Catherine K. Yang

CCAR1, a key regulator of Mediator complex recruitment to nuclear receptor transcription complexes

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.

Requirement of Cell Cycle and Apoptosis Regulator 1 for Target Gene Activation by Wnt and β-Catenin and for Anchorage-independent Growth of Human Colon Carcinoma Cells

Aberrant Wnt signaling promotes oncogenesis by increasing cellular levels of β-catenin, which associates with DNA-bound transcription factors and activates Wnt target genes. However, the molecular mechanism by which β-catenin mediates gene expression is still poorly understood. Here, we show that cell cycle and apoptosis regulator 1 (CCAR1), which was recently shown to function as a transcriptional coactivator for nuclear receptors, also interacts with β-catenin and enhances the ability of β-catenin to activate expression of transiently transfected reporter genes. Furthermore, association of CCAR1 with the promoter of an endogenous Wnt/β-catenin target gene in a colon cancer cell line depends on the presence of β-catenin. Depletion of CCAR1 inhibits expression of several Wnt/β-catenin target genes and suppresses anchorage-independent growth of the colon cancer cell line. Thus, CCAR1 is a novel component of Wnt/β-catenin signaling that plays an important role in transcriptional regulation by β-catenin and that, therefore, may represent a novel target for therapeutic intervention in cancers involving aberrantly activated Wnt/β-catenin signaling.

Downstream signaling mechanism of the C-terminal activation domain of transcriptional coactivator CoCoA

The coiled-coil coactivator (CoCoA) is a transcriptional coactivator for nuclear receptors and enhances nuclear receptor function by the interaction with the bHLH-PAS domain (AD3) of p160 coactivators. The C-terminal activation domain (AD) of CoCoA possesses strong transactivation activity and is required for the coactivator function of CoCoA with nuclear receptors. To understand how CoCoA AD transmits its activating signal to the transcription machinery, we defined specific subregions, amino acid motifs and protein binding partners involved in the function of CoCoA AD. The minimal transcriptional AD was mapped to approximately 91 C-terminal amino acids and consists of acidic, serine/proline-rich and phenylalanine-rich subdomains. Transcriptional activation by the CoCoA AD was p300-dependent, and p300 interacted physically and functionally with CoCoA AD and was recruited to a promoter by the interaction with CoCoA AD. The FYDVASAF motif in the CoCoA AD was critical for the transcriptional activity of CoCoA AD, the interaction of CoCoA with p300, the coactivator function of CoCoA for estrogen receptor α and GRIP1 and the transcriptional synergy among coactivators GRIP1, CARM1, p300 and CoCoA. Taken together these data extend our understanding of the mechanism of downstream signaling by the essential C-terminal AD of the nuclear receptor coactivator CoCoA; they indicate that p300 is a functionally important interaction partner of CoCoA AD and that their interaction potentiates transcriptional activation by the p160 coactivator complex.

Differential use of functional domains by CoCoA in its synergistic coactivator function with β-catenin or GRIP1

β-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 β-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 β-catenin as a coactivator for AR and TCF/LEF. CoCoA uses different domains to bind GRIP1 and β-catenin, and it uses different domains to transmit the activating signal to the transcription machinery, depending on whether it is bound to GRIP1 or β-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 β-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/β-catenin signaling pathway and the androgen signaling pathway.

Role of GAC63 in transcriptional activation mediated by β-catenin

β-Catenin is a key mediator in the canonical Wnt signaling pathway, which plays important roles in multiple developmental processes. Inappropriate activation of this pathway leads to developmental defects and development of certain cancers. Upon Wnt signaling, β-catenin binds TCF/LEF transcription factors. The TCF/LEF-β-catenin complex then recruits a variety of transcriptional coactivators to the promoter/enhancer region of Wnt-responsive genes and activates target gene transcription. In this article, we demonstrate that GRIP1-associated coactivator 63 (GAC63), a recently identified nuclear receptor (NR) coactivator, interacts with β-catenin. The N-terminus of GAC63 is the binding site for β-catenin, whereas a C-terminal fragment of β-catenin including armadillo repeats 10–12 binds to GAC63. Over-expression of GAC63 enhanced the transcriptional activity of β-catenin, and also greatly enhanced TCF/LEF-regulated reporter gene activity in a β-catenin-dependent manner. Endogenous GAC63 was recruited to TCF/LEF-responsive enhancer elements when β-catenin levels were induced by LiCl. In addition, reduction of endogenous GAC63 level by small interfering RNA (siRNA) inhibited TCF/LEF-mediated gene transcription. Our findings reveal a new function of GAC63 in transcriptional activation of Wnt-responsive genes.

SUMOylation of ZFP282 potentiates its positive effect on estrogen signaling in breast tumorigenesis.

Estrogen receptor α (ERα) has critical roles in the development and progression of breast cancer, and the coiled-coil co-activator (CoCoA) is an important ERα co-activator for estrogen-induced gene expression. The small ubiquitin-like modifier (SUMO) pathway is hyperactivated in breast cancer, but the mechanism by which SUMOylation regulates ERα-mediated transcription remains poorly understood. Here, we identified ZFP282 as a CoCoA-binding protein. ZFP282 associates directly with ERα and cooperates synergistically with CoCoA to enhance ERα function. ZFP282 is required for estrogen-induced expression of ERα target genes and estrogen-dependent breast cancer cell growth and tumorigenesis. In addition, we found that ZFP282 is SUMOylated and that SUMOylation positively regulates the co-activator activity of ZFP282 by increasing its binding affinity to ERα and CoCoA, and consequently increasing recruitment of ZFP282–CoCoA complex to the promoter of ERα target genes. These findings reveal essential roles for ZFP282 and its SUMOylation in estrogen signaling and breast tumorigenesis.

Differential regulation of the two transcriptional activation domains of the coiled-coil coactivator CoCoA by sumoylation

BackgroundThe coiled-coil coactivator (CoCoA) enhances transcriptional activity of nuclear receptors, the xenobiotic aryl hydrocarbon receptor, and the lymphocyte enhancer factors (LEF) in the Wnt/β-catenin signaling pathway. CoCoA is comprised of a large central coiled coil domain flanked by N-terminal and C-terminal activation domains (AD). The N-terminal AD of CoCoA is required for coactivator function with LEF and β-catenin, while the C-terminal AD of CoCoA is required for coactivator function with nuclear receptors. We explored the role of sumoylation in regulating the activities of the two ADs and the coactivator function of CoCoA.ResultsThe N-terminus of CoCoA is covalently modified by SUMO1 at Lys-29; both PIAS1 and ARIP3 function as E3 ligases. Fusion of SUMO1 to the N-terminus (mimicking sumoylation) reduced coactivator function of CoCoA with LEF1 and the activity of the N-terminal AD. The N- and C-termini of CoCoA can bind to each other, and C-terminal transactivation activity is attenuated in the presence of the N-terminus, indicating that the N-C interaction regulates the activity of the C-terminal AD. Fusion of SUMO1 to the N-terminal fragment of CoCoA reduced the N-C interaction and inhibition of C-terminal AD activity by the N-terminal fragment.ConclusionSumoylation of CoCoA differentially regulates the coactivator activity of CoCoA with nuclear receptors versus LEF1, by attenuating the N-terminal AD activity and enhancing the activity of the C-terminal AD.

Differential Use of Functional Domains by Coiled-coil Coactivator in Its Synergistic Coactivator Function with β-Catenin or GRIP1

β-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 β-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 β-catenin as a coactivator for AR and TCF/LEF. CoCoA uses different domains to bind GRIP1 and β-catenin, and it uses different domains to transmit the activating signal to the transcription machinery, depending on whether it is bound to GRIP1 or β-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 β-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/β-catenin signaling pathway and the androgen signaling pathway.