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1.  Differential CARM1 expression in prostate and colorectal cancers 
BMC Cancer  2010;10:197.
Background
Coactivator-associated arginine methyltransferase 1 (CARM1) functions as a transcriptional coactivator of androgen receptor (AR)-mediated signaling. Correspondingly, overexpression of CARM1 has been associated with the development of prostate cancer (PCa) and its progression to androgen-independent PCa. In our preliminary study, however, the promoting effects of CARM1, with regard to androgen-stimulated AR target gene expression were minimal. These results suggested that the AR target gene expression associated with CARM1 may result primarily from non-hormone dependent activity. The goal of this study was to confirm the pattern of expression of CARM1 in human tumors and determine the mechanism of action in CARM1 overexpressed tumors.
Methods
Tissue microarray was used to determine the pattern of expression of CARM1 in human cancers by immunohistochemistry. CARM1 expression was also evaluated in prostate and colorectal surgical specimens and the clinical records of all cases were reviewed. In addition, a reporter transcription assay using the prostate-specific antigen (PSA) promoter was used to identify the signaling pathways involved in non-hormone-mediated signal activation associated with CARM1.
Results
The tissue microarray showed that CARM1 was particularly overexpressed in the colorectal cancers while CARM1 expression was not prevalent in the prostate and breast cancers. Further studies using surgical specimens demonstrated that CARM1 was highly overexpressed in 75% of colorectal cancers (49 out of 65) but not in the androgen-independent PCa. In addition, CARM1's coactivating effect on the entire PSA promoter was very limited in both androgen-dependent and androgen-independent PCa cells. These results suggest that there are other factors associated with CARM1 expression in PSA regulation. Indeed, CARM1 significantly regulated both p53 and NF-κB target gene transcription.
Conclusions
The results of this study suggest that, in addition to its role in activation of steroid receptors, CARM1 functions as a transcriptional modulator by altering the activity of many transcriptional factors, especially with regard to androgen independent PCa and colorectal cancers.
doi:10.1186/1471-2407-10-197
PMCID: PMC2881889  PMID: 20462455
2.  Developmentally Essential Protein Flightless I Is a Nuclear Receptor Coactivator with Actin Binding Activity 
Molecular and Cellular Biology  2004;24(5):2103-2117.
Hormone-activated nuclear receptors (NR) activate transcription by recruiting multiple coactivator complexes to the promoters of target genes. One important coactivator complex includes a p160 coactivator (e.g., GRIP1, SRC-1, or ACTR) that binds directly to activated NR, the histone acetyltransferase p300 or CBP, and the arginine-specific histone methyltransferase CARM1. We previously demonstrated that the coactivator function of CARM1 depends both on the methyltransferase activity and on additional unknown proteins that bind to CARM1. In this study a yeast two-hybrid screen for proteins that bind CARM1 identified the protein Flightless I (Fli-I), which has essential roles in Drosophila and mouse development. Fli-I bound to CARM1, GRIP1, and NRs and cooperated synergistically with CARM1 and GRIP1 to enhance NR function. Fli-I bound poorly to and did not cooperate with PRMT1, a CARM1-related protein arginine methyltransferase that also functions as an NR coactivator. The synergy between GRIP1, CARM1, and Fli-I required the methyltransferase activity of CARM1. The C-terminal AD1 (binding site for p300/CBP) and AD2 (binding site for CARM1) activation domains of GRIP1 contributed to the synergy but were less stringently required than the N-terminal region of GRIP1, which is the binding site for Fli-I. Endogenous Fli-I was recruited to the estrogen-regulated pS2 gene promoter of MCF-7 cells in response to the hormone, and reduction of endogenous Fli-I levels by small interfering RNA reduced hormone-stimulated gene expression by the endogenous estrogen receptor. A fragment of Fli-I that is related to the actin binding protein gelsolin enhanced estrogen receptor activity, and mutations that reduced actin binding also reduced the coactivator function of this Fli-I fragment. These data suggest that Fli-I may facilitate interaction of the p160 coactivator complex with other coactivators or coactivator complexes containing actin or actin-like proteins.
doi:10.1128/MCB.24.5.2103-2117.2004
PMCID: PMC350567  PMID: 14966289
3.  The protein arginine methyltransferases CARM1 and PRMT1 cooperate in gene regulation 
Nucleic Acids Research  2008;36(10):3202-3213.
Protein arginine methyltransferases (PRMT) have been implicated in the regulation of transcription. They are recruited to promoters via interaction with transcription factors and exert their coactivator function by methylating arginine residues in histones and other chromatin proteins. Here, we employ an unbiased approach to identify novel target genes, which are under the control of two members of the enzyme family, PRMT1 and CARM1/PRMT4 (coactivator associated arginine methyltransferase 1). By using cDNA microarray analysis, we find that the siRNA-mediated single knockdown of neither CARM1 nor PRMT1 causes significant changes in gene expression. In contrast, double knockdown of both enzymes results in the deregulated expression of a large group of genes, among them the CITED2 gene. Cytokine-stimulated expression analysis indicates that transcriptional activation of CITED2 depends on STAT5 and the coactivation of both PRMTs. ChIP analysis identifies the CITED2 gene as a direct target gene of STAT5, CARM1 and PRMT1. In reporter gene assays, we show that STAT5-mediated transcription is cooperatively enhanced by CARM1 and PRMT1. Interaction assays reveal a cytokine-induced association of STAT5 and the two PRMTs. Our data demonstrate a widespread cooperation of CARM1 and PRMT1 in gene activation as well as repression and that STAT5-dependent transcription of the CITED2 gene is a novel pathway coactivated by the two methyltransferases.
doi:10.1093/nar/gkn166
PMCID: PMC2425501  PMID: 18413343
4.  Expression and purification of full-length mouse CARM1 from transiently transfected HEK293T cells using HaloTag technology 
Coactivator-associated arginine methyl transferase 1 (CARM1) is a protein arginine methyltransferase (PRMT) family member that functions as a coactivator in androgen and estrogen signaling pathways and plays a role in the progression of prostate and breast cancer. CARM1 catalyzes methylation of diverse protein substrates. Prior attempts to purify the full-length mouse CARM1 protein have proven unsatisfactory. The full-length protein expressed in E. coli forms insoluble inclusion bodies that are difficult to denature and to refold. The presented results demonstrate the use of a novel HaloTag™ technology to purify full-length CARM1 from both E. coli and mammalian HEK293T cells. A small amount of CARM1 was purified from E. coli; however, the protein was truncated on the N-terminus by 10–50 amino acids, most likely due to endogenous proteolytic activity. In contrast, substantial quantities of soluble full-length CARM1 were purified from transiently transfected HEK293T cells. The CARM1 from HEK293T cells was isolated alongside a number of co-purifying interacting proteins. The covalent bond formed between the HaloTag and the HaloLink resin allowed the use of stringent wash conditions without risk of eluting the CARM1 protein. The results also illustrate a highly effective approach for purifying and enriching both CARM1-associated proteins as well as substrates for CARM1’s methyltransferase activity.
doi:10.1016/j.pep.2010.11.010
PMCID: PMC3248244  PMID: 21126607
CARM1; PRMT family; methyl transferase; HaloTag; HaloLink resin; affinity purification
5.  A TR-FRET based functional assay for screening activators of CARM1 
Chembiochem : a European journal of chemical biology  2013;14(7):10.1002/cbic.201300029.
Epigenome is an emerging field that demands selective cell-permeable chemical probes to perturb, especially in vivo, the activity of specific enzymes involved in modulating the epigenetic codes. Coactivator Associated Arginine (R) Methyltransferase 1 (CARM1) is a coactivator of estrogen receptor α (ERα), the main target in human breast cancer. We previously showed that overexpression of CARM1 by two-fold in MCF7 breast cancer cells increased the expression of ERα-target genes involved in differentiation and reduced cell proliferation, leading to the hypothesis that activating CARM1 by chemical activators may be therapeutically effective in breast cancer. Selective, potent, cell-permeable CARM1 activators will be essential to test this hypothesis. Here we report the development of a cell-based, time-resolved Förster resonance energy transfer (TR-FRET) assay using poly (A) binding protein 1 (PABP1) methylation to monitor cellular activity of CARM1. The LanthaScreen® TR-FRET assay utilizes MCF7 cells expressing GFP-PABP1 fusion protein via BacMam gene delivery system, methyl-PABP1 specific antibody and terbium-labeled secondary antibody. This assay has been validated to reflect the expression and/or activity of CARM1 and optimized for high throughput screening to identify CARM1 allosteric activators. This TR-FRET platform serves as a generic tool for functional screening of cell-permeable, chemical modulators of CARM1 for elucidation of its in vivo functions.
doi:10.1002/cbic.201300029
PMCID: PMC3828750  PMID: 23585185
Arginine methylation; CARM1; PABP1; TR-FRET
6.  Differential CARM1 Isoform Expression in Subcellular Compartments and among Malignant and Benign Breast Tumors 
PLoS ONE  2015;10(6):e0128143.
Purpose
Coactivator-associated arginine methyltransferase 1 (CARM1) is a coactivator for ERα and cancer-relevant transcription factors, and can methylate diverse cellular targets including histones. CARM1 is expressed in one of two alternative splice isoforms, full-length CARM1 (CARM1FL) and truncated CARM1 (CARM1ΔE15). CARM1FL and CARM1ΔE15 function differently in transcriptional regulation, protein methylation, and mediation of pre-mRNA splicing in cellular models.
Methods
To investigate the functional roles and the prognosis potential of CARM1 alternative spliced isoforms in breast cancer, we used recently developed antibodies to detect differential CARM1 isoform expression in subcellular compartments and among malignant and benign breast tumors.
Results
Immunofluorescence in MDA-MB-231 and BG-1 cell lines demonstrated that CARM1ΔE15 is the dominant isoform expressed in the cytoplasm, and CARM1FL is more nuclear localized. CARM1ΔE15 was found to be more sensitive to Hsp90 inhibition than CARM1FL, indicating that the truncated isoform may be the oncogenic form. Clinical cancer samples did not have significantly higher expression of CARM1FL or CARM1ΔE15 than benign breast samples at the level of mRNA or histology. Furthermore neither CARM1FL nor CARM1ΔE15 expression correlated with breast cancer molecular subtypes, tumor size, or lymph node involvement.
Conclusions
The analysis presented here lends new insights into the possible oncogenic role of CARM1ΔE15. This study also demonstrates no obvious association of CARM1 isoform expression and clinical correlates in breast cancer. Recent studies, however, have shown that CARM1 expression correlates with poor prognosis, indicating a need for further studies of both CARM1 isoforms in a large cohort of breast cancer specimens.
doi:10.1371/journal.pone.0128143
PMCID: PMC4451767  PMID: 26030442
7.  Synergy among Nuclear Receptor Coactivators: Selective Requirement for Protein Methyltransferase and Acetyltransferase Activities 
Molecular and Cellular Biology  2002;22(11):3621-3632.
Hormone-activated nuclear receptors (NR) bind to specific regulatory DNA elements associated with their target genes and recruit coactivator proteins to remodel chromatin structure, recruit RNA polymerase, and activate transcription. The p160 coactivators (e.g., SRC-1, GRIP1, and ACTR) bind directly to activated NR and can recruit a variety of secondary coactivators. We have established a transient-transfection assay system under which the activity of various NR is highly or completely dependent on synergistic cooperation among three classes of coactivators: a p160 coactivator, the protein methyltransferase CARM1, and any of the three protein acetyltransferases, p300, CBP, or p/CAF. The three-coactivator functional synergy was only observed when low levels of NR were expressed and was highly or completely dependent on the methyltransferase activity of CARM1 and the acetyltransferase activity of p/CAF, but not the acetyltransferase activity of p300. Other members of the protein arginine methyltransferase family, which methylate different protein substrates than CARM1, could not substitute for CARM1 to act synergistically with p300 or p/CAF. A ternary complex of GRIP1, CARM1, and p300 or CBP was demonstrated in cultured mammalian cells, supporting a physiological role for the observed synergy. The transfection assay described here is a valuable new tool for investigating the mechanism of coactivator function and demonstrates the importance of multiple coactivators, including CARM1 and its specific protein methyltransferase activity, in transcriptional activation.
doi:10.1128/MCB.22.11.3621-3632.2002
PMCID: PMC133819  PMID: 11997499
8.  Xenoestrogens Regulate the Activity of Arginine Methyltransferases 
Chembiochem  2010;12(2):323-329.
Arginine methylation is a common posttranslational modification that has been strongly implicated in transcriptional regulation. The arginine methyltransferases (PRMTs) were first reported as transcriptional coactivators for the estrogen and androgen receptors. Compounds that inhibit these enzymes will provide us with valuable tools for dissecting the roles of these enzymes in cells, and will possibly also have therapeutic applications. In order to identify such inhibitors of the PRMTs, we performed a high throughput screen using a small molecule library a number of years ago. We termed these compounds AMIs (arginine methyltransferase inhibitors). The majority of these inhibitors were polyphenols, and one in particular (AMI-18) shared additional features with a group of known xenoestrogens. We thus tested a panel of xenoestrogens and found that a number of them possess the ability to inhibit PRMT activity in vitro. These inhibitors primarily target CARM1, and include licochalcone A, kepone, benzyl 4-hydroxybenzoate, and tamoxifen. We developed a cell-based reporter system for CARM1 activity, and showed that tamoxifen (IC50=30 µM) inhibits this PRMT. The ability of these compounds to regulate the activity of transcriptional coactivators may be an unappreciated mechanism of action for xenoestrogens and may also explain the efficacy of high-dose tamoxifen treatment on estrogen receptor negative cancers.
doi:10.1002/cbic.201000522
PMCID: PMC3142315  PMID: 21243720
PRMT1; CARM1; Arginine methylation; Xenoestrogens
9.  The Activity and Stability of the Transcriptional Coactivator p/CIP/SRC-3 Are Regulated by CARM1-Dependent Methylation▿  
Molecular and Cellular Biology  2006;27(1):120-134.
The transcriptional coactivator p/CIP(SRC-3/AIB1/ACTR/RAC3) binds liganded nuclear hormone receptors and facilitates transcription by directly recruiting accessory factors such as acetyltransferase CBP/p300 and the coactivator arginine methyltransferase CARM1. In the present study, we have established that recombinant p/CIP (p300/CBP interacting protein) is robustly methylated by CARM1 in vitro but not by other protein arginine methyltransferase family members. Metabolic labeling of MCF-7 breast cancer cells with S-adenosyl-L-[methyl-3H]methionine and immunoblotting using dimethyl arginine-specific antibodies demonstrated that p/CIP is specifically methylated in intact cells. In addition, methylation of full-length p/CIP is not supported by extracts derived from CARM1−/− mouse embryo fibroblasts, indicating that CARM1 is required for p/CIP methylation. Using mass spectrometry, we have identified three CARM1-dependent methylation sites located in a glutamine-rich region within the carboxy terminus of p/CIP which are conserved among all steroid receptor coactivator proteins. These results were confirmed by in vitro methylation of p/CIP using carboxy-terminal truncation mutants and synthetic peptides as substrates for CARM1. Analysis of methylation site mutants revealed that arginine methylation causes an increase in full-length p/CIP turnover as a result of enhanced degradation. Additionally, methylation negatively impacts transcription via a second mechanism by impairing the ability of p/CIP to associate with CBP. Collectively, our data highlight coactivator methylation as an important regulatory mechanism in hormonal signaling.
doi:10.1128/MCB.00815-06
PMCID: PMC1800659  PMID: 17043108
10.  Coactivator-Associated Arginine Methyltransferase 1 Enhances Transcriptional Activity of the Human T-Cell Lymphotropic Virus Type 1 Long Terminal Repeat through Direct Interaction with Tax 
Journal of Virology  2006;80(20):10036-10044.
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).
doi:10.1128/JVI.00186-06
PMCID: PMC1617284  PMID: 17005681
11.  Expression, purification, crystallization and preliminary crystallographic study of isolated modules of the mouse coactivator-associated arginine methyltransferase 1 
Isolated modules of mouse coactivator-associated arginine methyltransferase 1 encompassing the protein arginine N-methyltransferase catalytic domain have been overexpressed, purified and crystallized. X-ray diffraction data have been collected and have enabled determination of the structures by multiple isomorphous replacement using anomalous scattering.
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. CARM128–507 and two structural states of CARM1140–480 were expressed, purified and crystallized. Crystals of CARM128–507 belong to space group P6222, with unit-cell parameters a = b = 136.0, c = 125.3 Å; they diffract to beyond 2.5 Å resolution using synchrotron radiation and contain one monomer in the asymmetric unit. The structure of CARM128–507 was solved by multiple isomorphous replacement and anomalous scattering methods. Crystals of apo CARM1140–480 belong to space group I222, with unit-cell parameters a = 74.6, b = 99.0, c = 207.4 Å; they diffract to beyond 2.7 Å resolution and contain two monomers in the asymmetric unit. Crystals of CARM1140–480 in complex with S-­adenosyl-l-homocysteine belong to space P21212, with unit-cell parameters a = 74.6, b = 98.65, c = 206.08 Å; they diffract to beyond 2.6 Å resolution and contain four monomers in the asymmetric unit. The structures of apo and holo CARM1140–480 were solved by molecular-replacement techniques from the structure of CARM128–507.
doi:10.1107/S1744309107011785
PMCID: PMC2330207  PMID: 17401209
CARM1; gene expression
12.  Distinct Protein Arginine Methyltransferases Promote ATP-Dependent Chromatin Remodeling Function at Different Stages of Skeletal Muscle Differentiation▿  
Molecular and Cellular Biology  2009;29(7):1909-1921.
Temporal regulation of gene expression is a hallmark of cellular differentiation pathways, yet the mechanisms controlling the timing of expression for different classes of differentiation-specific genes are not well understood. We previously demonstrated that the class II arginine methyltransferase Prmt5 was required for skeletal muscle differentiation at the early stages of myogenesis (C. S. Dacwag, Y. Ohkawa, S. Pal, S. Sif, and A. N. Imbalzano, Mol. Cell. Biol. 27:384-394, 2007). Specifically, when Prmt5 levels were reduced, the ATP-dependent SWI/SNF chromatin-remodeling enzymes could not interact with or remodel the promoter of myogenin, an essential early gene. Here we investigated the requirement for Prmt5 and the class I arginine methyltransferase Carm1/Prmt4 in the temporal control of myogenesis. Both arginine methyltransferases could bind to and modify histones at late-gene regulatory sequences. However, the two enzymes showed sequential requirements for gene expression. Prmt5 was required for early-gene expression but dispensable for late-gene expression. Carm1/Prmt4 was required for late- but not for early-gene expression. The reason for the requirement for Carm1/Prmt4 at late genes was to facilitate SWI/SNF chromatin-remodeling enzyme interaction and remodeling at late-gene loci. Thus, distinct arginine methyltransferases are employed at different times of skeletal muscle differentiation for the purpose of facilitating ATP-dependent chromatin-remodeling enzyme interaction and function at myogenic genes.
doi:10.1128/MCB.00742-08
PMCID: PMC2655603  PMID: 19188441
13.  Genome wide comparative analysis of the effects of PRMT5 and PRMT4/CARM1 arginine methyltransferases on the Arabidopsis thaliana transcriptome 
BMC Genomics  2015;16(1):192.
Background
Methylation at arginine residues (R) is an important post-translational modification that regulates a myriad of essential cellular processes in eukaryotes, such as transcriptional regulation, RNA processing, signal transduction and DNA repair. Arginine methylation is catalyzed by a family of enzymes known as protein arginine methyltransferases (PRMTs). PRMTs are classified as Type I or Type II, depending on the position of the methyl group on the guanidine of the methylated arginine. Previous reports have linked symmetric R methylation to transcriptional repression, while asymmetric R methylation is generally associated with transcriptional activation. However, global studies supporting this conclusion are not available.
Results
Here we compared side by side the physiological and molecular roles of the best characterized plant PRMTs, the Type II PRMT5 and the Type I PRMT4, also known as CARM1 in mammals. We found that prmt5 and prmt4a;4b mutants showed similar alterations in flowering time, photomorphogenic responses and salt stress tolerance, while only prmt5 mutants exhibited alterations in circadian rhythms. An RNA-seq analysis revealed that expression and splicing of many differentially regulated genes was similarly enhanced or repressed by PRMT5 and PRMT4s. Furthermore, PRMT5 and PRMT4s co-regulated the expression and splicing of key regulatory genes associated with transcription, RNA processing, responses to light, flowering, and abiotic stress tolerance, being candidates to mediate the physiological alterations observed in the mutants.
Conclusions
Our global analysis indicates that two of the most important Type I and Type II arginine methyltransferases, PRTM4 and PRMT5, have mostly overlapping as well as specific, but not opposite, roles in the global regulation of gene expression in plants.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1399-2) contains supplementary material, which is available to authorized users.
doi:10.1186/s12864-015-1399-2
PMCID: PMC4381356  PMID: 25880665
14.  Identification of Novel Inhibitors against Coactivator Associated Arginine Methyltransferase 1 Based on Virtual Screening and Biological Assays 
BioMed Research International  2016;2016:7086390.
Overexpression of coactivator associated arginine methyltransferase 1 (CARM1), a protein arginine N-methyltransferase (PRMT) family enzyme, is associated with various diseases including cancers. Consequently, the development of small-molecule inhibitors targeting PRMTs has significant value for both research and therapeutic purposes. In this study, together with structure-based virtual screening with biochemical assays, two compounds DC_C11 and DC_C66 were identified as novel inhibitors of CARM1. Cellular studies revealed that the two inhibitors are cell membrane permeable and effectively blocked proliferation of cancer cells including HELA, K562, and MCF7. We further predicted the binding mode of these inhibitors through molecular docking analysis, which indicated that the inhibitors competitively occupied the binding site of the substrate and destroyed the protein-protein interactions between CARM1 and its substrates. Overall, this study has shed light on the development of small-molecule CARM1 inhibitors with novel scaffolds.
doi:10.1155/2016/7086390
PMCID: PMC5107250  PMID: 27872854
15.  Targeted disruption of the p160 coactivator interface of androgen receptor (AR) selectively inhibits AR activity in both androgen-dependent and castration-resistant AR-expressing prostate cancer cells 
The evidence that androgen blockade-resistant prostate cancer, termed castration resistant, remains androgen receptor (AR) dependent is compelling. AR is re-activated through multiple mechanisms including expression of constitutively active splice variants that lack hormone binding domains (HBD). This highlights the need to develop therapies that target regions other than the HBD. Because the p160 coactivators interact most strongly with the amino-terminus of AR, we examined the consequences of disrupting this interaction. We identified two overlapping SRC-1 peptides that interact with AR, but not with progesterone receptor. These peptides reduce AR and AR variant AR-V7 dependent induction of an AR responsive reporter. Using mammalian two hybrid assays, we found that the peptides interrupt the AR/SRC-1; AR/SRC-2 and AR N/C interactions, but not SRC-1/CARM-1 interactions. Consistent with the SRC-1 dependence of induced, but not repressed genes, in LNCaP cells, the peptides inhibited hormone dependent induction of endogenous target genes including PSA and TMPRSS2, but did not block AR dependent repression of UGT2B17 or inhibit vitamin D receptor activity. Simultaneous detection of SRC-1 peptides and PSA by double immunofluorescence in transfected LNCaP cells clearly demonstrated a strong reduction in PSA levels in cells expressing the peptides. The peptides also inhibited the AR dependent expression of PSA in castration resistant C4-2 cells,. Moreover they inhibited androgen dependent proliferation of LNCaP cells and proliferation of C4-2 cells in androgen depleted medium without affecting AR negative PC-3 cells. Thus, the p160 coactivator binding site is a novel potential therapeutic target to inhibit AR activity.
doi:10.1016/j.biocel.2012.12.012
PMCID: PMC3593820  PMID: 23270728
SRC-1; androgen receptor; prostate cancer; peptide; CRPC
16.  Identification of Small-Molecule Enhancers of Arginine Methylation Catalyzed by Coactivator-Associated Arginine Methyltransferase 1 
Journal of medicinal chemistry  2012;55(22):9875-9890.
Arginine methylation is a common post-translational modification that is crucial in modulating gene expression at multiple critical levels. The arginine methyltransferases (PRMTs) are envisaged as promising druggable targets but their role in physiological and pathological pathways is far from being clear, due to the limited number of modulators reported to date. In this effort, enzyme activators can be invaluable tools useful as gain-of-function reagents to interrogate the biological roles in cells and in vivo of PRMTs. Yet the identification of such molecules is rarely pursued. Herein we describe a series of aryl ureido acetamido indole carboxylates (dubbed “uracandolates”), able to increase the methylation of histone- (H3) or non-histone (polyadenylate-binding protein 1, PABP1) substrates induced by coactivator-associated arginine methyltransferase 1 (CARM1), both in in vitro and cellular settings. To the best of our knowledge, this is the first report of compounds acting as CARM1 activators.
doi:10.1021/jm301097p
PMCID: PMC3508294  PMID: 23095008
CARM1 activator; PRMT inhibitors; arginine methyltransferase; histone modifying enzyme; epigenetics
17.  Expression and sub-cellular localization of an epigenetic regulator, co-activator arginine methyltransferase 1 (CARM1), is associated with specific breast cancer subtypes and ethnicity 
Molecular Cancer  2013;12:40.
Background
Co-Activator Arginine Methyltransferase 1(CARM1) is an Estrogen Receptor (ER) cofactor that remodels chromatin for gene regulation via methylation of Histone3. We investigated CARM1 levels and localization across breast cancer tumors in a cohort of patients of either European or African ancestry.
Methods
We analyzed CARM1 levels using tissue microarrays with over 800 histological samples from 549 female cancer patients from the US and Nigeria, Africa. We assessed associations between CARM1 expression localized to the nucleus and cytoplasm for 11 distinct variables, including; ER status, Progesterone Receptor status, molecular subtypes, ethnicity, HER2+ status, other clinical variables and survival.
Results
We found that levels of cytoplasmic CARM1 are distinct among tumor sub-types and increased levels are associated with ER-negative (ER-) status. Higher nuclear CARM1 levels are associated with HER2 receptor status. EGFR expression also correlates with localization of CARM1 into the cytoplasm. This suggests there are distinct functions of CARM1 among molecular tumor types. Our data reveals a basal-like subtype association with CARM1, possibly due to expression of Epidermal Growth Factor Receptor (EGFR). Lastly, increased cytoplasmic CARM1, relative to nuclear levels, appear to be associated with self-identified African ethnicity and this result is being further investigated using quantified genetic ancestry measures.
Conclusions
Although it is known to be an ER cofactor in breast cancer, CARM1 expression levels are independent of ER. CARM1 has distinct functions among molecular subtypes, as is indicative of its sub-cellular localization and it may function in subtype etiology. These sub-cellular localization patterns, indicate a novel role beyond its ER cofactor function in breast cancer. Differential localization among ethnic groups may be due to ancestry-specific polymorphisms which alter the gene product.
doi:10.1186/1476-4598-12-40
PMCID: PMC3663705  PMID: 23663560
Tissue-microarray; Breast cancer; Molecular subtypes; CARM1; Epigenetic regulator; Subcellular localization; Ethnic disparities
18.  PRMT5 is required for cell-cycle progression and p53 tumor suppressor function 
Nucleic Acids Research  2009;37(15):4965-4976.
Protein arginine methyltransferases (PRMTs) mediate the transfer of methyl groups to arginines in proteins involved in signal transduction, transcriptional regulation and RNA processing. Tumor suppressor p53 coordinates crucial cellular processes, including cell-cycle arrest and DNA repair, in response to stress signals. Post-translational modifications and interactions with co-factors are important to regulate p53 transcriptional activity. To explore whether PRMTs modulate p53 function, we generated multiple cell lines in which PRMT1, CARM1 and PRMT5 are inducibly knocked down. Here, we showed that PRMT5, but not PRMT1 or CARM1, is essential for cell proliferation and PRMT5 deficiency triggers cell-cycle arrest in G1. In addition, PRMT5 is required for p53 expression and induction of p53 targets MDM2 and p21 upon DNA damage. Importantly, we established that PRMT5 knockdown prevents p53 protein synthesis. Furthermore, we found that PRMT5 regulates the expression of translation initiation factor eIF4E and growth suppression mediated upon PRMT5 knockdown is independent of p53 but is dependent on eIF4E. Taken together, we uncovered that arginine methyltransferase PRMT5 is a major pro-survival factor regulating eIF4E expression and p53 translation.
doi:10.1093/nar/gkp516
PMCID: PMC2731901  PMID: 19528079
19.  CARM1 is an important determinant of ERα-dependent breast cancer cell differentiation and proliferation in breast cancer cells 
Cancer research  2011;71(6):2118-2128.
Breast cancers expressing estrogen receptor α (ERα) are often more differentiated histologically than ERα-negative tumors, but the reasons for this difference are poorly understood. One possible explanation is that transcriptional co-factors 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 co-activator of ERα that can simultaneously block cell proliferation and induce differentiation through global regulation of ERα-regulated genes. CARM1 was evidenced as an ERα co-activator in cell-based assays, gene expression microarrays, and mouse xenograft models. In human breast tumors, CARM1 expression positively correlated with ERα levels in ER+ tumors but was inversely correlated with tumor grade. Our findings suggest that co-expression of CARM1 and ERα may provide a better biomarker of well-differentiated breast cancer. Further, our findings define an important functional role of this histone arginine methyltransferase in re-programming ERα-regulated cellular processes, implicating CARM1 as a putative epigenetic target in ER-positive breast cancers.
doi:10.1158/0008-5472.CAN-10-2426
PMCID: PMC3076802  PMID: 21282336
CARM1; histone methylation; breast cancer; differentiation; epigenetics
20.  The Methyltransferases PRMT4/CARM1 and PRMT5 Control Differentially Myogenesis in Zebrafish 
PLoS ONE  2011;6(10):e25427.
In vertebrates, skeletal myogenesis involves the sequential activation of myogenic factors to lead ultimately to the differentiation into slow and fast muscle fibers. How transcriptional co-regulators such as arginine methyltransferases PRMT4/CARM1 and PRMT5 control myogenesis in vivo remains poorly understood. Loss-of-function experiments using morpholinos against PRMT4/CARM1 and PRMT5 combined with in situ hybridization, quantitative polymerase chain reaction, as well as immunohistochemistry indicate a positive, but differential, role of these enzymes during myogenesis in vivo. While PRMT5 regulates myod, myf5 and myogenin expression and thereby slow and fast fiber formation, PRMT4/CARM1 regulates myogenin expression, fast fiber formation and does not affect slow fiber formation. However, our results show that PRMT4/CARM1 is required for proper slow myosin heavy chain localization. Altogether, our results reveal a combinatorial role of PRMT4/CARM1 and PRMT5 for proper myogenesis in zebrafish.
doi:10.1371/journal.pone.0025427
PMCID: PMC3189919  PMID: 22016767
21.  HISTONE H3 LYSINE 9 METHYLTRANSFERASE G9a IS A TRANSCRIPTIONAL COACTIVATOR FOR NUCLEAR RECEPTORS* 
The Journal of biological chemistry  2006;281(13):8476-8485.
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 GRIP1, 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 associates 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.
doi:10.1074/jbc.M511093200
PMCID: PMC1770944  PMID: 16461774
22.  Overexpression of CARM1 in breast cancer is correlated with poorly characterized clinicopathologic parameters and molecular subtypes 
Diagnostic Pathology  2013;8:129.
Background
Coactivator-associated arginine methyltransferase 1 (CARM1) belongs to the protein arginine methyltransferase family. CARM1 has been reported to be associated with high grade tumors in breast cancer. It still remains unknown the expression pattern of CARM1 in breast cancer and its relationships with clinicopathological characteristics and molecular subtypes.
Methods
Two hundred forty-seven invasive breast cancer cases were collected and prepared for tissue array. There were thirty-seven tumors with benign glandular epithelium adjacent to the tumors among these cases. Molecular subtype and CARM1 expression were investigated using immunohistochemistry.
Results
Cell staining was observed in the cytoplasm and/or nucleus. Staining for CARM1 was significantly stronger in adenocarcinoma compared with adjacent benign epithelium. There is a significant correlation between CARM1 overexpression with young age, high grade, estrogen receptor (ER) and progesterone receptor (PR) negative, increased p53 expression, and high Ki-67 index. Our study demonstrated CARM1 overexpression was associated with an increase in the protein expression of HER2. Furthermore, our data indicated CARM1-overexpression rate were remarkably higher in HER2 subtype (69.6%), luminal B subtype (59.6%) and TN subtype (57.1%) compared with luminal A subtype (41.3%).
Conclusions
CARM1 expression was increased in invasive breast cancer. CARM1 overexpression was associated with poorly characterized clinicopathologic parameters and HER2 overexpression. There were significant differences between different molecular subtypes in their relationship to CARM1 overexpression. Our results support the value of using CARM1 in prognostic stratification of breast cancer patients and its potential therapeutic implications in targeting treatment.
Virtual slides
The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/4116338491022965
doi:10.1186/1746-1596-8-129
PMCID: PMC3766166  PMID: 23915145
CARM1; Breast cancer; Clinicopathologic parameters; HER2; Molecular subtype
23.  Signaling within a Coactivator Complex: Methylation of SRC-3/AIB1 Is a Molecular Switch for Complex Disassembly▿  
Molecular and Cellular Biology  2006;26(21):7846-7857.
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.
doi:10.1128/MCB.00568-06
PMCID: PMC1636757  PMID: 16923966
24.  Regulation of Pluripotency-related Genes and Differentiation in Mouse Embryonic Stem Cells by Direct Delivery of Cell-penetrating Peptide-conjugated CARM1 Recombinant Protein 
Development & Reproduction  2013;17(1):9-16.
Coactivator-associated arginine methyltransferase 1 (CARM1) is included in the protein arginine methyltransferase (PRMT) family, which methylates histone arginine residues through posttranslational modification. It has been proposed that CARM1 may up-regulate the expression of pluripotency-related genes through the alteration of the chromatin structure. Mouse embryonic stem cells (mESCs) are pluripotent and have the ability to self-renew. The cells are mainly used to study the genetic function of novel genes, because the cells facilitate the transmission of the manipulated genes into target mice. Since the up-regulated methylation levels of histone arginine residue lead to the maintenance of pluripotency in embryos and stem cells, it may be suggested that CARM1 overexpressing mESCs elevate the expression of pluripotency-related genes in reconstituted embryos for transgenic mice and may resist the differentiation into trophectoderm (TE). We constructed a fusion protein by connecting CARM1 and 7X-arginine (R7). As a cell-penetrating peptide (CPP), can translocate CARM1 protein into mESCs. CPP-CARM1 protein was detected in the nuclei of the mESCs after a treatment of 24 hours. Accordingly, the expression of pluripotency-related genes was up-regulated in CPP-CARM1-treated mESCs. In addition, CPP-CARM1-treated mESC-derived embryoid bodies (EBs) showed an elevated expression of pluripotency-related genes and delayed spontaneous differentiation. This result suggests that the treatment of recombinant CPP-CARM1 protein elevates the expression of pluripotency-related genes of mESCs by epigenetic modification, and this protein-delivery system could be used to modify embryonic fate in reconstituted embryos with mESCs.
doi:10.12717/DR.2013.17.1.009
PMCID: PMC4282220  PMID: 25949116
CARM1; Cell-penetrating peptide; Pluripotent-related genes; Mouse embryonic stem cells; 3-germ layers
25.  CARM1 regulates astroglial lineage through transcriptional regulation of Nanog and posttranscriptional regulation by miR92a 
Molecular Biology of the Cell  2015;26(2):316-326.
CARM1-mediated H3R17 methylation in hESC-derived embryoid bodies regulates astrocyte fate commitment–associated genes and miRNAs directly by methylating the promoter or indirectly through miRNAs. This is the first report of the involvement of CARM1 in neural development, the absence of which is observed as sensory response defects in zebrafish embryos.
Coactivator-associated arginine methyltransferase (CARM1/PRMT4)–mediated transcriptional coactivation and arginine methylation is known to regulate various tissue-specific differentiation events. Although CARM1 is expressed in the neural crest region in early development, coinciding with early neuronal progenitor specification, the role of CARM1 in any neuronal developmental pathways has been unexplored. Using a specific small-molecule inhibitor of CARM1-mediated H3R17 methylation in human embryonic stem cell line, we find that H3R17 methylation contributes to the maintenance of the astroglial cell population. A network of regulation was observed on the miR92a promoter by which H3R17-responsive Nanog bound to the miR92a promoter decreased upon inhibition, resulting in an abnormal gene expression program influencing the glial lineage. This was also true in zebrafish, in which, with the help of CARM1 inhibitor and CARM1 morpholinos, we show that inhibition of H3R17 methylation results in defective glial cell morphology and a sensory defect in a subpopulation. A gain-of-function strategy in which mCARM1 was introduced in the morpholino-treated embryos exhibited recovery of the sensory defect phenotype. This study thus establishes the functional cooperation between arginine methylation and microRNA expression in the neuronal developmental process, with potential implications in sensory development pathways.
doi:10.1091/mbc.E14-01-0019
PMCID: PMC4294678  PMID: 25392304

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