Liuh-Yow Chen

Regulation of Androgen Receptor Activity by the Nuclear Receptor Corepressor SMRT

Androgen receptor (AR) is a hormone-regulated transcription factor that mediates a wide array of biological processes including sexual differentiation, spermatogenesis, and prostate cancer progression. The transcriptional activity of AR and other members of the nuclear receptor superfamily are modulated by coregulatory proteins. In this study, we have investigated the regulation of AR transcriptional activity by the silencing mediator for retinoid and thyroid hormone receptors (SMRT). We found that AR possesses an intrinsic transcriptional repression activity, and AR interacts directly with SMRT. One interacting surface on AR is mapped to the ligand-binding domain, and the presence of a DNA binding/hinge region enhances this interaction. The binding surface on SMRT is mapped to the C-terminal ID2 region, and mutation in the ID2 corepressor motif inhibits the interaction. Overexpression of SMRT inhibits dihydrotestosterone-dependent transactivation by AR and further suppresses the antiandrogen flutamide-mediated inhibition of AR activity. We provide evidence to suggest that the mechanisms of SMRT-mediated inhibition of AR activity involves inhibition of AR N/C interaction and competition with the p160 coactivator. Our data establish a significant role of SMRT in modulating AR transcriptional activity.

The human CST complex is a terminator of telomerase activity.

The lengths of human telomeres, which protect chromosome ends from degradation and end fusions, are crucial determinants of cell lifespan. During embryogenesis and in cancer, the telomerase enzyme counteracts telomeric DNA shortening. As shown in cancer cells, human telomerase binds the shelterin component TPP1 at telomeres during the S phase of the cell cycle, and adds ∼60 nucleotides in a single round of extension, after which telomerase is turned off by unknown mechanisms. Here we show that the human CST (CTC1, STN1 and TEN1) complex, previously implicated in telomere protection and DNA metabolism, inhibits telomerase activity through primer sequestration and physical interaction with the protection of telomeres 1 (POT1)–TPP1 telomerase processivity factor. CST competes with POT1–TPP1 for telomeric DNA, and CST–telomeric-DNA binding increases during late S/G2 phase only on telomerase action, coinciding with telomerase shut-off. Depletion of CST allows excessive telomerase activity, promoting telomere elongation. We propose that through binding of the telomerase-extended telomere, CST limits telomerase action at individual telomeres to approximately one binding and extension event per cell cycle. Our findings define the sequence of events that occur to first enable and then terminate telomerase-mediated telomere elongation.

Daxx Silencing Sensitizes Cells to Multiple Apoptotic Pathways

ABSTRACT Daxx is a nuclear protein involved in apoptosis and transcriptional repression, and it interacts with the death receptor Fas, promyelocytic leukemia protein (PML), and several transcriptional repressors. The function of Daxx in apoptosis is controversial because opposite results were obtained in transient overexpression and genetic knockout studies. Furthermore, the roles of PML and transcriptional repression in Daxx-regulated apoptosis are currently unknown. In this study, we investigated the role of Daxx in Fas- and stress-induced apoptosis by small interfering RNA-mediated Daxx silencing in mammalian cells. Daxx silencing had no apparent cytotoxic effects on mammalian cells within 72 h. Intriguingly, Daxx silencing strongly sensitized cells to Fas- and stress-induced apoptosis, which was accompanied by caspase activation, cytochrome c release, and Jun N-terminal kinase activation. Consistently, endogenous Daxx was degraded rapidly upon induction of apoptosis by stress or anti-Fas antibody. Finally, PML silencing had no effect on Daxx silencing-mediated apoptotic events, while caspase gene expression was upregulated in the absence of Daxx. These data strongly suggest that Daxx may inhibit Fas and stress-mediated apoptosis by suppressing proapoptotic gene expression outside of PML domains.

TRF2 functions as a protein hub and regulates telomere maintenance by recognizing specific peptide motifs

In mammalian cells, the telomeric repeat binding factor (TRF) homology (TRFH) domain–containing telomeric proteins TRF1 and TRF2 associate with a collection of molecules necessary for telomere maintenance and cell-cycle progression. However, the specificity and the mechanisms by which TRF2 communicates with different signaling pathways remain largely unknown. Using oriented peptide libraries, we demonstrate that the TRFH domain of human TRF2 recognizes [Y/F]XL peptides with the consensus motif YYHKYRLSPL. Disrupting the interactions between the TRF2 TRFH domain and its targets resulted in telomeric DNA-damage responses. Furthermore, our genome-wide target analysis revealed phosphatase nuclear targeting subunit (PNUTS) and microcephalin 1 (MCPH1) as previously unreported telomere-associated proteins that directly interact with TRF2 via the [Y/F]XL motif. PNUTS and MCPH1 can regulate telomere length and the telomeric DNA-damage response, respectively. Our findings indicate that an array of TRF2 molecules functions as a protein hub and regulates telomeres by recruiting different signaling molecules via a linear sequence code.

Telomere Maintenance through Spatial Control of Telomeric Proteins

ABSTRACT The six human telomeric proteins TRF1, TRF2, RAP1, TIN2, POT1, and TPP1 can form a complex called the telosome/shelterin, which is required for telomere protection and length control. TPP1 has been shown to regulate both POT1 telomere localization and telosome assembly through its binding to TIN2. It remains to be determined where such interactions take place and whether cellular compartmentalization of telomeric proteins is important for telomere maintenance. We systematically investigated here the cellular localization and interactions of human telomeric proteins. Interestingly, we found TIN2, TPP1, and POT1 to localize and interact with each other in both the cytoplasm and the nucleus. Unexpectedly, TPP1 contains a functional nuclear export signal that directly controls the amount of TPP1 and POT1 in the nucleus. Furthermore, binding of TIN2 to TPP1 promotes the nuclear localization of TPP1 and POT1. We also found that disrupting TPP1 nuclear export could result in telomeric DNA damage response and telomere length disregulation. Our findings highlight how the coordinated interactions between TIN2, TPP1, and POT1 in the cytoplasm regulate the assembly and function of the telosome in the nucleus and indicate for the first time the importance of nuclear export and spatial control of telomeric proteins in telomere maintenance.

Genome-wide YFP Fluorescence Complementation Screen Identifies New Regulators for Telomere Signaling in Human Cells

Detection of low-affinity or transient interactions can be a bottleneck in our understanding of signaling networks. To address this problem, we developed an arrayed screening strategy based on protein complementation to systematically investigate protein-protein interactions in live human cells, and performed a large-scale screen for regulators of telomeres. Maintenance of vertebrate telomeres requires the concerted action of members of the Telomere Interactome, built upon the six core telomeric proteins TRF1, TRF2, RAP1, TIN2, TPP1, and POT1. Of the ∼12,000 human proteins examined, we identified over 300 proteins that associated with the six core telomeric proteins. The majority of the identified proteins have not been previously linked to telomere biology, including regulators of post-translational modifications such as protein kinases and ubiquitin E3 ligases. Results from this study shed light on the molecular niche that is fundamental to telomere regulation in humans, and provide a valuable tool to investigate signaling pathways in mammalian cells.

Involvement of p38 Mitogen-activated Protein Kinase Signaling Pathway in the Rapid Induction of the 78-kDa Glucose-regulated Protein in 9L Rat Brain Tumor Cells

We have previously shown that treatment with okadaic acid (OA) followed by heat shock (HS) (termed OA → HS treatment) leads to rapid transactivation of the 78-kDa glucose-regulated protein gene (grp78) in 9L rat brain tumor cells. A cAMP-responsive element-like (CRE-like, TGACGTGA) promoter sequence and a protein kinase A signaling pathway are involved in this induction, and activation of both CRE binding protein (CREB) and activating transcription factor-2 (ATF-2) is required in the above process. Herein, we report that transactivation of grp78, as well as phosphorylation/activation of ATF-2, can be completely annihilated by SB203580, a highly specific inhibitor of p38 mitogen-activated protein kinase (p38MAPK). Activation of p38MAPK by OA → HS is also substantiated by its own phosphorylation as well as the phosphorylation and activation of MAPK activating protein kinase-2 in cells subjected to this treatment. The involvement of p38MAPK in the activation of ATF-2, which leads to the transactivation of rat grp78, is confirmed by electrophoretic mobility shift assay using a probe containing the CRE-like sequence as well as by transient transfection assays with a plasmid containing a 710-base pair stretch of the grp78promoter. Together with our previous studies, these results led us to conclude that phosphorylation/activation of CREB upon OA → HS treatment is mediated by cAMP-dependent protein kinase, whereas that of ATF-2 is mediated by p38MAPK. The transcription factors may bind to each other to form heterodimers that in turn transactivate grp78 by binding to the CRE-like element. This suggests that distinct signaling pathways converge on CREB-ATF-2, where each subunit is individually activated by a specific class of protein kinases. This may allow modulation ofgrp78 transactivation by diverse external stimuli.

Phosphorylation of TPP1 regulates cell cycle-dependent telomerase recruitment

Telomere maintenance is essential for organisms with linear chromosomes and is carried out by telomerase during cell cycle. The precise mechanism by which cell cycle controls telomeric access of telomerase and telomere elongation in mammals remains largely unknown. Previous work has established oligonucleotide/oligosaccharide binding (OB) fold-containing telomeric protein TPP1, formerly known as TINT1, PTOP, and PIP1, as a key factor that regulates telomerase recruitment and activity. However, the role of TPP1 in cell cycle-dependent telomerase recruitment is unclear. Here, we report that human TPP1 is phosphorylated at multiple sites during cell cycle progression and associates with higher telomerase activity at late S/G2/M. Phosphorylation of Ser111 (S111) within the TPP1 OB fold appears important for cell cycle-dependent telomerase recruitment. Structural analysis indicates that phosphorylated S111 resides in the telomerase-interacting domain within the TPP1 OB fold. Mutations that disrupt S111 phosphorylation led to decreased telomerase activity in the TPP1 complex and telomere shortening. Our findings provide insight into the regulatory pathways and structural basis that control cell cycle-dependent telomerase recruitment and telomere elongation through phosphorylation of TPP1.

Molecular basis of telomere syndrome caused by CTC1 mutations

Mutations in CTC1 lead to the telomere syndromes Coats Plus and dyskeratosis congenita (DC), but the molecular mechanisms involved remain unknown. CTC1 forms with STN1 and TEN1 a trimeric complex termed CST, which binds ssDNA, promotes telomere DNA synthesis, and inhibits telomerase-mediated telomere elongation. Here we identify CTC1 disease mutations that disrupt CST complex formation, the physical interaction with DNA polymerase α-primase (polα-primase), telomeric ssDNA binding in vitro, accumulation in the nucleus, and/or telomere association in vivo. While having diverse molecular defects, CTC1 mutations commonly lead to the accumulation of internal single-stranded gaps of telomeric DNA, suggesting telomere DNA replication defects as a primary cause of the disease. Strikingly, mutations in CTC1 may also unleash telomerase repression and telomere length control. Hence, the telomere defect initiated by CTC1 mutations is distinct from the telomerase insufficiencies seen in classical forms of telomere syndromes, which cause short telomeres due to reduced maintenance of distal telomeric ends by telomerase. Our analysis provides molecular evidence that CST collaborates with DNA polα-primase to promote faithful telomere DNA replication.

CST for the grand finale of telomere replication.

Telomeric DNA at eukaryotic chromosome ends terminates with single stranded 3′ G-rich overhangs. The overhang is generated by the interplay of several dynamic processes including semiconservative DNA replication, 3′ end elongation by telomerase, C-strand fill-in synthesis and nucleolytic processing. The mammalian CST (CTC1-STN1-TEN1) complex is directly involved at several stages of telomere end formation. Elucidation of its structural organization and identification of interaction partners support the notion that mammalian CST is, as its yeast counterpart, a RPA-like complex. CST binding at mammalian telomere 3′ overhangs increases upon their elongation by telomerase. Formation of a trimeric CST complex at telomeric 3′overhangs leads to telomerase inhibition and at the same time mediates a physical interaction with DNA polymerase-α. Thus CST seems to play critical roles in coordinating telomerase elongation and fill-in synthesis to complete telomere replication.