Hongwei Li's research while affiliated with University of Southern California and other places

beta-Catenin, a pivotal component of the Wnt-signaling pathway, binds to and serves as a transcriptional coactivator for the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of transcriptional activator proteins and for the androgen receptor (AR), a nuclear receptor. Three components of the p160 nuclear receptor coactivator complex, including CARM1, p300/CBP, and GRIP1 (one of the p160 coactivators), bind to and cooperate with beta-catenin to enhance transcriptional activation by TCF/LEF and AR. Here we report that another component of the p160 nuclear receptor coactivator complex, the coiled-coil coactivator (CoCoA), directly binds to and cooperates synergistically with beta-catenin as a coactivator for AR and TCF/LEF. CoCoA uses different domains to bind GRIP1 and beta-catenin, and it uses different domains to transmit the activating signal to the transcription machinery, depending on whether it is bound to GRIP1 or beta-catenin. CoCoA associated specifically with the promoters of transiently transfected and endogenous target genes of TCF/LEF, and reduction of the endogenous CoCoA level decreased the ability of TCF/LEF and beta-catenin to activate transcription of transient and endogenous target genes. Thus, CoCoA uses different combinations of functional domains to serve as a physiologically relevant component of the Wnt/beta-catenin signaling pathway and the androgen signaling pathway.

The p160 coactivators, such as GRIP1, bind nuclear receptors and help to mediate transcriptional activation. β-Catenin binds to and serves as a coactivator for the nuclear receptor, androgen receptor (AR), and the Lymphoid Enhancer Factor/T Cell Factor family member, Lef1. Here we report that GRIP1 and β-catenin can bind strongly to each other through the AD2 domain of GRIP1. Furthermore, GRIP1 and β-catenin can synergistically enhance the activity of both AR and Lef1, and both coactivators are recruited specifically to AR-driven and Lef1-driven promoters. However, the mechanism of β-catenin-GRIP1 coactivator function and synergy is different with AR and Lef1. While β-catenin can bind directly to both AR and Lef1, GRIP1 can only bind directly to AR; the ability of GRIP1 to associate with and function as a coactivator for Lef1 is entirely dependent on the presence of β-catenin. Thus, whereas GRIP1 coactivator function involves direct binding to nuclear receptors and most other classes of DNA-binding transcriptional activator proteins, the coactivator function of GRIP1 with Lef1 follows a novel paradigm where GRIP1 is recruited indirectly to Lef1 through their mutual association with β-catenin. The β-catenin-GRIP1 interaction represents another potential point of cross-talk between the AR and Wnt signaling pathways.

The p160 coactivators, such as GRIP1, bind nuclear receptors and help to mediate transcriptional activation. β-Catenin binds to and serves as a coactivator for the nuclear receptor, androgen receptor (AR), and the Lymphoid Enhancer Factor/T Cell Factor family member, Lef1. Here we report that GRIP1 and β-catenin can bind strongly to each other through the AD2 domain of GRIP1. Furthermore, GRIP1 and β-catenin can synergistically enhance the activity of both AR and Lef1, and both coactivators are recruited specifically to AR-driven and Lef1-driven promoters. However, the mechanism of β-catenin-GRIP1 coactivator function and synergy is different with AR and Lef1. While β-catenin can bind directly to both AR and Lef1, GRIP1 can only bind directly to AR; the ability of GRIP1 to associate with and function as a coactivator for Lef1 is entirely dependent on the presence of β-catenin. Thus, whereas GRIP1 coactivator function involves direct binding to nuclear receptors and most other classes of DNA-binding transcriptional activator proteins, the coactivator function of GRIP1 with Lef1 follows a novel paradigm where GRIP1 is recruited indirectly to Lef1 through their mutual association with β-catenin. The β-catenin-GRIP1 interaction represents another potential point of cross-talk between the AR and Wnt signaling pathways.

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.

The RNA-binding protein HuR stabilizes labile mRNAs carrying AU-rich instability elements. This mRNA stabilization can be induced by hypoxia, lipopolysaccharide, and UV light. The mechanism by which these stimuli activate HuR is unclear and might be related to post-translational modification of this protein. Here we show that HuR can be methylated on arginine. However, HuR is not a substrate for PRMT1, the most prominent protein-arginine methyltransferase in mammalian cells, which methylates a number of heterogeneous nuclear ribonucleoproteins. Instead, HuR is specifically methylated by coactivator-associated arginine methyltransferase 1 (CARM1), a protein-arginine methyltransferase previously shown to serve as a transcriptional coactivator. By analyzing methylation of specific HuR arginine-to-lysine mutants and by sequencing radioactively methylated HuR peptides, Arg(217) was identified as the major HuR methylation site. Arg(217) is located in the hinge region between the second and third of the three HuR RNA recognition motif domains. Antibodies against a methylated HuR peptide were used to demonstrate in vivo methylation of HuR. HuR methylation increased in cells that overexpressed CARM1. Importantly, lipopolysaccharide stimulation of macrophages, which leads to HuR-mediated stabilization of tumor necrosis factor alpha mRNA in these cells, caused increased methylation of endogenous HuR. Thus, CARM1, which plays a role in transcriptional activation through histone H3 methylation, may also play a role in post-transcriptional gene regulation by methylating HuR.

The androgen receptor (AR) binds to and activates transcription of specific genes in response to its cognate steroid hormone, dihydrotestosterone. Transcriptional activation by the DNA-bound AR is accomplished with the help of a variety of coactivator proteins. For example, the p160 coactivators bind directly to AR and recruit additional coactivators such as the histone acetyltransferase p300 and the histone methyltransferase CARM1. The current study tested whether CARM1 can cooperate with other types of coactivator proteins. Recently it was shown that β-catenin can also bind directly to and serve as a coactivator for AR. Here it is shown that CARM1 binds to β-catenin and can function in synergy with β-catenin and p300 as coactivators for AR. The methyltransferase activity of CARM1 is important for its synergistic coactivator function with β-catenin. The synergistic coactivator function of β-catenin and CARM1 is not restricted to steroid receptors because these two coactivators can also act synergistically with another type of DNA binding transcriptional activator, LEF-1/TCF-4.

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.