the context of adipocyte differentiation process.Support or Funding InformationTKK is recipient of JC Bose fellowship from Dept. of Science and technology, Govt. of India (SR/S2/JCB‐28/2010). AKB is a research associate of JNCASR. AB is a senior research fellow of UGC, India. This work was supported by funding from Dept. of Biotchnology, Govt. of India (BT/01/CEIB/10/III/01) and JNCASR, India.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of coactivator-associated arginine methyltransferase 1 modulates dendritic arborization and spine maturation of cultured hippocampal neurons

An improved understanding of the molecular mechanisms in synapse formation provides insight into both learning and memory and the etiology of neurodegenerative disorders. Coactivator-associated arginine methyltransferase 1 (CARM1) is a protein methyltransferase that negatively regulates synaptic gene expression and inhibits neuronal differentiation. Despite its regulatory function in neurons, little is known about the CARM1 cellular location and its role in dendritic maturation and synapse formation. Here, we examined the effects of CARM1 inhibition on dendritic spine and synapse morphology in the rat hippocampus. CARM1 was localized in hippocampal post-synapses, with immunocytochemistry and electron microscopy revealing co-localization of CARM1 with post-synaptic density (PSD)-95 protein, a post-synaptic marker. Specific siRNA-mediated suppression of CARM1 expression resulted in precocious dendritic maturation, with increased spine width and density at sites along dendrites and induction of mushroom-type spines. These changes were accompanied by a striking increase in the cluster size and number of key synaptic proteins, including N-methyl-d-aspartate receptor subunit 2B (NR2B) and PSD-95. Similarly, pharmacological inhibition of CARM1 activity with the CARM1-specific inhibitor AMI-1 significantly increased spine width and mushroom-type spines and also increased the cluster size and number of NR2B and cluster size of PSD-95. These results suggest that CARM1 is a post-synaptic protein that plays roles in dendritic maturation and synaptic formation and that spatiotemporal regulation of CARM1 activity modulates neuronal connectivity and improves synaptic dysfunction.

The Overexpression of CARM1 Promotes Human Osteosarcoma Cell Proliferation through the pGSK3β/β-Catenin/cyclinD1 Signaling Pathway.

Osteosarcoma (OS) is a kind of malignant bone tumor that occurs frequently in the region surrounding the knee joint and poses a threat to the health of teenagers. Since the application of chemotherapy to treat OS, 5-year survival rate in patients has improved from 10% to 70%, but patient survival has not changed over the past four decades. Coactivator-associated arginine methyltransferase 1 (CARM1) is a member of the PRMT protein family; it acts as an oncogene in many cancers, but its function in OS is still unknown. In this study, we found that CARM1 is overexpressed in OS and its expression is correlated with the Enneking stage. CCK-8 and colony forming assays showed that proliferation in OS cell lines was downregulated when siRNA was used to knockdown CARM1 expression. The cell cycle was inhibited in S phase after si-CARM1 transfection in OS cell lines. An antibody array indicated that Erk1/2 (Thr202/Tyr204), PARS40 (Thr246), and GSK3β (Ser9) expression are affected by CARM1, and western blotting verified that CARM1 promotes OS cell proliferation via pGSK3β/β-catenin/cyclinD1 signaling. Accordingly, CARM1 is a crucial gene in OS and is a potential new treatment target.

Ubiquitination-dependent CARM1 degradation facilitates Notch1-mediated podocyte apoptosis in diabetic nephropathy

Podocyte apoptosis induced by hyperglycemia is considered a critical factor in the development of diabetic nephropathy. Recent studies have implicated Notch signaling in podocyte apoptosis; however, its regulatory mechanisms are not fully understood. In this study, we found that high-glucose treatment increased Notch1 and Jagged-1 expression, the transcriptional activity of Hes, and podocyte apoptosis, and decreased the expression of coactivator-associated arginine methyltransferase 1 (CARM1) in rat podocytes. Transient transfection of CARM1 reversed high-glucose-induced Notch1 expression, the transcriptional activity of Hes, and podocyte apoptosis. Moreover, the silencing of CARM1 using siRNA increased Notch1 expression, the transcriptional activity of Hes, and podocyte apoptosis. However, the Glu266-mediated enzymatic activity of CARM1 was not necessary for Notch signaling activation and podocyte apoptosis. Here, we demonstrate that AMP-activated protein kinase alpha (AMPKα) and cannabinoid receptor 1 (CB1R) are regulated by CARM1 and that high-glucose-induced podocyte apoptosis is mediated by a CARM1–AMPKα–Notch1–CB1R signaling axis. We also show that high-glucose-induced CARM1 downregulation is due to ubiquitination-dependent CARM1 degradation. Finally, we demonstrate that CARM1 expression in podocytes was diminished in rats with streptozotocin-induced diabetes compared to vehicle-treated rats. Together, our data provide evidence that ubiquitination-dependent CARM1 degradation in podocytes in diabetes promotes podocyte apoptosis via Notch1 activation. Strategies to preserve CARM1 expression or reduce the enzymatic activity of a ubiquitin ligase specific for CARM1 could be used to prevent podocyte loss in diabetic nephropathy.

Elevated expression of coactivator-associated arginine methyltransferase 1 is associated with early hepatocarcinogenesis

Aberrant expression of regulators for epigenetics is involved in tumorigenesis. There is an urgent need to identify and characterize regulators concerned with epigenetics in the early stages of hepatocarcinogenesis. In the present study, we found that the expression of coactivator-associated arginine methyltransferase 1 (CARM1), a histone methyltransferase that functions as a cofactor for nuclear hormone receptors and several transcription factors, was elevated in adenomas and aberrant in carcinomas during hepatocellular carcinogenesis. In addition to RNA expression, immunohistochemical staining of liver sections revealed that CARM1 was highly expressed in the nucleus of tumor marker glutathione S-transferase placental form (GST-P)-positive foci. Neoplastic transformation of GST-P-positive foci guides the formation of hepatocellular carcinomas. CARM1 expression was not elevated in GST-P-negative regions. Furthermore, a luciferase reporter analysis revealed that CARM1 activated the Gst-p promoter in H4IIE, a hepatocellular carcinoma cell line. This activation was mediated by the enhancer element responsible for the carcinogenic-specific expression of Gst-p and nuclear factor E2-related factor2. Knockdown of Carm1 by shRNA in H4IIE cells inhibited cell proliferation. These findings suggest that aberrantly expressed CARM1 in tumor marker-positive cells promotes tumorigenesis in the early stages of hepatocarcinogenesis.

The oestrogen receptor coactivator CARM1 has an oncogenic effect and is associated with poor prognosis in breast cancer

The coactivator-associated arginine methyltransferase-1 (CARM1) is implicated in regulation of oestrogen receptor (ER) α-mediated gene pathways in response to ER activation. It plays an important role in breast cancer growth by regulating the E2F1 expression suggesting that CARM1 could be a target in the subclassification of oestrogen-dependent breast cancer. This study aims to investigate the clinical and biological importance of CARM1 protein expression in a large (1,130 patients), well-characterised and annotated series of invasive breast cancers using tissue microarrays and immunohistochemistry. In the whole series, increased CARM1 expression is correlated with features associated with aggressive behaviour such as young age, premenopausal status, large tumour size and high tumour grade. There is a positive correlation between CARM1 expression and biomarkers associated with non-luminal phenotype and poor prognosis such as HER2, basal cytokeratins, EGFR, p53 and the proliferation markers Ki67, TK1, CD71 and Cyclin E. Negative associations with the luminal-associated markers including steroid receptors and luminal cytokeratins are found. Similar associations are identified in the ER-positive/luminal subgroup (n=767). Outcome analyses indicate that CARM1 expression is an independent predictor of shorter breast cancer-specific survival and disease-free interval in the whole series and in the ER-positive subgroup. CARM1 shows an oncogenic effect in breast cancer and its expression is associated with poor prognosis. CARM1 could be a potential marker of luminal class subclassification and for target therapy, particularly in the ER-positive luminal-like subgroup.

CARM1 Is an Important Determinant of ERα-Dependent Breast Cancer Cell Differentiation and Proliferation in Breast Cancer Cells

Breast cancers with estrogen receptor α (ERα) expression are often more differentiated histologically than ERα-negative tumors, but the reasons for this difference are poorly understood. One possible explanation is that transcriptional cofactors associated with ERα determine the expression of genes which promote a more differentiated phenotype. In this study, we identify one such cofactor as coactivator-associated arginine methyltransferase 1 (CARM1), a unique coactivator of ERα that can simultaneously block cell proliferation and induce differentiation through global regulation of ERα-regulated genes. CARM1 was evidenced as an ERα coactivator in cell-based assays, gene expression microarrays, and mouse xenograft models. In human breast tumors, CARM1 expression positively correlated with ERα levels in ER-positive tumors but was inversely correlated with tumor grade. Our findings suggest that coexpression of CARM1 and ERα may provide a better biomarker of well-differentiated breast cancer. Furthermore, our findings define an important functional role of this histone arginine methyltransferase in reprogramming ERα-regulated cellular processes, implicating CARM1 as a putative epigenetic target in ER-positive breast cancers.

HuD interacts with survival motor neuron protein and can rescue spinal muscular atrophy-like neuronal defects

Spinal muscular atrophy is an autosomal-recessive neuromuscular disease caused by disruption of the survival of motor neuron (SMN) gene, which promotes cytoplasmic assembly of the splicing core machinery. It remains unclear how a deficiency in SMN results in a disorder leading to selective degeneration of lower motor neurons. We report here that SMN interacts with RNA-binding protein HuD in neurites of motorneuron-derived MN-1 cells. This interaction is mediated through the Tudor domain of SMN and, importantly, naturally occurring Tudor mutations found in patients with severe spinal muscular atrophy (SMA) completely abrogate the interaction, underscoring its relevance to the disease process. We also characterized a regulatory pathway involving coactivator-associated arginine methyltransferase 1 (CARM1) and HuD. Specifically, we show that CARM1 expression is rapidly downregulated, at the protein level, following induction of differentiation through retinoid and neurotrophic signaling. Using purified proteins, we demonstrate that methylation of HuD by CARM1 reduces its interaction with the p21(cip1/waf1) mRNA, showing that CARM1 can directly influence RNA-binding activity. We further demonstrate that this CARM1-dependent regulatory switch mainly controls the activity of HuD in promoting cell-cycle exit, whereas the interaction between HuD and SMN is required for proper recruitment of HuD and its mRNA targets in neuronal RNA granules. Finally, we were able to rescue SMA-like defects in a hypomorphic Smn knockdown MN-1 cell line through overexpression of HuD. Together, these findings extend our understanding of specific role(s) of SMN in motor neurons and provide crucial insights into potential new avenues for SMA therapeutic strategies.

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.

Control of CBP co-activating activity by arginine methylation

The histone acetyltransferases CREB binding protein (CBP) and the related p300 protein function as key transcriptional co-activators in multiple pathways. In the case of transcriptional activation by nuclear receptors, ligand promotes the recruitment of co-activators of the p160 family, such as GRIP-1. Subsequently, the p160 co-activators recruit other co-activators via two activation domains, AD1 and AD2. AD1 binds CBP or p300, whereas AD2 has been shown to activate transcription through the recruitment of the arginine methyltransferase CARM1. Recently, the KIX domain of CBP has been shown to be methylated by CARM1 in vitro. Here, we report that another domain of CBP is specifically methylated by CARM1 on conserved arginine residues in vitro and in vivo. We also provide functional evidence that arginine residues methylated by CARM1 play a critical role in GRIP-1-dependent transcriptional activation and in hormone-induced gene activation. Altogether, our data provide strong evidence that arginine methylation represents an important mechanism for modulating co-activator transcriptional activity.