Han Ma

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.

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.

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.

The roles of protein-protein interactions and protein methylation in transcriptional activation by nuclear receptors and their coactivators

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.

An additional region of coactivator GRIP1 required for interaction with the hormone-binding domains of a subset of nuclear receptors.

Transcriptional coactivators of the p160 family (SRC-1, GRIP1, and p/CIP) associate with DNA-bound nuclear receptors (NRs) and help the NRs to recruit an active transcription initiation complex to the promoters of target genes. Previous studies have demonstrated the importance of the NR interaction domain (NID) of p160 proteins containing three NR box motifs (LXXLL) for the interaction with the hormone-binding domains of NRs. Here we report that, in addition to NID, another region of coactivator GRIP1 (amino acids 1011–1121), called the auxiliary NID (NIDaux), is requiredin vitro and in vivo for efficient interaction with a subset of NRs, including the glucocorticoid receptor (GR), androgen receptor, and retinoic acid receptor α. A second group of NRs, which includes the progesterone receptor, retinoid X receptor α, thyroid hormone receptor β1, and vitamin D receptor, required only NID for efficient interaction. For binding to GR, the NID and NIDaux of GRIP1 must act in cis, but deletion of up to 144 amino acids between the two regions did not reduce binding efficiency. Amino acids 1011–1121 of GRIP1 also contain a p300 interaction domain, but mutational analysis indicated that the p300 interaction function within this region is separable from the ability to contribute to GR hormone-binding domain binding. SRC-1 lacks an NIDaux activity equivalent to that in GRIP1.

Study of Nuclear Receptor-Induced Transcription Complex Assembly and Histone Modification by Chromatin Immunoprecipitation Assays

Publisher Summary This chapter focuses on the study of nuclear receptor-induced transcription complex assembly and histone modification by chromatin immunoprecipitation (ChIP) assays. Nuclear receptor-mediated transcriptional regulation is a complex process involving the dynamic assembly of multiprotein complexes and modifications of chromosomal proteins, such as histones, at the target gene promoter. An increasingly used method to study this process in the context of natural chromosome structure is the chromatin immunoprecipitation assay. The ChIP assay employs a combination of formaldehyde induced in vivo cross-linking, immunoprecipitation, and sequence-specific DNA detection methods to observe the specific proteins associated with specific gene promoter regions in tissues or cultured cells. During the procedure of the ChIP assay a nuclear lysate is prepared from formaldehyde cross-linked cells that have been previously treated with or without hormones. ChIP assays allow the accurate assessment of the protein complexes associated with a given promoter during transcriptional regulation, spatially and temporally. The proteins of interest that can be examined by the use of specific antibodies include transcription factors that bind directly to the DNA, coactivators, corepressors, RNA polymerase and its associated proteins, and modified histones. ChIP assays have been applied to study nuclear receptor-mediated cofactor complex assembly and histone modifications at target gene promoters. The amount of antibody used in the immunoprecipitation depends on the affinity of the specific antibody, as well as the abundance of the chromatin-associated proteins and protein modifications.