Budding yeast Cdc13, Stn1, and Ten1 form the CST complex to protect telomeres from lethal DNA degradation. It remains unknown whether similar complexes are conserved in higher eukaryotes or not. Here we isolated mammalian STN1 and TEN1 homologs and CTC1 (conserved telomere maintenance component 1). The three proteins contain putative OB-fold domains and form a complex called CST, which binds to single-stranded DNA with high affinity in a sequence-independent manner. CST associates with a fraction of telomeres consistently during the cell cycle, in quiescent cells and Pot1-knockdown cells. It does not colocalize with replication foci in S phase. Significant increases in the abundance of single-stranded G-strand telomeric DNA were observed in Stn1-knockdown cells. We propose that CST is a replication protein A (RPA)-like complex that is not directly involved in conventional DNA replication at forks but plays a role in DNA metabolism frequently required by telomeres.

Telomeres are specialized chromatin structures that protect chromosomal ends. Protection of telomeres 1 (Pot1) binds to the telomeric G-rich overhang, thereby protecting telomeres and regulating telomerase. Mammalian POT1 and TPP1 interact and constitute part of the six-protein shelterin complex. Here we report that Tpz1, the TPP1 homolog in fission yeast, forms a complex with Pot1. Tpz1 binds to Ccq1 and the previously undiscovered protein Poz1 (Pot1-associated in Schizosaccharomyces pombe), which protect telomeres redundantly and regulate telomerase in positive and negative manners, respectively. Thus, the Pot1-Tpz1 complex accomplishes its functions by recruiting effector molecules Ccq1 and Poz1. Moreover, Poz1 bridges Pot1-Tpz1 and Taz1-Rap1, thereby connecting the single-stranded and double-stranded telomeric DNA regions. Such molecular architectures are similar to those of mammalian shelterin, indicating that the overall DNA-protein architecture is conserved across evolution.

Telomerase is a specialized type of reverse transcriptase which catalyzes the synthesis and extension of telomeric DNA (for review, see ref.1). This enzyme is highly active in most cancer cells, but is inactive in most somatic cells. This striking observation led to the suggestion that telomerase might be important for the continued growth or progression of cancer cells. However, little is known about the molecular mechanism of telomerase activation in cancer cells. Human telomerase reverse transcriptase (hTRT) has recently been identified as a putative human telomerase catalytic subunit. We transfected the gene encoding hTRT into telomerase-negative human normal fibroblast cells and demonstrated that expression of wild-type hTRT induces telomerase activity, whereas hTRT mutants containing mutations in regions conserved among other reverse transcriptases did not. Hepatocellular carcinoma (20 samples) and non-cancerous liver tissues (19 samples) were examined for telomerase activity and expression of hTRT, the human telomerase RNA component (hTR; encoded by TERC) and the human telomerase-associated protein (hTLP1; encoded by TEP1). A significant correlation between hTRT expression and telomerase activity was observed. These results indicate that the hTRT protein is the catalytic subunit of human telomerase, and that it plays a key role in the activation of telomerase in cancer cells.

We have cloned and characterized the rat telomerase protein component 1 gene (TLP1), which is related to the gene for Tetrahymena p80. The cDNA encodes a 2629 amino acid sequence and produces the TLP1 proteins p240 and p230. The anti-TLP1 antibody specifically immunoprecipitated the telomerase activity. Moreover, p240 and p230 were copurified with telomerase activity in a series of extensive purification experiments. These results strongly suggest that the TLP1 proteins are components of, or are closely associated with, the rat telomerase. A pulse-chase experiment showed that p240 is modified to p230 in vivo. p230 was the dominant form in telomerase-positive cells, suggesting that modification of the TLP1 protein may regulate telomerase activity in vivo.

Oct-3/4 is a key transcriptional factor whose expression level governs the fate of primitive inner cell mass and embryonic stem (ES) cells. Previously, an upstream 3.3-kb distal enhancer (DE) fragment was identified to be responsible for the specific expression of mouse Oct-3/4 in the inner cell mass and ES cells. However, little is known about the cis-elements and trans-factors required for DE activity. In this study, we identified a novel cis-element, called Site 2B here, located ϳ30 bp downstream from Site 2A, which was previously revealed in DE by an in vivo chemical modification experiment. Using the luciferase reporter assay, we demonstrated that both Site 2A and Site 2B are necessary and sufficient for activating DE in the contexts of both the native Oct-3/4 promoter and the heterologous thymidine kinase minimal promoter. In an electrophoretic mobility shift assay we showed that Site 2B specifically binds to Oct-3/4 and Sox2 when ES-derived cell extracts were used, whereas Site 2A binds to a factor(s) present in both ES and NIH 3T3 cells. Furthermore, we showed that the physiological level of Oct-3/4 in ES cells is required for Site 2B-mediated DE activity using the inducible knockout system of Oct-3/4 in ES cells. These results indicate that Oct-3/4 is a member of the gene family regulated by Oct-3/4 and Sox2, as reported before for the FGF-4, UTF1, Sox2, and Fbx15 genes. Thus, Oct-3/4 and Sox2 comprise a regulatory complex that controls the expression of genes important for the maintenance of the primitive state, including themselves. This autoregulatory circuit of the Sox2⅐Oct-3/4 complex may contribute to maintaining robustly the precise expression level of Oct-3/4 in primitive cells.

Dendrites allow neurons to integrate sensory or synaptic inputs, and the spatial disposition and local density of branches within the dendritic arbor limit the number and type of inputs. Drosophila melanogaster dendritic arborization (da) neurons provide a model system to study the genetic programs underlying such geometry in vivo. Here we report that mutations of motor-protein genes, including a dynein subunit gene (dlic) and kinesin heavy chain (khc), caused not only downsizing of the overall arbor, but also a marked shift of branching activity to the proximal area within the arbor. This phenotype was suppressed when dominant-negative Rab5 was expressed in the mutant neurons, which deposited early endosomes in the cell body. We also showed that 1) in dendritic branches of the wild-type neurons, Rab5-containing early endosomes were dynamically transported and 2) when Rab5 function alone was abrogated, terminal branches were almost totally deleted. These results reveal an important link between microtubule motors and endosomes in dendrite morphogenesis.

Cellular senescence is a tumor-suppressing mechanism that is accompanied by characteristic chromatin condensation called senescence-associated heterochromatic foci (SAHFs). We found that individual SAHFs originate from individual chromosomes. SAHFs do not show alterations of posttranslational modifications of core histones that mark condensed chromatin in mitotic chromosomes, apoptotic chromatin, or transcriptionally inactive heterochromatin. Remarkably, SAHF-positive senescent cells lose linker histone H1 and exhibit increased levels of chromatin-bound high mobility group A2 (HMGA2). The expression of N-terminally enhanced green fluorescent protein (EGFP)–tagged histone H1 induces premature senescence phenotypes, including increased levels of phosphorylated p53, p21, and hypophosphorylated Rb, and a decrease in the chromatin-bound endogenous histone H1 level but not in p16 level accumulation or SAHF formation. However, the simultaneous ectopic expression of hemagglutinin-tagged HMGA2 and N-terminally EGFP-tagged histone H1 leads to significant SAHF formation (P < 0.001). It is known that histone H1 and HMG proteins compete for a common binding site, the linker DNA. These results suggest that SAHFs are a novel type of chromatin condensation involving alterations in linker DNA–binding proteins.

Although mammalian MBD3 contains the mCpG-binding domain (MBD) and is highly homologous with the authentic mCpG-binding protein MBD2, it was reported that the protein does not bind to mCpG specifically. Using recombinant human wild type and mutant MBD3 proteins, we demonstrated that atypical amino acids found in MBD3 MBD, namely, His-30 and Phe-34, are responsible for the inability of MBD3 to bind to mCpG. Interestingly, although H30K/F34Y MBD3 mutant protein binds to mCpG efficiently in vitro, it was not localized at the mCpG-rich pericentromeric regions in mouse cells. We also showed that Y34F MBD2b MBD, which possesses not the mCpG-specific DNA-binding activity but the nonspecific DNA-binding activity, was localized at the pericentromeric regions. These results suggested that the mCpG-specific DNA-binding activity is largely dispensable, and another factor(s) is required for the localization of MBD proteins in vivo. MBD3 was identified as a component of the NuRD/Mi2 complex that shows chromatin remodeling and histone deacetylase activities. We demonstrated that MBD3 MBD is necessary and sufficient for binding to HDAC1 and MTA2, two components of the NuRD/Mi2 complex. It was therefore suggested that mCpG-binding-defective MBD3 has evolutionarily conserved its MBD because of the secondary role played by the MBD in protein-protein interactions.DNA methylation is the major modification in eukaryote genomes. This modification occurs predominantly at position 5 of cytosine when followed by guanosine (CpG site) in vertebrates. Approximately 60 -90% of the total CpG sites is methylated in vertebrates. One of the direct consequences of mCpG is the transcriptional repression of nearby genes. Several molecular mechanisms are thought to be responsible for this methylcytosine-mediated gene repression. Among them, the repression mediated by mCpG-binding proteins has been most extensively studied. Five mCpG-binding proteins, MBD1-4 1 and MeCP2, have been identified in mammals and are collectively called MBD family proteins because these proteins share the mCpG-binding domain (1, 2). MBD4 recognizes the T/G mismatched base pair, and is a component of the DNA repair machinery (3, 4). MBD1, MBD2, and MeCP2 have been well characterized for their gene-repressing activities. These proteins bind to mCpG via MBD and recruit histone deacetylase (HDAC) complexes to the binding sites via their separate domain called TRD (transcriptional repression domain). The deacetylation by HDAC of the core histones H3 and H4 is partly responsible for the gene repression mediated by these MBD proteins (1, 5-7). In contrast to these well characterized MBD proteins, the function of MBD3 remains unclear. Although MBD2 and MBD3 are highly homologous in and outside MBD, experiments performed in vitro and in vivo failed to demonstrate the mCpG-binding activity of recombinant mouse MBD3 (2). A controversy arose when Xenopus MBD3 was isolated and demonstrated to possess mCpG-binding activity (8). However, recent studies have pointed out that amino acids in...