Protein arginine methyltransferase 6 regulates multiple aspects of gene expression

It is well established that transcription and alternative splicing events are functionally coupled during gene expression. Here, we report that protein arginine N-methyltransferase 6 (PRMT6) may play a key role in this coupling process by functioning as a transcriptional coactivator that can also regulate alternative splicing. PRMT6 coactivates the progesterone, glucocorticoid and oestrogen receptors in luciferase reporter assays in a hormone-dependent manner. In addition, small interfering RNA (siRNA) oligonucleotide duplex knockdown of PRMT6 disrupts oestrogen-stimulated transcription of endogenous GREB1 and progesterone receptor in MCF-7 breast cancer cells, demonstrating the importance of PRMT6 in hormone-dependent transcription. In contrast, the regulation of alternative splicing by PRMT6 is hormone independent. siRNA knockdown of PRMT6 increases the exon inclusion:skipping ratio of alternatively spliced exons in endogenous vascular endothelial growth factor and spleen tyrosine kinase RNA transcripts in both the presence and absence of oestrogen. These results demonstrate that PRMT6 has a dual role in regulating gene expression and that these two functions can occur independently of each other.

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title: The RXR heterodimers and orphan receptors
abstract: The nuclear receptor superfamily is comprised of over 150 different proteins that have evolved to mediate a complex array of extracellular signals into transcriptional responses. Many, but not all, of these proteins directly bind to signaling molecules, which, because of their small lipophilic character, can easily enter the target cell. Thus, unlike membrane-bound receptors, the nuclear receptors are intracellular and function to control the activity of target genes directly. In aggregate, these target genes comprise a genetic network whose coordinate activity defines the physiologic response. The purpose of this review is to establish the historical links between the steroid and nonsteroid receptor signaling systems, to chart the explosive development of the retinoid X receptor (RXR) heterodimer and orphan receptor family, to explain the impact of these discoveries on our understanding of the mechanisms of hormonal signaling, and, finally, to present emerging issues and implications of these studies for animal development, physiology, and human disease.

title: Sensors and signals: a coactivator/corepressor/epigenetic code for integrating signal-dependent programs of transcriptional response.
abstract: A decade of intensive investigation of coactivators and corepressors required for regulated actions of DNA-binding transcription factors has revealed a network of sequentially exchanged cofactor complexes that execute a series of enzymatic modifications required for regulated gene expression. These coregulator complexes possess "sensing" activities required for interpretation of multiple signaling pathways. In this review, we examine recent progress in understanding the functional consequences of "molecular sensor" and "molecular adaptor" actions of corepressor/coactivator complexes in integrating signal-dependent programs of transcriptional responses at the molecular level. This strategy imposes a temporal order for modifying programs of transcriptional regulation in response to the cellular milieu, which is used to mediate developmental/homeostatic and pathological events.

title: The roles of protein–protein interactions and protein methylation in transcriptional activation by nuclear receptors and their coactivators
abstract: Hormone-activated nuclear receptors (NR) bind to the promoters of their target genes and recruit coactivator proteins to help activate transcription. The p160 coactivators bind directly to activated NRs and recruit secondary coactivators CBP/p300 with protein acetyltransferase activity and CARM1 with protein methyltransferase activity. To further investigate the components of the p160 coactivator complex and their mechanisms of action, we have used two guiding assumptions. First, the coactivators constitute a signal transduction pathway that convey the signal from DNA-bound NRs to the transcription machinery. Second, each coactivator has signal input and signal output domains that facilitate signal transduction. These assumptions were used to address the mechanism by which CARM1 and the N-terminal region of p160 coactivators transmit activating signals to the transcription machinery. The p160-binding activity of CARM1 is in the same centrally located structural domain as the methyltransferase activity; the p160-binding domain anchors CARM1 to the target gene promoter and thereby serves as its signal input domain. CARM1 has two signal output mechanisms: the protein methyltransferase activity, which methylates histones and other proteins in the transcription initiation complex; and a strong autonomous activation function in the C-terminal region. We identified a protein, CCCP, which binds to the C-terminal region of CARM1 and cooperates synergistically with CARM1 to enhance NR function. We also defined the N-terminal region of p160 coactivators as another signal output domain, which binds a novel coactivator called coiled-coil coactivator (CoCoA). CoCoA acts synergistically with p160 coactivators to enhance NR function.

title: Nuclear Receptor Coactivator ACTR Is a Novel Histone Acetyltransferase and Forms a Multimeric Activation Complex with P/CAF and CBP/p300
abstract: We report here the identification of a novel cofactor, ACTR, that directly binds nuclear receptors and stimulates their transcriptional activities in a hormone-dependent fashion. ACTR also recruits two other nuclear factors, CBP and P/CAF, and thus plays a central role in creating a multisubunit coactivator complex. In addition, and unexpectedly, we show that purified ACTR is a potent histone acetyltransferase and appears to define a distinct evolutionary branch to this recently described family. Thus, hormonal activation by nuclear receptors involves the mutual recruitment of at least three classes of histone acetyltransferases that may act cooperatively as an enzymatic unit to reverse the effects of histone deacetylase shown to be part of the nuclear receptor corepressor complex.

title: The nuclear hormone receptor coactivator SRC-1 is a specific target of p300.
abstract: p300 and its family member, CREB-binding protein (CBP), function as key transcriptional coactivators by virtue of their interaction with the activated forms of certain transcription factors. In a search for additional cellular targets of p300/CBP, a protein-protein cloning strategy, surprisingly identified SRC-1, a coactivator involved in nuclear hormone receptor transcriptional activity, as a p300/CBP interactive protein. p300 and SRC-1 interact, specifically, in vitro and they also form complexes in vivo. Moreover, we show that SRC-1 encodes a new member of the basic helix-loop-helix-PAS domain family and that it physically interacts with the retinoic acid receptor in response to hormone binding. Together, these results implicate p300 as a component of the retinoic acid signaling pathway, operating, in part, through specific interaction with a nuclear hormone receptor coactivator, SRC-1.

title: Regulation of transcription by a protein methyltransferase.
abstract: The p160 family of coactivators, SRC-1, GRIP1/TIF2, and p/CIP, mediate transcriptional activation by nuclear hormone receptors. Coactivator-associated arginine methyltransferase 1 (CARM1), a previously unidentified protein that binds to the carboxyl-terminal region of p160 coactivators, enhanced transcriptional activation by nuclear receptors, but only when GRIP1 or SRC-1a was coexpressed. Thus, CARM1 functions as a secondary coactivator through its association with p160 coactivators. CARM1 can methylate histone H3 in vitro, and a mutation in the putative S-adenosylmethionine binding domain of CARM1 substantially reduced both methyltransferase and coactivator activities. Thus, coactivator-mediated methylation of proteins in the transcription machinery may contribute to transcriptional regulation.

title: Synergistic Enhancement of Nuclear Receptor Function by p160 Coactivators and Two Coactivators with Protein Methyltransferase Activities*
abstract: Nuclear receptors (NRs) activate gene transcription by binding to specific enhancer elements and recruiting coactivators of the p160 family to promoters of target genes. The p160 coactivators in turn enhance transcription by recruiting secondary coactivators, including histone acetyltransferases such as CREB-binding protein (CBP) and p300/CBP-associated factor (p/CAF), as well as the recently identified protein methyltransferase, coactivator-associated arginine methyltransferase 1 (CARM1). In the current study, protein arginine methyltransferase 1 (PRMT1), another arginine-specific protein methyltransferase that shares a region of high homology with CARM1, was also found to act as a coactivator for NRs. PRMT1, like CARM1, bound to the C-terminal AD2 activation domain of p160 coactivators and thereby enhanced the activity of NRs in transient transfection assays. The shape of the graphs of reporter gene activity versus the amounts of CARM1 or PRMT1 expression vector indicated a cooperative relationship between coactivator concentration and activity. Moreover, CARM1 and PRMT1 acted in a synergistic manner to enhance reporter gene activation by both hormone-dependent and orphan NRs. The synergy was most evident at low levels of transfected NR expression vectors, where activation of reporter genes was almost completely dependent on the presence of NR and all three exogenously supplied coactivators, i.e. GRIP1, CARM1, and PRMT1. In contrast, with the higher levels of NR expression vectors typically used in transient transfection assays, NR activity was much less dependent on the combination of coactivators, suggesting that target gene activation occurs by different mechanisms at high versus low cellular concentrations of NR. Because multiple coactivators are presumably required to mediate transcriptional activation of native genes in vivo, the low-NR conditions may provide a more physiologically relevant assay for coactivator function.

title: RNA and protein interactions modulated by protein arginine methylation.
abstract: This review summarizes the current status of protein arginine N-methylation reactions. These covalent modifications of proteins are now recognized in a number of eukaryotic proteins and their functional significance is beginning to be understood. Genes that encode those methyltransferases specific for catalyzing the formation of asymmetric dimethylarginine have been identified. The enzyme modifies a number of generally nuclear or nucleolar proteins that interact with nucleic acids, particularly RNA. Postulated roles for these reactions include signal transduction, nuclear transport, or a direct modulation of nucleic acid interactions. A second methyltransferase activity that symmetrically dimethylates an arginine residue in myelin basic protein, a major component of the axon sheath, has also been characterized. However, a gene encoding this activity has not been identified to date and the cellular function for this methylation reaction has not been clearly established. From the analysis of the sequences surrounding known arginine methylation sites, we have determined consensus methyl-accepting sequences that may be useful in identifying novel substrates for these enzymes and may shed further light on their physiological role.

title: The protein arginine methyltransferase family: an update about function, new perspectives and the physiological role in humans
abstract: Information about the family of protein arginine methyltransferases (PRMTs) has been growing rapidly over the last few years and the emerging role of arginine methylation involved in cellular processes like signaling, RNA processing, gene transcription, and cellular transport function has been investigated. To date, 11 PRMTs gene transcripts have been identified in humans. Almost all PRMTs have been shown to have enzymatic activity and to catalyze arginine methylation. This review will summarize the overall function of human PRMTs and include novel highlights on each family member.

title: Methylation at arginine 17 of histone H3 is linked to gene activation
abstract: The nuclear hormone receptor co‐activator CARM1 has the potential to methylate histone H3 at arginine residues in vitro. The methyltransferase activity of CARM1 is necessary for its co‐activator functions in transient transfection assays. However, the role of this methyltransferase in vivo is unclear, given that methylation of arginines is not easily detectable on histones. We have raised an antibody that specifically recognizes methylated arginine 17 (R17) of histone H3, the major site of methylation by CARM1. Using this antibody we show that methylated R17 exists in vivo. Chromatin immunoprecipitation analysis shows that R17 methylation on histone H3 is dramatically upregulated when the estrogen receptor‐regulated pS2 gene is activated. Coincident with the appearance of methylated R17, CARM1 is found associated with the histones on the pS2 gene. Together these results demonstrate that CARM1 is recruited to an active promoter and that CARM1‐mediated R17 methylation on histone H3 takes place in vivo during this active state.

title: Hormone-dependent, CARM1-directed, arginine-specific methylation of histone H3 on a steroid-regulated promoter
abstract: Activation of gene transcription involves chromatin remodeling by coactivator proteins that are recruited by DNA-bound transcription factors. Local modification of chromatin structure at specific gene promoters by ATP-dependent processes and by posttranslational modifications of histone N-terminal tails provides access to RNA polymerase II and its accompanying transcription initiation complex. While the roles of lysine acetylation, serine phosphorylation, and lysine methylation of histones in chromatin remodeling are beginning to emerge, low levels of arginine methylation of histones have only recently been documented, and its physiological role is unknown. The coactivator CARM1 methylates histone H3 at Arg17 and Arg26 in vitro and cooperates synergistically with p160-type coactivators (e.g., GRIP1, SRC-1, ACTR) and coactivators with histone acetyltransferase activity (e.g., p300, CBP) to enhance gene activation by steroid and nuclear hormone receptors (NR) in transient transfection assays. In the current study, CARM1 cooperated with GRIP1 to enhance steroid hormone-dependent activation of stably integrated mouse mammary tumor virus (MMTV) promoters, and this coactivator function required the methyltransferase activity of CARM1. Chromatin immunoprecipitation assays and immunofluorescence studies indicated that CARM1 and the CARM1-methylated form of histone H3 specifically associated with a large tandem array of MMTV promoters in a hormone-dependent manner. Thus, arginine-specific histone methylation by CARM1 is an important part of the transcriptional activation process.

title: Methylation of histone H3 by coactivator-associated arginine methyltransferase 1.
abstract: The preferential in vitro methylation of histone H3 by coactivator-associated arginine methyltransferase 1 (CARM1) has been proposed as a basis for its ability to enhance gene transcription [Chen, D., et al. (1999) Science 284, 2174-2177]. To further evaluate the significance of H3 methylation, we studied the kinetics and site specificity of its modification by CARM1. Affinity-purified CARM1 methylated recombinant chick H3, which is free of posttranslational modifications, and calf thymus H3, which is heterogeneous with regard to preexisting modifications, equally well, exhibiting a V(max) of 4500 pmol min(-1) (mg of enzyme)(-1) and an apparent K(m) for H3 of < or = 0.2 microM. The catalytic efficiency (k(cat)/K(m)) of CARM1 toward H3 was at least 1000 times that toward R1 (GGFGGRGGFGG-amide), a highly effective substrate for protein arginine methyltransferase 1. Peptide mapping of 3H-methyl-labeled H3 indicated methylation at Arg-2, Arg-17, and Arg-26 in the N-terminal region and at one or more of four arginines (128/129/131/134) at the C-terminus. Two of the N-terminal sites, Arg-17 and Arg-26, occur in the sequence KAXRK and appear to be more efficiently methylated than Arg-2. CARM1 catalyzed formation of N(G),N(G)-dimethylarginine (asymmetric) but little or no N(G),N'(G)-dimethylarginine (symmetric) and no form of methyllysine. Amino acid analysis of untreated calf thymus H3 revealed that 3.7% of the molecules naturally contain asymmetric dimethylarginine and/or monomethylarginine. Our findings support the hypothesis that methylation of H3 may be involved in the mechanism of transcriptional coactivation by CARM1 of genes whose expression is under the control of nuclear receptors.

title: Minireview: protein arginine methylation of nonhistone proteins in transcriptional regulation.
abstract: Endocrine regulation frequently culminates in altered transcription of specific genes. The signal transduction pathways, which transmit the endocrine signal from cell surface to the transcription machinery, often involve posttranslational modifications of proteins. Although phosphorylation has been by far the most widely studied protein modification, recent studies have indicated important roles for other types of modification, including protein arginine methylation. Ten different protein arginine methyltransferase (PRMT) family members have been identified in mammalian cells, and numerous substrates are being identified for these PRMTs. Whereas major attention has been focused on the methylation of histones and its role in chromatin remodeling and transcriptional regulation, there are many nonhistone substrates methylated by PRMTs. This review primarily focuses on recent progress on the roles of the nonhistone protein methylation in transcription. Protein methylation of coactivators, transcription factors, and signal transducers, among other proteins, plays important roles in transcriptional regulation. Protein methylation may affect protein-protein interaction, protein-DNA or protein-RNA interaction, protein stability, subcellular localization, or enzymatic activity. Thus, protein arginine methylation is critical for regulation of transcription and potentially for various physiological/pathological processes.

title: Control of CBP co‐activating activity by arginine methylation
abstract: 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.

title: Regulation of coactivator complex assembly and function by protein arginine methylation and demethylimination.
abstract: Nuclear receptors activate transcription by recruiting multiple coactivators to the promoters of specific target genes. The functional synergy of the p160 coactivators [steroid receptor coactivator-1, glucocorticoid receptor interacting protein (GRIP1), or the activator for thyroid hormone and retinoid receptors], the histone acetyltransferases cAMP response element binding protein binding protein (CBP) and p300 and the histone methyltransferase coactivator-associated arginine methyltransferase (CARM1) depends on the methyltransferase activity of CARM1. CARM1 methylates histone H3 and other factors including the N-terminal region of p300. Here, we report that CARM1 also methylates Arg-2142 within the C-terminal GRIP1 binding domain (GBD) of p300. In the GBD, both Arg-2088 and Arg-2142 are important for binding GRIP1. Methylation of Arg-2142 inhibits the bimolecular interaction of GRIP1 to p300 in vitro and in vivo. This methylation mark of p300 GBD is removed by peptidyl deiminase 4, thereby enhancing the p300-GRIP1 interaction. These methylation and demethylimination events also alter the conformation and activity of the coactivator complex and regulate estrogen receptor-mediated transcription, and they thus represent unique mechanisms for regulating coactivator complex assembly, conformation, and function.

title: The Activity and Stability of the Transcriptional Coactivator p/CIP/SRC-3 Are Regulated by CARM1-Dependent Methylation
abstract: 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.

title: Signaling within a Coactivator Complex: Methylation of SRC-3/AIB1 Is a MolecularSwitch for Complex Disassembly
abstract: 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.

title: GAC63, a GRIP1-Dependent Nuclear Receptor Coactivator
abstract: Nuclear receptors (NRs) regulate target gene transcription through the recruitment of multiple coactivator complexes to the promoter regions of target genes. One important coactivator complex includes a p160 coactivator (GRIP1, SRC-1, or ACTR) and its downstream coactivators (e.g., p300, CARM1, CoCoA, and Fli-I), which contribute to transcriptional activation by protein acetylation, protein methylation, and protein-protein interactions. In this study, we identified a novel NR coactivator, GAC63, which binds to the N-terminal region of p160 coactivators as well as the ligand binding domains of some NRs. GAC63 enhanced transcriptional activation by NRs in a hormone-dependent and GRIP1-dependent manner in transient transfection assays and cooperated synergistically and selectively with other NR coactivators, including GRIP1 and CARM1, to enhance estrogen receptor function. Endogenous GAC63 was recruited to the estrogen-responsive pS2 gene promoter of MCF-7 cells in response to the hormone. Reduction of the endogenous GAC63 level by small interfering RNA inhibited transcriptional activation by the hormone-activated estrogen receptor. Thus, GAC63 is a physiologically relevant part of the p160 coactivator signaling pathway that mediates transcriptional activation by NRs.

title: CoCoA, a nuclear receptor coactivator which acts through an N-terminal activation domain of p160 coactivators.
abstract: The p160 coactivators bind to and potentiate transcriptional activation by nuclear receptors by recruiting secondary coactivators such as the histone acetyltransferases p300 and CBP and the protein methyltransferase CARM1. The function of the highly conserved N-terminal basic-helix-loop-helix/Per-Arnt-Sim (bHLH-PAS) domain of p160 coactivators is unknown. This region is required for coactivator synergy among p160, p300, and CARM1 coactivators. We identified a coactivator, coiled-coil coactivator (CoCoA), which binds to this domain and thereby enhances transcriptional activation by the estrogen receptor and other nuclear receptors. Endogenous CoCoA was found simultaneously with p160 coactivators on the promoter of an endogenous estrogen-responsive gene. Reduction of endogenous cellular CoCoA levels inhibited the estrogen-stimulated expression of transiently transfected and endogenous genes. Moreover, CoCoA cooperated synergistically with GRIP1, CARM1, and p300 to enhance ER-mediated transcription. Thus, the N-terminal region of p160 coactivators contains an additional activation domain which contributes to coactivator function by recruitment of CoCoA.

title: Developmentally Essential Protein Flightless I Is a Nuclear Receptor Coactivator with Actin Binding Activity
abstract: 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.

title: Synergy among Nuclear Receptor Coactivators: Selective Requirement for Protein Methyltransferase and Acetyltransferase Activities
abstract: 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.

title: SNF2-related CBP activator protein (SRCAP) functions as a coactivator of steroid receptor-mediated transcription through synergistic interactions with CARM-1 and GRIP-1.
abstract: SRCAP (SNF2-related CBP activator protein) is a 350-kDa protein that shares homology with the SNF2 family of proteins whose members function in various aspects of transcriptional regulation. In various cell types, SRCAP is found in distinct multiprotein complexes that include proteins found in SWI/SNF chromatin remodeling complexes. SRCAP was identified by its ability to bind to CBP and was found to potentiate the ability of CBP to activate transcription. Studies in our laboratory have demonstrated that SRCAP functions as a coactivator for CREB-mediated transcription of a number of promoters, including that of the phosphoenolpyruvate carboxykinase gene. Our current studies demonstrate that SRCAP enhances phosphoenolpyruvate carboxykinase promoter transcription induced by glucocorticoids. SRCAP also enhances glucocorticoid receptor-mediated transcription of a simple promoter containing only two glucocorticoid response elements, indicating that SRCAP functions as a glucocorticoid receptor coactivator. In similar studies, SRCAP was also found to serve as a coactivator for the androgen receptor. SRCAP exhibits synergistic activation with nuclear receptor coactivators and functionally interacts in vivo with glucocorticoid receptor-interacting protein-1 and coactivator-associated arginine methyltransferase-1. We propose that SRCAP, by virtue of its ability to interact with CBP, functions as a coactivator to regulate transcription initiated by several signaling pathways.

title: Synergistic, p160 Coactivator-dependent Enhancement of Estrogen Receptor Function by CARM1 and p300*
abstract: Members of the p160 coactivator family (steroid receptor coactivator-1 (SRC-1), glucocorticoid receptor interacting protein 1 (GRIP1), and activator of thyroid and retinoic acid receptors (ACTR)) mediate transcriptional activation by nuclear receptors. After being recruited to the promoter by nuclear receptors, the p160 coactivator transmits the activating signal via two C-terminal activation domains, AD1 and AD2. AD1 is a binding site for the related coactivators cAMP-response element binding protein binding protein (CBP) and p300, whereas AD2 binds to another coactivator, coactivator-associated arginine methyltransferase 1 (CARM1), a protein-arginine methyltransferase. The current study explored the cooperative functional and mechanistic relationships among GRIP1, CARM1, and p300 in transient transfection assays, where they enhanced the ability of the estrogen receptor (ER) to activate transcription of a reporter gene. The coactivator functions of p300 and CARM1 depended on the co-expression of GRIP1. Simultaneous co-expression of all three coactivators caused a synergistic enhancement of ER function. Deletion of the AD1 domain of GRIP1 abolished the ability of p300 to potentiate ER activity but had no effect on CARM1-mediated stimulation. In contrast, when the AD2 domain of GRIP1 was deleted, p300 still stimulated ER function through the mutant GRIP1, but CARM1 failed to do so. Thus, both binding of p300 to AD1 and binding of CARM1 to AD2 are required for their respective coactivator functions and for their synergy. Furthermore, CARM1 and p300 function independently through different activating domains of GRIP1, and their synergy suggests that they enhance transcription by different, complementary mechanisms.

title: Transcriptional intermediary factor 1alpha mediates physical interaction and functional synergy between the coactivator-associated arginine methyltransferase 1 and glucocorticoid receptor-interacting protein 1 nuclear receptor coactivators.
abstract: In previous studies transcriptional intermediary factor 1alpha (TIF1alpha) was identified as a direct binding partner and potential transcriptional coactivator for nuclear receptors (NRs) but its overexpression inhibited, rather than enhanced, transcriptional activation by NRs. Here we show that TIF1alpha bound to and enhanced the function of the C-terminal activation domain (AD) of coactivator associated arginine methyltransferase 1 (CARM1) and the N-terminal AD of glucocorticoid receptor-interacting protein 1 (GRIP1). Furthermore, although TIF1alpha had little or no NR coactivator activity by itself, it cooperated synergistically with GRIP1 and CARM1 to enhance NR-mediated transcription. Inhibition of endogenous TIF1alpha expression reduced transcriptional activation by the GRIP1 N-terminal domain but not by the CARM1 C-terminal domain, suggesting that TIF1alpha may be more important for mediating the activity of the former than the latter. Reduction of endogenous TIF1alpha levels also compromised the androgen-dependent induction of an endogenous target gene of the androgen receptor. Finally, TIF1alpha formed a ternary complex with the GRIP1 N-terminal and CARM1 C-terminal domains. Thus, we conclude that TIF1alpha cooperates with NR coactivators GRIP1 and CARM1 by forming a stable ternary complex with them and enhancing the AD function of one or both of them.

title: CCAR1, a key regulator of mediator complex recruitment to nuclear receptor transcription complexes.
abstract: DNA-bound transcription factors recruit many coactivator proteins to remodel chromatin and activate transcription. The Mediator complex is believed to recruit RNA polymerase II to most protein-encoding genes. It is generally assumed that interaction of Mediator subunits with DNA-binding transcription factors is responsible for Mediator recruitment to promoters. However, we report here that Mediator recruitment by nuclear receptors (NR) requires a coactivator protein, CCAR1 (cell-cycle and apoptosis regulator 1). CCAR1 associates with components of the Mediator and p160 coactivator complexes and is recruited to endogenous NR target genes in response to the appropriate hormone. Reduction of endogenous CCAR1 levels inhibited hormone-induced expression of endogenous NR target genes, hormone-induced recruitment of Mediator components and RNA polymerase II to target gene promoters, and estrogen-dependent growth of breast cancer cells. Thus, CCAR1 regulates expression of key proliferation-inducing genes. CCAR1 also functions as a p53 coactivator, suggesting a broader role in transcriptional regulation.

title: Coordinate Regulation of Transcription and Splicing by Steroid Receptor Coregulators
abstract: Recent observations indicating that promoter identity influences alternative RNA-processing decisions have created interest in the regulatory interactions between RNA polymerase II transcription and precursor messenger RNA (pre-mRNA) processing. We examined the impact of steroid receptor–mediated transcription on RNA processing with reporter genes subject to alternative splicing driven by steroid-sensitive promoters. Steroid hormones affected the processing of pre-mRNA synthesized from steroid-sensitive promoters, but not from steroid-unresponsive promoters, in a steroid receptor–dependent and receptor-selective manner. Several nuclear receptor coregulators showed differential splicing effects, suggesting that steroid hormone receptors may simultaneously control gene transcription activity and exon content of the product mRNA by recruiting coregulators involved in both processes.

title: Steroid Hormone Receptor Coactivation and Alternative RNA Splicing by U2AF65-Related Proteins CAPERα and CAPERβ
abstract: Increasing evidence indicates that transcription and pre-mRNA processing are functionally coupled to modulate gene expression. Here, we report that two members of the U2AF 65 family of proteins, hCC1.3, which we call CAPERα, and a related protein, CAPERβ, regulate both steroid hormone receptor-mediated transcription and alternative splicing. The CAPER proteins coactivate the progesterone receptor in luciferase transcription reporter assays and alter alternative splicing of a calcitonin/calcitonin gene-related peptide minigene in a hormone-dependent manner. The importance of CAPER coactivators in the regulation of alternative RNA splicing of an endogenous cellular gene (VEGF) was substantiated by siRNA knockdown of CAPERα. Mutational analysis of CAPERβ indicates that the transcriptional and splicing functions are located in distinct and separable domains of the protein. These results indicate that steroid hormone receptor-regulated transcription and pre-mRNA splicing can be directly linked through dual function coactivator molecules such as CAPERα and CAPERβ.

title: The arginine methyltransferase CARM1 regulates the coupling of transcription and mRNA processing.
abstract: 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.

title: The RNA‐binding and adaptor protein Sam68 modulates signal‐dependent splicing and transcriptional activity of the androgen receptor
abstract: The RNA‐binding protein Sam68 has been reported to be up‐regulated in clinical cases of prostate cancer (PCa), where it is thought to contribute to cell proliferation and survival. Consistent with this, we observed over‐expression of Sam68 in a panel of clinical prostate tumours as compared with benign controls. Since Sam68 is implicated in a number of signalling pathways, we reasoned that its role in PCa may involve modulation of the androgen receptor (AR) signalling cascade, which drives the onset and progression of PCa. We found that Sam68 interacts with the AR in vivo in LNCaP cells, and is dynamically recruited to androgen response elements within the promoter region of the prostate‐specific antigen (PSA) gene. Based on its known functions and nuclear location, Sam68 might either: (a) co‐regulate AR‐dependent transcription positively or negatively; or (b) modulate AR‐dependent alternative splicing by enhancing incorporation of a Sam68‐responsive exon transcribed under the control of an androgen‐responsive promoter. We tested these possibilities using functional assays. Both wild‐type Sam68 protein and the Sam68V229F mutant, which is impaired in RNA binding, functioned as a ligand‐dependent AR co‐activator on an androgen‐regulated reporter gene. In contrast, splicing of a Sam68‐responsive variable exon, transcribed under control of an androgen‐responsive promoter, was strongly repressed in the presence of AR and androgens. This splicing inhibition was reversed by ectopic expression of Sam68 but enhanced by Sam68V229F. These results demonstrate that Sam68 has separable effects on AR‐regulated transcriptional activity and alternative splicing, both of which may affect PCa phenotypes.

title: Differential recruitment of nuclear receptor coactivators may determine alternative RNA splice site choice in target genes.
abstract: The biological consequences of steroid hormone-mediated transcriptional activation of target genes might be difficult to predict because alternative splicing of a single neosynthesized precursor RNA can result in production of different protein isoforms with opposite biological activities. Therefore, an important question to address is the manner in which steroid hormones affect the splicing of their target gene transcripts. In this report, we demonstrate that individual steroid hormones had different and opposite effects on alternative splicing decisions, stimulating the production of different spliced variants produced from genes driven by steroid hormone-dependent promoters. Steroid hormone transcriptional effects are mediated by steroid hormone receptor coregulators that also modify alternative splicing decisions. Our data suggest that activated steroid hormone receptors recruit coregulators to the target promoter that participate in both the production and the splicing of the target gene transcripts. Because different coregulators activating transcription can have opposite effects on alternative splicing decisions, we conclude that the precise nature of the transcriptional coregulators recruited by activated steroid receptors, depending on the promoter and cellular contexts, may play a major role in regulating the nature of the spliced variants produced from certain target genes in response to steroid hormones.

title: The RNA helicase p68 is a novel androgen receptor coactivator involved in splicing and is overexpressed in prostate cancer.
abstract: The androgen receptor (AR) is a member of the nuclear steroid hormone receptor family and is thought to play an important role in the development of both androgen-dependent and androgen-independent prostatic malignancy. Elucidating roles by which cofactors regulate AR transcriptional activity may provide therapeutic advancement for prostate cancer (PCa). The DEAD box RNA helicase p68 (Ddx5) was identified as a novel AR-interacting protein by yeast two-hybrid screening, and we sought to examine the involvement of p68 in AR signaling and PCa. The p68-AR interaction was verified by colocalization of overexpressed protein by immunofluorescence and confirmed in vivo by coimmunoprecipitation in the PCa LNCaP cell line. Chromatin immunoprecipitation in the same cell line showed AR and p68 recruitment to the promoter region of the androgen-responsive prostate-specific antigen (PSA) gene. Luciferase reporter, minigene splicing assays, and RNA interference (RNAi) were used to examine a functional role of p68 in AR-regulated gene expression, whereby p68 targeted RNAi reduced AR-regulated PSA expression, and p68 enhanced AR-regulated repression of CD44 splicing (P = 0.008). Tyrosine phosphorylation of p68 was found to enhance coactivation of ligand-dependent transcription of AR-regulated luciferase reporters independent of ATP-binding. Finally, we observe increased frequency and expression of p68 in PCa compared with benign tissue using a comprehensive prostate tissue microarray (P = 0.003; P = 0.008). These findings implicate p68 as a novel AR transcriptional coactivator that is significantly overexpressed in PCa with a possible role in progression to hormone-refractory disease.

title: A Subset of Nuclear Receptor Coregulators Act as Coupling Proteins during Synthesis and Maturation of RNA Transcripts
abstract: Gene expression is a multistep process starting in the cell nucleus with the synthesis of the primary transcripts that undergo several modifications (including capping, splicing, and polyadenylation) leading to the export of the mature mRNAs into the cytoplasm for translation into proteins. Although an emerging view is that all the steps from transcription to translation are mechanically and functionally coupled, the proteins that are involved in this coupled process are still poorly characterized. In recent years, a growing list of proteins known to control gene expression at the transcriptional level, named transcriptional coregulators, have been independently shown to play additional roles in other steps of gene expression. In this review we compile these emerging data suggesting that a subset of transcriptional coregulators play a major role in the coordination of the individual steps of the gene expression process.

title: Arginine Methylation of Scaffold Attachment Factor A by Heterogeneous Nuclear Ribonucleoprotein Particle-associated PRMT1*
abstract: Components of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex and other nucleic acid-binding proteins are subject to methylation on specific arginine residues by the catalytic activity of arginine methyltransferases. The methylation has been implicated in transcriptional regulation and RNA and protein trafficking and signal transduction, but the mechanism by which these functions are achieved has remained undetermined. We show here that the predominant arginine methyltransferase in human cells, protein arginine methyltransferase 1 (PRMT1), is associated with hnRNP complexes, dependent on the methylation status of the cell, and that it methylates its preferred substrates in situ. Binding of PRMT1 occurs through physical interaction with scaffold attachment factor A (SAF-A), also known as hnRNP-U, which is quantitatively methylated by PRMT1 in all investigated cell lines as determined by a novel, highly specific, methylation-sensitive antibody.

title: Arginine Methylation of RNA Helicase A Determines Its Subcellular Localization*
abstract: RNA helicase A (RHA) undergoes nuclear translocation via a classical import mechanism utilizing karyopherin beta. The nuclear transport domain (NTD) of RHA is known to be necessary and sufficient for its bi-directional nuclear trafficking. We report here that arginine methylation is a novel requirement for NTD-mediated nuclear import. Nuclear translocation of glutathione S-transferase (GST)-NTD fusion proteins is abrogated by arginine-methylation inhibitors. However, in vitro arginine-methylation of GST-NTD prior to injection allows the fusion protein to localize to the nucleus in the presence of methylation inhibitors. Removal of the arginine-rich C-terminal region negates the effects of the methylation inhibitors on NTD import, suggesting that methylation of the NTD C terminus the relieves the cytoplasmic retention of RHA. The NTD physically interacts with PRMT1, the major protein arginine methyltransferase. These findings provide evidence for a novel arginine methylation-dependent regulatory pathway controlling the nuclear import of RHA.

title: Sam68 RNA binding protein is an in vivo substrate for protein arginine N-methyltransferase 1.
abstract: RNA binding proteins often contain multiple arginine glycine repeats, a sequence that is frequently methylated by protein arginine methyltransferases. The role of this posttranslational modification in the life cycle of RNA binding proteins is not well understood. Herein, we report that Sam68, a heteronuclear ribonucleoprotein K homology domain containing RNA binding protein, associates with and is methylated in vivo by the protein arginine N-methyltransferase 1 (PRMT1). Sam68 contains asymmetrical dimethylarginines near its proline motif P3 as assessed by using a novel asymmetrical dimethylarginine-specific antibody and mass spectrometry. Deletion of the methylation sites and the use of methylase inhibitors resulted in Sam68 accumulation in the cytoplasm. Sam68 was also detected in the cytoplasm of PRMT1-deficient embryonic stem cells. Although the cellular function of Sam68 is unknown, it has been shown to export unspliced human immunodeficiency virus RNAs. Cells treated with methylase inhibitors prevented the ability of Sam68 to export unspliced human immunodeficiency virus RNAs. Other K homology domain RNA binding proteins, including SLM-1, SLM-2, QKI-5, GRP33, and heteronuclear ribonucleoprotein K were also methylated in vivo. These findings demonstrate that RNA binding proteins are in vivo substrates for PRMT1, and their methylation is essential for their proper localization and function.

title: The RGG domain in hnRNP A2 affects subcellular localization.
abstract: The heterogeneous nuclear ribonucleoproteins (hnRNP) associate with pre-mRNA in the nucleus and play an important role in RNA processing and splice site selection. In addition, hnRNP A proteins function in the export of mRNA to the cytoplasm. Although the hnRNP A proteins are predominantly nuclear, hnRNP A1 shuttles rapidly between the nucleus and the cytoplasm. HnRNP A2, whose cytoplasmic overexpression has been identified as an early biomarker of lung cancer, has been less well studied. Cytosolic hnRNP A2 overexpression has also been noted in brain tumors, in which it has been correlated with translational repression of Glucose Transporter-1 expression. We now examine the role of arginine methylation on the nucleocytoplasmic localization of hnRNP A2 in the HEK-293 and NIH-3T3 mammalian cell lines. Treatment of either cell line with the methyltransferase inhibitor adenosine dialdehyde dramatically shifts hnRNP A2 localization from the nuclear to the cytoplasmic compartment, as shown both by immunoblotting and by immunocytochemistry. In vitro radiolabeling with [(3)H]AdoMet of GST-tagged hnRNP A2 RGG mutants, using recombinant protein arginine methyltransferase (PRMT1), shows (i) that hnRNP A2 is a substrate for PRMT1 and (ii) that methylated residues are found only in the RGG domain. Deletion of the RGG domain (R191-G253) of hnRNP A2 results in a cytoplasmic localization phenotype, detected both by immunoblotting and by immunocytochemistry. These studies indicate that the RGG domain of hnRNP A2 contains sequences critical for cellular localization of the protein. The data suggest that hnRNP A2 may contain a novel nuclear localization sequence, regulated by arginine methylation, that lies in the R191-G253 region and may function independently of the M9 transportin-1-binding region in hnRNP A2.

title: PRMT6-mediated methylation of R2 in histone H3 antagonizes H3 K4 trimethylation.
abstract: The arginine methyltransferase PRMT6 (protein arginine methyltransferase 6) has been shown recently to regulate DNA repair and gene expression. As arginine methylation of histones is an important mechanism in transcriptional regulation, we asked whether PRMT6 possesses activity toward histones. We show here that PRMT6 methylates histone H3 at R2 and histones H4/H2A at R3 in vitro. Overexpression and knockdown analysis identify PRMT6 as the major H3 R2 methyltransferase in vivo. We find that H3 R2 methylation inhibits H3 K4 trimethylation and recruitment of WDR5, a subunit of the MLL (mixed lineage leukemia) K4 methyltransferase complex, to histone H3 in vitro. Upon PRMT6 overexpression, transcription of Hox genes and Myc-dependent genes, both well-known targets of H3 K4 trimethylation, decreases. This transcriptional repression coincides with enhanced occurrence of H3 R2 methylation and PRMT6 as well as reduced levels of H3 K4 trimethylation and MLL1/WDR5 recruitment at the HoxA2 gene. Upon retinoic acid-induced transcriptional activation of HoxA2 in a cell model of neuronal differentiation, PRMT6 recruitment and H3 R2 methylation are diminished and H3 K4 trimethylation increases at the gene. Our findings identify PRMT6 as the mammalian methyltransferase for H3 R2 and establish the enzyme as a crucial negative regulator of H3 K4 trimethylation and transcriptional activation.

title: Arginine Methylation of the Histone H3 Tail Impedes Effector Binding*
abstract: Histone tail post-translational modification results in changes in cellular processes, either by generating or blocking docking sites for histone code readers or by altering the higher order chromatin structure. H3K4me3 is known to mark the promoter regions of active transcription. Proteins bind H3K4 in a methyl-dependent manner and aid in the recruitment of histone-remodeling enzymes and transcriptional cofactors. The H3K4me3 binders harbor methyl-specific chromatin binding domains, including plant homeodomain, Chromo, and tudor domains. Structural analysis of the plant homeodomains present in effector proteins, as well as the WD40 repeats of WDR5, reveals critical contacts between residues in these domains and H3R2. The intimate contact between H3R2 and these domain types leads to the hypothesis that methylation of this arginine residue antagonizes the binding of effector proteins to the N-terminal tail of H3. Here we show that H3 tail binding effector proteins are indeed sensitive to H3R2 methylation and that PRMT6, not CARM1/PRMT4, is the primary methyltransferase acting on this site. We have tested the expression of a select group of H3K4 effector-regulated genes in PRMT6 knockdown cells and found that their levels are altered. Thus, PRMT6 methylates H3R2 and is a negative regulator of N-terminal H3 tail binding.

title: Methylation of histone H3R2 by PRMT6 and H3K4 by an MLL complex are mutually exclusive
abstract: Eukaryotic genomes are organized into active (euchromatic) and inactive (heterochromatic) chromatin domains. Post-translational modifications of histones (or ‘marks’) are key in defining these functional states, particularly in promoter regions. Mutual regulatory interactions between these marks—and the enzymes that catalyse them—contribute to the shaping of this epigenetic landscape, in a manner that remains to be fully elucidated. We previously observed that asymmetric di-methylation of histone H3 arginine 2 (H3R2me2a) counter-correlates with di- and tri- methylation of H3 lysine 4 (H3K4me2, H3K4me3) on human promoters. Here we show that the arginine methyltransferase PRMT6 catalyses H3R2 di-methylation in vitro and controls global levels of H3R2me2a in vivo. H3R2 methylation by PRMT6 was prevented by the presence of H3K4me3 on the H3 tail. Conversely, the H3R2me2a mark prevented methylation of H3K4 as well as binding to the H3 tail by an ASH2/WDR5/MLL-family methyltransferase complex. Chromatin immunoprecipitation showed that H3R2me2a was distributed within the body and at the 3′ end of human genes, regardless of their transcriptional state, whereas it was selectively and locally depleted from active promoters, coincident with the presence of H3K4me3. Hence, the mutual antagonism between H3R2 and H3K4 methylation, together with the association of MLL-family complexes with the basal transcription machinery, may contribute to the localized patterns of H3K4 tri-methylation characteristic of transcriptionally poised or active promoters in mammalian genomes.

title: Methylation of Tat by PRMT6 Regulates Human Immunodeficiency Virus Type 1 Gene Expression
abstract: The human immunodeficiency virus (HIV) transactivator protein, Tat, stimulates transcription from the viral long terminal repeats via an arginine-rich transactivating domain. Since arginines are often known to be methylated, we investigated whether HIV type 1 (HIV-1) Tat was a substrate for known protein arginine methyltransferases (PRMTs). Here we identify Tat as a substrate for the arginine methyltransferase, PRMT6. Tat is specifically associated with and methylated by PRMT6 within cells. Overexpression of wild-type PRMT6, but not a methylase-inactive PRMT6 mutant, decreased Tat transactivation of an HIV-1 long terminal repeat luciferase reporter plasmid in a dose-dependent manner. Knocking down PRMT6 consistently increased HIV-1 production in HEK293T cells and also led to increased viral infectiousness as shown in multinuclear activation of a galactosidase indicator assays. Our study demonstrates that arginine methylation of Tat negatively regulates its transactivation activity and that PRMT6 acts as a restriction factor for HIV replication.

title: PRMT6 diminishes HIV-1 Rev binding to and export of viral RNA
abstract: BackgroundThe HIV-1 Rev protein mediates nuclear export of unspliced and partially spliced viral RNA through interaction with the Rev response element (RRE) by means of an arginine rich motif that is similar to the one found in Tat. Since Tat is known to be asymmetrically arginine dimethylated by protein arginine methyltransferase 6 (PRMT6) in its arginine rich motif, we investigated whether the Rev protein could act as a substrate for this enzyme.ResultsHere, we report the methylation of Rev due to a single arginine dimethylation in the N-terminal portion of its arginine rich motif and the association of Rev with PRMT6 in vivo. Further analysis demonstrated that the presence of increasing amounts of wild-type PRMT6, as well as a methylation-inactive mutant PRMT6, dramatically down-regulated Rev protein levels in concentration-dependent fashion, which was not dependent on the methyltransferase activity of PRMT6. Quantification of Rev mRNA revealed that attenuation of Rev protein levels was due to a posttranslational event, carried out by a not yet defined activity of PRMT6. However, no relevant protein attenuation was observed in subsequent chloramphenicol acetyltransferase (CAT) expression experiments that screened for RNA export and interaction with the RRE. Binding of the Rev arginine rich motif to the RRE was reduced in the presence of wild-type PRMT6, whereas mutant PRMT6 did not exert this negative effect. In addition, diminished interactions between viral RNA and mutant Rev proteins were observed, due to the introduction of single arginine to lysine substitutions in the Rev arginine rich motif. More importantly, wild-type PRMT6, but not mutant methyltransferase, significantly decreased Rev-mediated viral RNA export from the nucleus to the cytoplasm in a dose-dependent manner.ConclusionThese findings indicate that PRMT6 severely impairs the function of HIV-1 Rev.

title: Arginine methylation of the HIV-1 nucleocapsid protein results in its diminished function
abstract: The HIV-1 nucleocapsid protein (NC) is involved in transfer RNALys3 annealing to the primer binding site of viral genomic RNA by means of two basic regions that are similar to the N-terminal portion of the arginine-rich motif (ARM) of Tat. As Tat is known to be asymmetrically arginine dimethylated by protein arginine methyltransferase 6 (PRMT6) in its ARM, we investigated whether NC could also act as a substrate for this enzyme. Arginine methylation of NC was demonstrated in vitro and in vivo, and sites of methylation were determined by mutational analysis. The impact of the arginine methylation of NC was measured in RNA annealing and reverse transcription initiation assays. An arginine methyltransferase inhibitor (AMI)3.4 was tested for its effects on viral infectivity and replication in vivo. NC is a substrate for PRMT6 both in vitro and in vivo. NC possesses arginine dimethylation sites in each of its two basic regions at positions R10 and R32, and methylated NC was less able than wild-type to promote RNA annealing and participate in the initiation of reverse transcription. Exposure of HIV-1-infected MT2 and primary cord blood mononuclear cells to AMI3.4 led to increased viral replication, whereas viral infectivity was not significantly affected in multinuclear-activation galactosidase indicator assays. NC is an in-vivo target of PRMT6, and arginine methylation of NC reduces RNA annealing and the initiation of reverse transcription. These findings may lead to ways of driving HIV-infected cells out of latency with drugs that inhibit PRMT6.

title: The steroid receptor coactivator, GRIP-1, is necessary for MEF-2C-dependent gene expression and skeletal muscle differentiation.
abstract: Nuclear receptor-mediated activation of transcription involves coactivation by cofactors collectively denoted the steroid receptor coactivators (SRCs). The process also involves the subsequent recruitment of p300/CBP and PCAF to a complex that synergistically regulates transcription and remodels the chromatin. PCAF and p300 have also been demonstrated to function as critical coactivators for the muscle-specific basic helix-loop-helix (bHLH) protein MyoD during myogenic commitment. Skeletal muscle differentiation and the activation of muscle-specific gene expression is dependent on the concerted action of another bHLH factor, myogenin, and the MADS protein, MEF-2, which function in a cooperative manner. We examined the functional role of one SRC, GRIP-1, in muscle differentiation, an ideal paradigm for the analysis of the determinative events that govern the cell's decision to divide or differentiate. We observed that the mRNA encoding GRIP-1 is expressed in proliferating myoblasts and post-mitotic differentiated myotubes, and that protein levels increase during differentiation. Exogenous/ectopic expression studies with GRIP-1 sense and antisense vectors in myogenic C2C12 cells demonstrated that this SRC is necessary for (1) induction/activation of myogenin, MEF-2, and the crucial cell cycle regulator, p21, and (2) contractile protein expression and myotube formation. Furthermore, we demonstrate that the SRC GRIP-1 coactivates MEF-2C-mediated transcription. GRIP-1 also coactivates the synergistic transactivation of E box-dependent transcription by myogenin and MEF-2C. GST-pulldowns, mammalian two-hybrid analysis, and immunoprecipitation demonstrate that the mechanism involves direct interactions between MEF-2C and GRIP-1 and is associated with the ability of the SRC to interact with the MADS domain of MEF-2C. The HLH region of myogenin mediates the direct interaction of myogenin and GRIP-1. Interestingly, interaction with myogenic factors is mediated by two regions of GRIP-1, an amino-terminal bHLH-PAS region and the carboxy-terminal region between amino acids 1158 and 1423 (which encodes an activation domain, has HAT activity, and interacts with the coactivator-associated arginine methyltransferase). This work demonstrates that GRIP-1 potentiates skeletal muscle differentiation by acting as a critical coactivator for MEF-2C-mediated transactivation and is the first study to ascribe a function to the amino-terminal bHLH-PAS region of SRCs.

title: Minigene reporter for identification and analysis of cis elements and trans factors affecting pre-mRNA splicing.
abstract: All human genes contain a diverse array of cis-acting elements within introns and exons that are required for correct and efficient precursor messenger RNA (pre-mRNA) splicing. Recent computational analyses predict that most human exons contain elements required for splicing coinciding with an appreciation for the high frequency with which mutations that disruption pre-mRNA splicing cause disease. Minigenes provide a means to directly determine whether disease-causing mutations or single nucleotide polymorphisms (SNPs) affect splicing efficiency. Minigenes have also been instrumental in investigations of alternative splicing to identify cis elements required for cell-specific splicing events, demonstrating regulation of individual splicing events by specific RNA binding proteins, and correlating binding of these splicing regulators with splicing regulation. Here we present a versatile minigene plasmid vector designed for rapid cloning and analysis of cis elements and trans-acting factors that influence splicing efficiency or regulate cell-specific splicing. Ubiquitous expression and unique restriction sites allow for straightforward replacement of a variety of gene segments to analyze the effects of nucleotide substitutions on splicing, to identify tissue-specific regulatory elements, or to determine responsiveness to coexpressed proteins or small molecules.

title: Specific reverse transcription-PCR quantification of vascular endothelial growth factor (VEGF) splice variants by LightCycler technology.
abstract: Overexpression of vascular endothelial growth factor (VEGF) is associated with increased angiogenesis, growth and invasion in solid tumors, and hematologic malignancies. The expression of isoforms of VEGF, which mediate different effects, can be discriminated by splice-variant-specific quantitative reverse transcription-PCR (RT-PCR), but current methods have only modest sensitivity and precision and suffer from heteroduplex formation.



We used a real-time RT-PCR assay on the LightCycler system. Applicability for detection of different VEGF mRNAs and total VEGF message was tested on seven healthy tissues (each pooled from healthy donors) and seven correlated malignant tissues. Results were normalized to beta(2)-microglobulin mRNA. Amplification of VEGF splice variants was performed exclusively with variant-specific reverse primers, whereas forward primer and fluorescent probe were common to obtain similar RT-PCR kinetics.



Highly specific detection of VEGF splice variants was achieved with minor intra- and interassay variation (<0.22 threshold cycle). Total VEGF expression was higher in malignant tissues. In healthy tissues, the mRNA encoding diffusible variants VEGF(121) and VEGF(165) constituted on average 78% (SD = 9.3%) of the total VEGF message, and the cell-adherent variant VEGF(189) constituted on average 22% (SD = 5.4%). In contrast, in malignant tissues VEGF(121) and VEGF(165) accounted for 94% (SD = 7.6%) and VEGF(189) only 6% (SD = 3.7%).



Because of the ability for quantification of VEGF splice variants with high specificity, sensitivity, and reproducibility, this new LightCycler assay is superior to conventional semiquantitative competitive RT-PCR and immunological assays and may contribute to better understanding of VEGF-mediated angiogenesis.

title: Long-range activation of GREB1 by estrogen receptor via three distal consensus estrogen-responsive elements in breast cancer cells.
abstract: The estrogen receptor (ER) binds to estrogen-responsive elements (EREs) to activate gene transcription. The best characterized EREs are located in proximal gene promoters, but recent data indicate that only a minority of ER binding sites lie within proximal promoter regions. GREB1 (gene regulated by estrogen in breast cancer 1) is an ER target gene that regulates estrogen-induced proliferation in breast cancer cells. We identified three consensus EREs, located at -21.2, -9.5, and -1.6 kb upstream of the closest GREB1a transcription start site that appear to mediate long-range GREB1 gene activation by ER. All three ERE sites nucleate ER, steroid receptor coactivator-3 (SRC-3), and RNA polymerase II (Pol II) and undergo histone acetylation in response to estradiol. Estrogen-stimulated ER binding at all three EREs was cyclic and synchronous. SRC-3 and Pol II recruitment to all three EREs was activated by estrogen but not tamoxifen. In contrast, estrogen stimulated only Pol II and not ER or SRC-3 recruitment to the GREB1 core promoter regions. Long-range histone acetylation, centered on the three ERE motifs and the GREB1 core promoters, was observed in response to estrogen but not to tamoxifen. These data suggest that estrogen-stimulated GREB1 transcription may involve coordinated ER binding to all three distal consensus ERE motifs. Long-range activation by ER acting at multiple EREs may be more common than previously appreciated.

title: Regulation of estrogen receptor alpha by the SET7 lysine methyltransferase.
abstract: Estrogen receptor alpha (ER) is a ligand-dependent transcription factor. Upon binding estrogen, ER recruits coactivator complexes with histone acetyltransferase or methyltransferase activities to activate downstream target genes. In addition to histones, coactivators can modify ER itself and other proteins in the transactivation complex. Here, we show that ER is directly methylated at lysine 302 (K302) by the SET7 methyltransferase. SET7-mediated methylation stabilizes ER and is necessary for the efficient recruitment of ER to its target genes and for their transactivation. The SET7-ER complex structure reveals the molecular basis for ER peptide recognition and predicts that modifications or mutations of nearby residues would affect K302 methylation. Indeed, a breast cancer-associated mutation at K303 (K303R) alters methylation at K302 in vitro and in vivo. These findings raise the possibility that generation, recognition, and removal of modifications within the ER hinge region generate "ER modification cassettes" that yield distinct patterns for signaling downstream events.

title: Transcription factor accessibility and histone acetylation of the progesterone receptor gene differs between parental MCF-7 cells and a subline that has lost progesterone receptor expression.
abstract: The human progesterone receptor (PgR) gene has a complex promoter that produces alternate mRNAs encoding the PgRA (94 kDa) and PgRB (120 kDa) protein isoforms. Expression of PgR is induced by estradiol (E(2)) in the breast, reproductive tract and many cell lines despite the lack of a classical estrogen responsive element (ERE) in the promoter regions. We employed chromatin immunoprecipitation (ChIP) to analyze the sites of estrogen receptor alpha (ERalpha) and Sp1 occupancy of the PgR promoters in vivo. We also assessed the functional relevance of histone acetylation levels on the accessibility of transcription factors to the promoter and subsequent hormone-induced transcription. We utilized MCF-7 human breast cancer cells that express PgR in response to E(2) and the MCF-7 derived C4 cell strain that has lost PgR expression as a model system. We found that promoter-wide levels of histone acetylation were not decreased in C4 cells, but that access was partially blocked for Sp1 and completely blocked for ERalpha. The basal level of histone acetylation at six localized regions of the promoter did show some differences between cell lines, but it did not correlate with transcription factor binding. Furthermore, we found only a modest and highly localized change in histone acetylation levels in response to E(2) at only one of three sites of ERalpha binding in MCF-7 cells. This was at the B1 site at the distal 5' end of the promoter. This site also showed a significant decrease in basal histone acetylation in C4 compared to MCF-7 cells. We speculate that the histone acetylation level at this site may be a marker for chromatin structure that affects the access of transcription factors to the whole promoter.

title: The Novel Human Protein Arginine N-Methyltransferase PRMT6 Is a Nuclear Enzyme Displaying Unique Substrate Specificity*
abstract: Protein arginine methylation is a prevalent posttranslational modification in eukaryotic cells that has been implicated in signal transduction, the metabolism of nascent pre-RNA, and the transcriptional activation processes. In searching the human genome for protein arginine N-methyltransferase (PRMT) family members, a novel gene has been found on chromosome 1 that encodes for an apparent methyltransferase, PRMT6. The polypeptide chain of PRMT6 is 41.9 kDa consisting of a catalytic core sequence common to other PRMT enzymes. Expressed as a glutathioneS-transferase fusion protein, PRMT6 demonstrates type I PRMT activity, capable of forming both ω-NG -monomethylarginine and asymmetric ω-NG ,NG -dimethylarginine derivatives on the recombinant glycine- and arginine-rich substrate in a processive manner with a specific activity of 144 pmol methyl groups transferred min−1 mg−1 enzyme. A comparison of substrate specificity reveals that PRMT6 is functionally distinct from two previously characterized type I enzymes, PRMT1 and PRMT4. In addition, PRMT6 displays automethylation activity; it is the first PRMT to do so. This novel human PRMT, which resides solely in the nucleus when fused to the green fluorescent protein, joins a family of enzymes with diverse functions within cells.

title: Genome-wide analysis of estrogen receptor binding sites
abstract: The estrogen receptor is the master transcriptional regulator of breast cancer phenotype and the archetype of a molecular therapeutic target. We mapped all estrogen receptor and RNA polymerase II binding sites on a genome-wide scale, identifying the authentic cis binding sites and target genes, in breast cancer cells. Combining this unique resource with gene expression data demonstrates distinct temporal mechanisms of estrogen-mediated gene regulation, particularly in the case of estrogen-suppressed genes. Furthermore, this resource has allowed the identification of cis-regulatory sites in previously unexplored regions of the genome and the cooperating transcription factors underlying estrogen signaling in breast cancer.

title: The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing.
abstract: Vascular endothelial growth factor (VEGF) is an apparently endothelial cell-specific mitogen that is structurally related to platelet-derived growth factor. By Northern blot and protein analyses, we show that VEGF is produced by cultured vascular smooth muscle cells. Analysis of VEGF transcripts in these cells by polymerase chain reaction and cDNA cloning revealed three different forms of the VEGF coding region, as had been reported in HL60 cells. The three forms of the human VEGF protein chain predicted from these coding regions are 189, 165, and 121 amino acids in length. Comparison of cDNA nucleotide sequences with sequences derived from human VEGF genomic clones indicates that the VEGF gene is split among eight exons and that the various VEGF coding region forms arise from this gene by alternative splicing: the 165-amino-acid form of the protein is missing the residues encoded by exon 6, whereas the 121-amino-acid form is missing the residues encoded by exons 6 and 7. Analysis of the VEGF gene promoter region revealed a single major transcription start, which lies near a cluster of potential Sp1 factor binding sites. The promoter region also contains several potential binding sites for the transcription factors AP-1 and AP-2; consistent with the presence of these sites, Northern blot analysis demonstrated that the level of VEGF transcripts is elevated in cultured vascular smooth muscle cells after treatment with the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate.

title: Role of the Protein Tyrosine Kinase Syk in Regulating Cell-Cell Adhesion and Motility in Breast Cancer Cells
abstract: The expression of the Syk protein tyrosine kinase in breast cancer cells is inversely correlated with invasive growth and metastasis. The expression of Syk inhibits cell motility while supporting the formation of cell clusters by enhancing cell-cell contacts and promoting the redistribution of the adhesion proteins cortactin and vinculin to these contacts. Syk associates physically with cortactin and catalyzes its phosphorylation on tyrosine. The clustering of integrins leads to the phosphorylation of Syk and of numerous cellular proteins in a manner dependent on the activity of the kinase and on the presence of tyrosine 342 located in the linker B region. The ability of Syk to participate in integrin-mediated protein tyrosine phosphorylation correlates well with its ability to inhibit cell motility.

title: Cloning of the cDNA for the deleted syk kinase homologous to ZAP-70 from human basophilic leukemia cell line (KU812).
abstract: Syk kinase is one of the protein tyrosine kinases and forms a family with ZAP-70. We isolated two different sized cDNA clones of syk (Syk11 and Syk41) from a cDNA library of human KU812 (human basophilic leukemia cell line). The obtained two clones carried different messages, that Syk41 had a 69 bp-long insertion between the SH2 domain and the kinase domain compared with Syk11. Alignment of the two human syk predicted polypeptides with those of the porcine syk and ZAP-70 revealed that human syk was 5 amino acid longer than the porcine syk at the N-termini and that the insertion of the 23 amino acid found in Syk41 was present in the porcine syk and absent in ZAP-70. Reverse transcribed polymerase chain reaction targeting this region showed that both forms of the polyA RNA were expressed in Jurkat cells, human peripheral leukocytes and also KU812 cells and that the inserted form was dominant.

title: Differential Intrinsic Enzymatic Activity of Syk and Zap-70 Protein-tyrosine Kinases*
abstract: Syk and Zap-70 are related protein-tyrosine kinases implicated in antigen and Fc receptor signaling. While Zap-70 is restricted to T-cells and natural killer cells, Syk accumulates in B-cells, mast cells, platelets, and immature T-cells. In addition, we found that an isoform of Syk (SykB), which carries a 23-amino acid deletion in the “linker” region, is prominently expressed in bone marrow. To better understand the relative impact of Syk, SykB, and Zap-70 on signal transduction, we compared their intrinsic enzymatic properties in transiently transfected COS-1 cells and in hemopoietic cells. Using modified versions of these enzymes bearing a common Myc epitope at the amino terminus, we determined that the ability of Syk and SykB to undergo autophosphorylation and to phosphorylate erythrocyte band 3 in immune complex kinase reactions was at least 100-fold greater than that of Zap-70. Similarly, Syk and SykB, but not Zap-70, caused prominent tyrosine phosphorylation of p120c-cbl in COS-1 cells. A similar pattern of activity was also noted for endogenous Syk and Zap-70 from hemopoietic cells. To understand the structural basis for these characteristics, we also created and analyzed a series of chimeras between Syk and Zap-70. These studies indicated that the catalytic domain of Syk and Zap-70, but not their SH2 domains, linker region or carboxyl-terminal tail, was responsible for their respective activity. Taken together, these data demonstrated that the intrinsic enzymatic activity of Syk and SykB is superior to that of Zap-70 and that such a distinction relates to structural variations in the catalytic domain.

title: Molecular Cloning of Rodent p72Syk.
abstract: Northern blot analysis of polyadenylated RNA prepared from RBL-2H3 cells revealed the presence of three distinct mRNAs encoding p72Syk, a protein-tyrosine kinase previously shown to be associated with the high affinity IgE receptor present on the surface of these cells (Hutchcroft, J. E., Geahlen, R. L., Deanin, G. G., and Oliver, J. M.(1992) ). Here we report the full-length nucleotide sequence of two of these messages, as well as the complete predicted amino acid sequence of the rodent p72Syk protein-tyrosine kinase. In addition, we report evidence indicating alternative splicing of p72Syk mRNAs within RBL-2H3 cells. This splicing event results in the expression of two distinct protein isoforms that differ with respect to the presence of a 23-amino acid insert located within the region of the protein that separates the two SH2 domains from the catalytic domain. Both mRNAs arising from this splicing event appear to encode functional protein-tyrosine kinases, as expression of the corresponding cDNAs in COS cells results in the production of proteins of the expected sizes that possess intrinsic tyrosine specific kinase activity.

title: An extensive network of coupling among gene expression machines
abstract: Gene expression in eukaryotes requires several multi-component cellular machines. Each machine carries out a separate step in the gene expression pathway, which includes transcription, several pre-messenger RNA processing steps and the export of mature mRNA to the cytoplasm. Recent studies lead to the view that, in contrast to a simple linear assembly line, a complex and extensively coupled network has evolved to coordinate the activities of the gene expression machines. The extensive coupling is consistent with a model in which the machines are tethered to each other to form ‘gene expression factories’ that maximize the efficiency and specificity of each step in gene expression.

title: Direct coupling of transcription and mRNA processing through the thermogenic coactivator PGC-1.
abstract: Transcription and mRNA processing are coupled events in vivo, but the mechanisms that coordinate these processes are largely unknown. PGC-1 is a transcriptional coactivator that plays a major role in the regulation of adaptive thermogenesis. PGC-1 also has certain motifs characteristic of splicing factors. We demonstrate here that mutations in the serine- and arginine-rich domain and RNA recognition motif of PGC-1 interfere with the ability of PGC-1 to induce mRNAs of target genes. These mutations also disrupt the ability of PGC-1 to co-localize and associate with RNA processing factors. PGC-1 can alter the processing of an mRNA, but only when it is loaded onto the promoter of the gene. These data demonstrate the coordinated regulation of RNA transcription and processing through PGC-1.

title: CoAA, a Nuclear Receptor Coactivator Protein at the Interface of Transcriptional Coactivation and RNA Splicing
abstract: We have shown that steroid hormones coordinately control gene transcriptional activity and splicing decisions in a promoter-dependent manner. Our hypothesis is that a subset of hormonally recruited coregulators involved in regulation of promoter transcriptional activity also directly participate in alternative RNA splicing decisions. To gain insight into the molecular mechanisms by which transcriptional coregulators could control splicing decisions, we focused our attention on a recently identified coactivator, CoAA. This heterogeneous nuclear ribonucleoprotein (hnRNP)-like protein interacts with the transcriptional coregulator TRBP, a protein recruited to target promoters through interactions with activated nuclear receptors. Using transcriptional and splicing reporter genes driven by different promoters, we observed that CoAA mediates transcriptional and splicing effects in a promoter-preferential manner. We compared the activity of CoAA to the activity of other hnRNP-related proteins that, like CoAA, contain two N-terminal RNA recognition motifs (RRMs) followed by a C-terminal auxiliary domain and either have or have not been implicated in transcriptional control. By swapping either CoAA RRMs or the CoAA auxiliary domain with the corresponding domains of the proteins selected, we showed that depending on the promoter, the RRMs and the auxiliary domain of CoAA are differentially engaged in transcription. This contributes to the promoter-preferential effects mediated by CoAA on RNA splicing during the course of steroid hormone action.

title: Methylation of histone H4 at arginine 3 occurs in vivo and is mediated by the nuclear receptor coactivator PRMT1
abstract: Posttranslational modifications of histone amino termini play an important role in modulating chromatin structure and function. Lysine methylation of histones has been well documented, and recently this modification has been linked to cellular processes involving gene transcription and heterochromatin assembly. However, the existence of arginine methylation on histones has remained unclear. Recent discoveries of protein arginine methyltransferases, CARM1 and PRMT1, as transcriptional coactivators for nuclear receptors suggest that histones may be physiological targets of these enzymes as part of a poorly defined transcriptional activation pathway. Here we show by using mass spectrometry that histone H4, isolated from asynchronously growing human 293T cells, is methylated at arginine 3 (Arg-3) in vivo. In support, a novel antibody directed against histone H4 methylated at Arg-3 independently demonstrates the in vivo occurrence of this modification and reveals that H4 Arg-3 methylation is highly conserved throughout eukaryotes. Finally, we show that PRMT1 is the major, if not exclusive, H4 Arg-3 methyltransfase in human 293T cells. These findings suggest a role for arginine methylation of histones in the transcription process.

title: SET-mediated Promoter Hypoacetylation Is a Prerequisite for Coactivation of the Estrogen-responsive pS2 Gene by PRMT1*
abstract: Induction of transcription requires an ordered recruitment of coregulators and specific combinations of histone modifications at the promoter. Occurrence of histone H4 arginine (Arg) 3 methylation by protein arginine methyltransferase 1 (PRMT1) represents an early promoter event in ER (estrogen receptor)-regulated gene activation. However, its in vivo significance in ER signaling and the prerequisites for PRMT1 recruitment to promoters have not been established yet. We show here that endogenous PRMT1 is a crucial and non-redundant coactivator of ER-mediated pS2 gene induction in MCF7 cells. By investigating promoter requirements for PRMT1 recruitment we find that the patient SE translocation (SET) protein, which was reported to protect histone tails from acetylation, associates with the uninduced pS2 gene promoter and dissociates early upon estrogen treatment. Knockdown of SET or trichostatin A (TSA) treatment causes premature acetylation of H4 and abrogation of H4 Arg3 methylation at the pS2 gene promoter resulting in diminished transcriptional induction. Thus, SET prevents promoter acetylation and is a prerequisite for the initial acetylation-sensitive steps of pS2 gene activation, namely PRMT1 function. Similar to pS2 we identify lactoferrin as a PRMT1-dependent and TSA-sensitive ER target gene. In contrast, we find that the C3 gene, another ER target, is activated in a PRMT1-independent manner and that SET is involved in C3 gene repression. These findings establish the existence of PRMT1-dependent and -independent ER target genes and show that proteins guarding promoter hypoacetylation, like SET, execute a key function in the coactivation process by PRMT1.

title: Protein arginine methyltransferase 6 specifically methylates the nonhistone chromatin protein HMGA1a.
abstract: The HMGA family proteins HMGA1a and HMGA1b are nuclear nonhistone species implicated in a wide range of cellular processes including inducible gene transcription, modulation of chromosome structure through nucleosome and chromosome remodeling, and neoplastic transformation. HMGA proteins are highly modified, and changes in their phosphorylation states have been correlated with the phase of the cell cycle and changes in their transcriptional activity. HMGA1a is also methylated in the first DNA-binding AT-hook at Arg25 and other sites, although the enzyme or enzymes responsible have not been identified. We demonstrate here that a GST fusion of protein arginine methyltransferase 6 (PRMT6) specifically methylates full-length recombinant HMGA1a protein in vitro. Although GST fusions of PRMT1 and PRMT3 were also capable of methylating the full-length HMGA1a polypeptide, they recognize its proteolytic degradation products much better. GST fusions of PRMT4 or PRMT7 were unable to methylate the full-length protein or its degradation products. We conclude that PRMT6 is a good candidate for the endogenous enzyme responsible for HGMA1a methylation.

title: The AT-hook of the Chromatin Architectural Transcription Factor High Mobility Group A1a Is Arginine-methylated by Protein Arginine Methyltransferase 6*
abstract: 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 Arg57 and Arg59, 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.

title: A mass spectrometric study on the in vitro methylation of HMGA1a and HMGA1b proteins by PRMTs: methylation specificity, the effect of binding to AT-rich duplex DNA, and the effect of C-terminal phosphorylation.
abstract: HMGA1a and HMGA1b are members of one subfamily of non-histone chromosomal high-mobility group (HMG) proteins. They bind to various DNA-related substrates, including the minor groove of AT-rich duplex DNA sequences, and have been postulated to be architectural transcription factors functioning in a wide variety of cellular processes. Post-translational modifications of HMGA1 proteins, such as phosphorylation, acetylation, and methylation, are widely observed in tumor cells in vivo and correlated with the modulation of protein function. Here, we investigated the in vitro methylation of recombinant human HMGA1a and HMGA1b proteins by three members of the protein arginine methyltransferase (PRMT) family: PRMT1, PRMT3, and PRMT6. PRMT1 and PRMT3 showed a preference for methylating arginine residues in the first AT-hook of HMGA1 proteins, whereas PRMT6 methylated mainly residues in the second AT-hook. The initial sites of methylation catalyzed by PRMT1 and PRMT3 were mapped by tandem mass spectrometry to be Arg25 and Arg23, respectively, while we confirmed that the initial sites of methylation catalyzed by PRMT6 were at Arg57 and Arg59. Our results also revealed that binding of HMGA1 proteins to AT-rich duplex DNA, but not GC-rich duplex DNA, significantly inhibited the methylation efficiency of all of the PRMTs toward HMGA1 proteins. Moreover, C-terminal constitutive phosphorylation of HMGA1 proteins induced by protein kinase CK2 did not have any appreciable effect on the in vitro methylation of HMGA1. Our results suggest that PRMT1 might be involved in the previously reported methylation of Arg25 in HMGA1a in vivo.

title: Histone arginine methylation regulates pluripotency in the early mouse embryo
abstract: It has been generally accepted that the mammalian embryo starts its development with all cells identical, and only when inside and outside cells form do differences between cells first emerge. However, recent findings show that cells in the mouse embryo can differ in their developmental fate and potency as early as the four-cell stage. These differences depend on the orientation and order of the cleavage divisions that generated them. Because epigenetic marks are suggested to be involved in sustaining pluripotency, we considered that such developmental properties might be achieved through epigenetic mechanisms. Here we show that modification of histone H3, through the methylation of specific arginine residues, is correlated with cell fate and potency. Levels of H3 methylation at specific arginine residues are maximal in four-cell blastomeres that will contribute to the inner cell mass (ICM) and polar trophectoderm and undertake full development when combined together in chimaeras. Arginine methylation of H3 is minimal in cells whose progeny contributes more to the mural trophectoderm and that show compromised development when combined in chimaeras. This suggests that higher levels of H3 arginine methylation predispose blastomeres to contribute to the pluripotent cells of the ICM. We confirm this prediction by overexpressing the H3-specific arginine methyltransferase CARM1 in individual blastomeres and show that this directs their progeny to the ICM and results in a dramatic upregulation of Nanog and Sox2. Thus, our results identify specific histone modifications as the earliest known epigenetic marker contributing to development of ICM and show that manipulation of epigenetic information influences cell fate determination.

title: Histone Methylation-Dependent Mechanisms Impose Ligand Dependency for Gene Activation by Nuclear Receptors
abstract: Nuclear receptors undergo ligand-dependent conformational changes that are required for corepressor-coactivator exchange, but whether there is an actual requirement for specific epigenetic landmarks to impose ligand dependency for gene activation remains unknown. Here we report an unexpected and general strategy that is based on the requirement for specific cohorts of inhibitory histone methyltransferases (HMTs) to impose gene-specific gatekeeper functions that prevent unliganded nuclear receptors and other classes of regulated transcription factors from binding to their target gene promoters and causing constitutive gene activation in the absence of stimulating signals. This strategy, based at least in part on an HMT-dependent inhibitory histone code, imposes a requirement for specific histone demethylases, including LSD1, to permit ligand- and signal-dependent activation of regulated gene expression. These events link an inhibitory methylation component of the histone code to a broadly used strategy that circumvents pathological constitutive gene induction by physiologically regulated transcription factors.

title: Dynamic methylation of histone H3 at lysine 4 in transcriptional regulation by the androgen receptor.
abstract: The methylation of histone H3 correlates with either gene expression or silencing depending on the residues modified. Methylated lysine 4 (H3-K4) is associated with transcription at active gene loci. Furthermore, it was reported that trimethylated but not dimethylated H3-K4 is exclusively associated with active chromatin in Saccharomyces cerevisiae. In the present study, we investigated the H3-K4 methylation at the human prostate specific antigen (PSA) locus following gene activation and repression via androgen receptor (AR). We show that ligand-induced, AR-mediated transcription was accompanied by rapid decreases in di- and trimethylated H3-K4 at the PSA enhancer and promoter. Moreover, the observed decreases in H3-K4 methylation were reversed when AR was inhibited by a specific AR antagonist, bicalutamide. In contrast to the decreases in methylation at the 5' transcriptional control regions of the PSA gene, H3-K4 methylation in the coding region steadily increased after a lag period of approximately 4 h. The results suggest a novel role of methylated H3-K4 in transcriptional regulation.

title: The autoimmune regulator PHD finger binds to non-methylated histone H3K4 to activate gene expression
abstract: Mutations in the gene autoimmune regulator (AIRE) cause autoimmune polyendocrinopathy candidiasis ectodermal dystrophy. AIRE is expressed in thymic medullary epithelial cells, where it promotes the expression of tissue‐restricted antigens. By the combined use of biochemical and biophysical methods, we show that AIRE selectively interacts with histone H3 through its first plant homeodomain (PHD) finger (AIRE–PHD1) and preferentially binds to non‐methylated H3K4 (H3K4me0). Accordingly, in vivo AIRE binds to and activates promoters containing low levels of H3K4me3 in human embryonic kidney 293 cells. We conclude that AIRE–PHD1 is an important member of a newly identified class of PHD fingers that specifically recognize H3K4me0, thus providing a new link between the status of histone modifications and the regulation of tissue‐restricted antigen expression in thymus.