Michael R. Stallcup

GRIP1, a transcriptional coactivator for the AF-2 transactivation domain of steroid, thyroid, retinoid, and vitamin D receptors.

After binding to enhancer elements, transcription factors require transcriptional coactivator proteins to mediate their stimulation of transcription initiation. A search for possible coactivators for steroid hormone receptors resulted in identification of glucocorticoid receptor interacting protein 1 (GRIP1). The complete coding sequence for GRIP1, isolated from a mouse brain cDNA library, contains an open reading frame of 1,462 codons. GRIP1 is the probable ortholog of the subsequently identified human protein transcription intermediary factor 2 (TIF2) and is also partially homologous to steroid receptor coactivator 1 (SRC-1). The full-length GRIP1 interacted with the hormone binding domains (HBDs) of all five steroid receptors in a hormone-dependent manner and also with HBDs of class II nuclear receptors, including thyroid receptor alpha, vitamin D receptor, retinoic acid receptor alpha, and retinoid X receptor alpha. In contrast to agonists, glucocorticoid antagonists did not promote interaction between the glucocorticoid receptor and GRIP1. In yeast cells, GRIP1 dramatically enhanced the transcriptional activation function of proteins containing the HBDs of any of the above-named receptors fused to the GAL4 DNA binding domain and thus served as a transcriptional coactivator for them. This finding contrasts with previous reports of TIF2 and SRC-1, which in mammalian cells enhanced the transactivation activities of only a subset of the steroid and nuclear receptors that they physically interacted with. GRIP1 also enhanced the hormone-dependent transactivation activity of intact glucocorticoid receptor, estrogen receptor, and mineralocorticoid receptor. Experiments with glucocorticoid receptor truncation and point mutants indicated that GRIP1 interacted with and enhanced the activity of the C-terminal AF-2 but not the N-terminal AF-1 transactivation domain of the glucocorticoid receptor. These results demonstrate directly that AF-1 and AF-2 domains accomplish their transactivation activities through different mechanisms: AF-2 requires GRIP1 as a coactivator, but AF-1 does not.

Methylation of histone H4 at arginine 3 occurs in vivo and is mediated by the nuclear receptor coactivator PRMT1

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.

Hormone-independent Transcriptional Activation and Coactivator Binding by Novel Orphan Nuclear Receptor ERR3

Orphan nuclear receptors share sequence homology with members of the nuclear receptor superfamily, but ligands are unknown or unnecessary. A novel orphan receptor, estrogen receptor-related protein 3 (ERR3), was identified by yeast two-hybrid screening, using the transcriptional coactivator glucocorticoid receptor interacting protein 1 (GRIP1) as bait. The putative full-length mouse ERR3 contains 458 amino acids and is closely related to two known orphan receptors ERR1 and ERR2. All the ERR family members share an almost identical DNA-binding domain, which has 68% amino acid identity with that of estrogen receptor. ERR3 bound specifically to an estrogen response element and activated reporter genes controlled by estrogen response elements, both in yeast and in mammalian cells, in the absence of any added ligand. A conserved AF-2 activation domain located in the hormone-binding domain of ERR3 was primarily responsible for transcriptional activation. The ERR3 AF-2 domain bound GRIP1 in a ligand-independent manner both in vitro and in vivo, through the LXXLL motifs of GRIP1, and GRIP1 functioned as a transcriptional coactivator for ERR3 in both yeast and mammalian cells. Expression of ERR3 in adult mouse was restricted; highest expression was observed in heart, kidney, and brain. In the mouse embryo no expression was observed at day 7, and highest expression occurred around the 11–15 day stages. Although ERR3 is much more closely related to ERR2 than to ERR1, the expression pattern for ERR3 was similar to that of ERR1 and distinct from that for ERR2, suggesting a unique role for ERR3 in development.

Multiple signal input and output domains of the 160-kilodalton nuclear receptor coactivator proteins.

ABSTRACT Members of the 160-kDa nuclear receptor coactivator family (p160 coactivators) bind to the conserved AF-2 activation function found in the hormone binding domains of nuclear receptors (NR) and are potent transcriptional coactivators for NRs. Here we report that the C-terminal region of p160 coactivators glucocorticoid receptor interacting protein 1 (GRIP1), steroid receptor coactivator 1 (SRC-1a), and SRC-1e binds the N-terminal AF-1 activation function of the androgen receptor (AR), and p160 coactivators can thereby enhance transcriptional activation by AR. While they all interact efficiently with AR AF-1, these same coactivators have vastly different binding strengths with and coactivator effects on AR AF-2. p160 activation domain AD1, which binds secondary coactivators CREB binding protein (CBP) and p300, was previously implicated as the principal domain for transmitting the activating signal to the transcription machinery. We identified a new highly conserved motif in the AD1 region which is important for CBP/p300 binding. Deletion of AD1 only partially reduced p160 coactivator function, due to signaling through AD2, another activation domain located at the C-terminal end of p160 coactivators. C-terminal coactivator fragments lacking AD1 but containing AD2 and the AR AF-1 binding site served as efficient coactivators for full-length AR and AR AF-1. The two signal input domains (one that binds NR AF-2 domains and one that binds AF-1 domains of some but not all NRs) and the two signal output domains (AD1 and AD2) of p160 coactivators played different relative roles for two different NRs: AR and thyroid hormone receptor.

Molecular Pathogenesis of Chronic Wounds : The Role of β-Catenin and c-myc in the Inhibition of Epithelialization and Wound Healing

Lack of understanding of the molecular mechanisms and pathogenesis of impaired healing in chronic ulcers is a serious health issue that contributes to excessive limb amputations and mortality. Here we show that beta-catenin and its downstream targets in keratinocytes, c-myc, and keratins K6 and K16, play important roles in the development of chronic wounds. In contrast to normal epidermis, we observed a significant nuclear presence of beta-catenin and elevated c-myc expression at the nonhealing wound edge of chronic ulcers from 10 patients. In vitro studies indicated that stabilization of nuclear beta-catenin inhibited wound healing and keratinocyte migration by blocking epidermal growth factor response, inducing c-myc and repressing the K6/K16 keratins (cytoskeletal components important for migration). The molecular mechanism of K6/K16 repression involved beta-catenin and arginine methyltransferase (CARM-1) acting as co-repressors of glucocorticoid receptor monomers. We conclude that activation of the beta-catenin/c-myc pathway(s) contributes to impaired healing by inhibiting keratinocyte migration and altering their differentiation. The presence of activated beta-catenin and c-myc in the epidermis of chronic wounds may serve as a molecular marker of impaired healing and may provide future targets for therapeutic intervention.

Synergistic, p160 Coactivator-dependent Enhancement of Estrogen Receptor Function by CARM1 and p300

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.

The ankyrin repeats of G9a and GLP histone methyltransferases are mono- and dimethyllysine binding modules.

Histone modifications have important roles in transcriptional control, mitosis and heterochromatin formation. G9a and G9a-like protein (GLP) are euchromatin-associated methyltransferases that repress transcription by mono- and dimethylating histone H3 at Lys9 (H3K9). Here we demonstrate that the ankyrin repeat domains of G9a and GLP bind with strong preference to N-terminal H3 peptides containing mono- or dimethyl K9. X-ray crystallography revealed the basis for recognition of the methylated lysine by a partial hydrophobic cage with three tryptophans and one acidic residue. Substitution of key residues in the cage eliminated the H3 tail interaction. Hence, G9a and GLP contain a new type of methyllysine binding module (the ankyrin repeat domains) and are the first examples of protein (histone) methyltransferases harboring in a single polypeptide the activities that generate and read the same epigenetic mark.

Hormone-dependent, CARM1-directed, arginine-specific methylation of histone H3 on a steroid-regulated promoter

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.

Role of protein methylation in chromatin remodeling and transcriptional regulation.

Recent findings suggest that lysine and arginine-specific methylation of histones may cooperate with other types of post-translational histone modification to regulate chromatin structure and gene transcription. Proteins that methylate histones on arginine residues can collaborate with other coactivators to enhance the activity of specific transcriptional activators such as nuclear receptors. Lysine methylation of histones is associated with transcriptionally active nuclei, regulates other types of histone modifications, and is necessary for proper mitotic cell divisions. The fact that some transcription factors and proteins involved in RNA processing can also be methylated suggests that protein methylation may also contribute in other ways to regulation of transcription and post-transcriptional steps in gene regulation. In future work, it will be important to develop methods for evaluating the precise roles of protein methylation in the regulation of native genes in physiological settings, e.g. by using chromatin immunoprecipitation assays, differentiating cell culture systems, and genetically altered cells and animals. It will also be important to isolate additional protein methyltransferases by molecular cloning and to characterize new methyltransferase substrates, the regulation of methyltransferase activities, and the roles of new methyltransferases and substrates.

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.