In Kwon Chung

Eukaryotic topoisomerase II preferentially cleaves alternating purine-pyrimidine repeats

Alternating purine-pyrimidine sequences (RY repeats) demonstrate considerable homology to the consensus sequence for vertebrate topoisomerase II (Spitzner and Muller (1988) Nucleic Acids Res. 16: 1533-1556). This is shown below and positions that can match are underscored. RYRYRYRYRYRYRYRYRY = alternating purine-pyrimidine 18 bp RNYNNCNNGYNGKTNYNY = topoisomerase II consensus sequence (R is purine, Y is pyrimidine, K is G or T.) Topoisomerase II cleavage reactions were performed (in the absence of inhibitors) on a plasmid containing a 54 base RY repeat and the single strong cleavage site mapped to the RY repeat. Analysis of this DNA on sequencing gels showed that the enzyme cleaved a number of sites, all within the 54 base pair RY repeat. Topoisomerase II also made clustered cleavages within other RY repeats that were examined. Quantitative analysis of homology to the consensus sequence, as measured by the match of a site to a matrix of base proportions from the consensus data base (the matrix mean), showed that both the locations and the frequencies of cleavage sites within RY repeats were proportional to homology scores. However, topoisomerase II cleaved RY repeats preferentially in comparison to non-RY sites with similar homology scores. The activity of the enzyme at RY repeats appears to be proportional to the length of the repeat; additionally, GT, AC and AT repeats were better substrates for cleavage than GC repeats.

Sequence-specific DNA Recognition by the Myb-like Domain of Plant Telomeric Protein RTBP1

We have identified a rice gene encoding a DNA-binding protein that specifically recognizes the telomeric repeat sequence TTTAGGG found in plants. This gene, which we refer to asRTBP1 (rice telomere-binding protein 1), encodes a polypeptide with a predicted molecular mass of 70 kDa. RTBP1 is ubiquitously expressed in various organs and binds DNA with two or more duplex TTTAGGG repeats. The predicted protein sequence includes a single domain at the C terminus with extensive homology to Myb-like DNA binding motif. The Myb-like domain of RTBP1 is very closely related to that of other telomere-binding proteins, including TRF1, TRF2, Taz1p, and Tbf1p, indicating that DNA-binding domains of telomere-binding proteins are well conserved among evolutionarily distant species. To obtain precise information on the sequence of the DNA binding site recognized by RTBP1, we analyzed the sequence-specific binding properties of the isolated Myb-like domain of RTBP1. The isolated Myb-like domain was capable of sequence-specific DNA binding as a homodimer. Gel retardation analysis with a series of mutated telomere probes revealed that the internal GGGTTT sequence in the two-telomere repeats is critical for binding of Myb-like domain of RTBP1, which is consistent with the model of the TRF1·DNA complex showing that base-specific contacts are made within the sequence GGGTTA. To the best of our knowledge, RTBP1 is the first cloned gene in which the product is able to bind double-stranded telomeric DNA in plants. Because the Myb-like domain appears to be a significant motif for a large class of proteins that bind the duplex telomeric DNA, RTBP1 may play important roles in plant telomere function in vivo.

Sequence-specific binding property of Arabidopsis thaliana telomeric DNA binding protein 1 (AtTBP1)

We have identified an Arabidopsis thaliana cDNA, designated as AtTBP1, encoding a protein with a predicted size of 70.6 kDa that specifically binds to the plant telomeric repeat sequence TTTAGGG. AtTBP1 is present as a single‐copy gene in Arabidopsis genome and is expressed ubiquitously in various organs. AtTBP1 has a single Myb telomeric DNA binding domain at the C‐terminus and an extensive homology with other known telomere‐binding proteins. The isolated C‐terminus of AtTBP1 is capable of sequence‐specific DNA binding to plant duplex telomeric DNA. These results suggest that AtTBP1 may play important roles in plant telomere function in vivo.

Nuclear import of hTERT requires a bipartite nuclear localization signal and Akt-mediated phosphorylation

Sustained cell proliferation requires telomerase to maintain functional telomeres that are essential for chromosome integrity and protection. Although nuclear import of telomerase transcriptase (hTERT) is required for telomerase activity to elongate telomeres in vivo, the molecular mechanism regulating nuclear localization of hTERT is unclear. We have identified a bipartite nuclear localization signal (NLS; amino acid residues 222–240) that is responsible for nuclear import of hTERT. Immunofluorescence imaging of hTERT revealed that mutations in any of the bipartite NLS sequences result in decreased nuclear fluorescence intensity compared with wild-type hTERT. We also show that Akt-mediated phosphorylation at serine 227 is necessary for directing nuclear translocation of hTERT. Interestingly, serine 227 is located between two clusters of basic amino acids in the bipartite NLS. Inactivation of Akt activity by a dominant-negative mutant or wortmannin treatment attenuated nuclear localization of hTERT. We further show that both bipartite NLS and serine 227 in hTERT are required for cell immortalization of normal human foreskin fibroblast cells. Taken together, our findings reveal a previously unknown regulatory mechanism for nuclear import of hTERT through a bipartite NLS mediated by Akt phosphorylation, which represents an alternative pathway for modulating telomerase activity in cancer.

CHIP Promotes Human Telomerase Reverse Transcriptase Degradation and Negatively Regulates Telomerase Activity

The maintenance of eukaryotic telomeres requires telomerase, which is minimally composed of a telomerase reverse transcriptase (TERT) and an associated RNA component. Telomerase activity is tightly regulated by expression of human (h) TERT at both the transcriptional and post-translational levels. The Hsp90 and p23 molecular chaperones have been shown to associate with hTERT for the assembly of active telomerase. Here, we show that CHIP (C terminus of Hsc70-interacting protein) physically associates with hTERT in the cytoplasm and regulates the cellular abundance of hTERT through a ubiquitin-mediated degradation. Overexpression of CHIP prevents nuclear translocation of hTERT and promotes hTERT degradation in the cytoplasm, thereby inhibiting telomerase activity. In contrast, knockdown of endogenous CHIP results in the stabilization of cytoplasmic hTERT. However, it does not affect the level of nuclear hTERT and has no effect on telomerase activity and telomere length. We further show that the binding of CHIP and Hsp70 to hTERT inhibits nuclear translocation of hTERT by dissociating p23. However, Hsp90 binding to hTERT was not affected by CHIP overexpression. These results suggest that CHIP can remodel the hTERT-chaperone complexes. Finally, the amount of hTERT associated with CHIP peaks in G2/M phases but decreases during S phase, suggesting a cell cycle-dependent regulation of hTERT. Our data suggest that CHIP represents a new pathway for modulating telomerase activity in cancer.

Identification of a quinoxaline derivative that is a potent telomerase inhibitor leading to cellular senescence of human cancer cells.

Telomere maintenance is essential for the continued proliferation of dividing cells, and is implicated in chromosome stability and cell immortalization. Telomerase activity allows cells to maintain their telomeric DNA and contributes to the indefinite replicative capacity of cancer cells. Telomerase is expressed in most cancer cells, but not in normal somatic cells, suggesting that telomerase is an attractive target for cancer chemotherapy. Here we screened a chemical library for inhibition of human telomerase, and identified 2,3,7-trichloro-5-nitroquinoxaline (TNQX) as a potent inhibitor. TNQX showed a potent inhibitory effect, with 50% inhibition at approximately 1.4 microM, and did not inhibit DNA and RNA polymerases, including retroviral reverse trancriptase. A series of enzyme kinetic experiments suggested that TNQX is a mixed-type non-competitive inhibitor, with an inhibitor-binding site distinct from the binding sites for the telomeric substrate (TS) primer and the dNTPs. Long-term cultivation of the MCF7 cell line with a drug concentration that did not cause acute cytotoxicity resulted in progressive telomere erosion followed by an increased incidence of chromosome abnormalities and induction of the senescence phenotype. The results presented here indicate that TNQX is a highly potent and selective anti-telomerase agent with good potential for further development as a promising anti-cancer agent.

Curcumin inhibits nuclear localization of telomerase by dissociating the Hsp90 co-chaperone p23 from hTERT

The molecular chaperone complex Hsp90-p23 interacts with the rate-limiting catalytic subunit of telomerase, hTERT. Although their interactions are required for proper folding of nascent hTERT as well as the assembly of active telomerase, the precise role of the chaperone proteins in regulation of nuclear localization of hTERT remains unclear. Here we demonstrate that curcumin inhibits telomerase activity in a time- and dose-dependent manner by decreasing the level of hTERT expression. Following curcumin treatment, we observed a clear accumulation of hTERT in the cytoplasmic compartment of the cell. The curcumin-induced cytoplasmic retention of hTERT could be due to failure of nuclear import, and the resulting cytoplasmic hTERT protein was rapidly ubiquitinated and degraded by the proteasome. We also report that curcumin treatment results in a substantial decrease in association of p23 and hTERT but does not affect the Hsp90 binding to hTERT. In contrast, the treatment of the Hsp90 inhibitor geldanamycin promotes dissociation of both Hsp90 and p23 proteins from hTERT. Taken together, these results demonstrate that the interaction of the Hsp90-p23 complex with hTERT is critical for regulation of the nuclear localization of telomerase, and that down-regulation of hTERT by curcumin involves dissociating the binding of hTERT with p23. Thus, inhibition of nuclear translocation of hTERT by curcumin may provide new perspectives for regulation of telomerase activity during tumorigenic progression.

Interaction of an Arabidopsis RNA-binding Protein with Plant Single-stranded Telomeric DNA Modulates Telomerase Activity

Telomeres are the specialized structures at the end of linear chromosomes and terminate with a single-stranded 3′ overhang of the G-rich strand. The primary role of telomeres is to protect chromosome ends from recombination and fusion and from being recognized as broken DNA ends. This protective function can be achieved through association with specific telomere-binding proteins. Although proteins that bind single-stranded G-rich overhang regulate telomere length and telomerase activity in mammals and lower eukaryotes, equivalent factors have yet to be identified in plants. Here we have identified proteins capable of interacting with the G-rich single-stranded telomeric repeat from the Arabidopsis extracts by affinity chromatography. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis indicates that the isolated protein is a chloroplast RNA-binding protein (and a truncated derivative). The truncated derivative, which we refer to as STEP1 (single-stranded telomere-binding protein 1), binds specifically the single-stranded G-rich plant telomeric DNA sequences but not double-stranded telomeric DNA. Unlike the chloroplast-localized full-length RNA-binding protein, STEP1 localizes exclusively to the nucleus, suggesting that it plays a role in plant telomere biogenesis. We also demonstrated that the specific binding of STEP1 to single-stranded telomeric DNA inhibits telomerase-mediated telomere extension. The evidence presented here suggests that STEP1 is a telomere-end binding protein that may contribute to telomere length regulation by capping the ends of chromosomes and thereby repressing telomerase activity in plants.

Ubiquitin Ligase RLIM Modulates Telomere Length Homeostasis through a Proteolysis of TRF1.

The telomeric protein TRF1 negatively regulates telomere length by inhibiting telomerase access at the telomere termini, suggesting that the protein level of TRF1 at telomeres is tightly regulated. Regulation of TRF1 protein abundance is essential for proper telomere function and occurs primarily through post-translational modifications of TRF1. Here we describe RLIM, a RING H2 zinc finger protein with intrinsic ubiquitin ligase activity, as a TRF1-interacting protein. RLIM increases TRF1 turnover by targeting it for degradation by the proteasome in a ubiquitin-dependent manner, independently of Fbx4, which is known to interact with and negatively regulate TRF1. Whereas overexpression of RLIM decreases the level of TRF1 protein, depletion of endogenous RLIM expression by small hairpin RNA increases the level of TRF1 and leads to telomere shortening, thereby impairing cell growth. These results demonstrate that RLIM is involved in the negative regulation of TRF1 function through physical interaction and ubiquitin-mediated proteolysis. Hence, RLIM represents a new pathway for telomere maintenance by modulating the level of TRF1 at telomeres.

Restoration of senescent human diploid fibroblasts by modulation of the extracellular matrix

Human diploid fibroblasts have the capacity to complete a finite number of cell divisions before entering a state of replicative senescence characterized by growth arrest, changes in morphology, and altered gene expression. Herein, we report that interaction with extracellular matrix (ECM) from young cells is sufficient to restore aged, senescent cells to an apparently youthful state. The identity of the restored cells as having been derived from senescent cells has been confirmed by a variety of methods, including time lapse live cell imaging and DNA finger print analysis. In addition to cell morphology, phenotypic restoration was assessed by resumption of proliferative potential, growth factor responsiveness, reduction of intracellular reactive oxygen species levels, recovery of mitochondrial membrane potential, and increased telomere length. Mechanistically, we find that both Ku and SIRT1 are induced during restoration and are required for senescent cells to return to a youthful phenotype. These observations demonstrate that human cellular senescence is profoundly influenced by cues from the ECM, and that senescent cell plasticity is much greater than that was previously believed to be the case.