CTC1 OB-B interaction with TPP1 terminates telomerase and prevents telomere overextension.

CST (CTC1-STN1-TEN1) is a telomere associated complex that binds ssDNA and is required for multiple steps in telomere replication, including termination of G-strand extension by telomerase and synthesis of the complementary C-strand. CST contains seven OB-folds which appear to mediate CST function by modulating CST binding to ssDNA and the ability of CST to recruit or engage partner proteins. However, the mechanism whereby CST achieves its various functions remains unclear. To address the mechanism, we generated a series of CTC1 mutants and studied their effect on CST binding to ssDNA and their ability to rescue CST function in CTC1-/- cells. We identified the OB-B domain as a key determinant of telomerase termination but not C-strand synthesis. CTC1-ΔB expression rescued C-strand fill-in, prevented telomeric DNA damage signaling and growth arrest. However, it caused progressive telomere elongation and the accumulation of telomerase at telomeres, indicating an inability to limit telomerase action. The CTC1-ΔB mutation greatly reduced CST-TPP1 interaction but only modestly affected ssDNA binding. OB-B point mutations also weakened TPP1 association, with the deficiency in TPP1 interaction tracking with an inability to limit telomerase action. Overall, our results indicate that CTC1-TPP1 interaction plays a key role in telomerase termination.

Mutations in the insulator protein Suppressor of Hairy wing induce genome instability.

Insulator proteins orchestrate the three-dimensional organization of the genome. Insulators function by facilitating communications between regulatory sequences and gene promoters, allowing accurate gene transcription regulation during embryo development and cell differentiation. However, the role of insulator proteins beyond genome organization and transcription regulation remains unclear. Suppressor of Hairy wing [Su(Hw)] is a Drosophila insulator protein that plays an important function in female oogenesis. Here we find that su(Hw) has an unsuspected role in genome stability during cell differentiation. We show that su(Hw) mutant developing egg chambers have poorly formed microtubule organization centers (MTOCs) in the germarium and display mislocalization of the anterior/posterior axis specification factor gurken in later oogenesis stages. Additionally, eggshells from partially rescued su(Hw) mutant female germline exhibit dorsoventral patterning defects. These phenotypes are very similar to phenotypes found in the important class of spindle mutants or in piRNA pathway mutants in Drosophila, in which defects generally result from the failure of germ cells to repair DNA damage. Similarities between mutations in su(Hw) and spindle and piRNA mutants are further supported by an excess of DNA damage in nurse cells, and because Gurken localization defects are partially rescued by mutations in the ATR (mei-41) and Chk1 (grapes) DNA damage response genes. Finally, we also show that su(Hw) mutants produce an elevated number of chromosome breaks in dividing neuroblasts from larval brains. Together, these findings suggest that Su(Hw) is necessary for the maintenance of genome integrity during Drosophila development, in both germline and dividing somatic cells.

CTC1-STN1 terminates telomerase while STN1-TEN1 enables C-strand synthesis during telomere replication in colon cancer cells.

Telomerase elongates the telomeric G-strand to prevent telomere shortening through conventional DNA replication. However, synthesis of the complementary C-strand by DNA polymerase α is also required to maintain telomere length. Polymerase α cannot perform this role without the ssDNA binding complex CST (CTC1-STN1-TEN1). Here we describe the roles of individual CST subunits in telomerase regulation and G-overhang maturation in human colon cancer cells. We show that CTC1-STN1 limits telomerase action to prevent G-overhang overextension. CTC1-/- cells exhibit telomeric DNA damage and growth arrest due to overhang elongation whereas TEN1-/- cells do not. However, TEN1 is essential for C-strand synthesis and TEN1-/- cells exhibit progressive telomere shortening. DNA binding analysis indicates that CTC1-STN1 retains affinity for ssDNA but TEN1 stabilizes binding. We propose CTC1-STN1 binding is sufficient to terminate telomerase action but altered DNA binding dynamics renders CTC1-STN1 unable to properly engage polymerase α on the overhang for C-strand synthesis.

CTC1-mediated C-strand fill-in is an essential step in telomere length maintenance.

To prevent progressive telomere shortening as a result of conventional DNA replication, new telomeric DNA must be added onto the chromosome end. The de novo DNA synthesis involves elongation of the G-rich strand of the telomere by telomerase. In human cells, the CST complex (CTC1-STN1-TEN1) also functions in telomere replication. CST first aids in duplication of the telomeric dsDNA. Then after telomerase has extended the G-rich strand, CST facilitates fill-in synthesis of the complementary C-strand. Here, we analyze telomere structure after disruption of human CTC1 and demonstrate that functional CST is essential for telomere length maintenance due to its role in mediating C-strand fill-in. Removal of CTC1 results in elongation of the 3΄ overhang on the G-rich strand. This leads to accumulation of RPA and telomeric DNA damage signaling. G-overhang length increases with time after CTC1 disruption and at early times net G-strand growth is apparent, indicating telomerase-mediated G-strand extension. In contrast, C-strand length decreases continuously, indicating a deficiency in C-strand fill-in synthesis. The lack of C-strand maintenance leads to gradual shortening of the telomeric dsDNA, similar to that observed in cells lacking telomerase. Thus, telomerase-mediated G-strand extension and CST-mediated C-strand fill-in are equally important for telomere length maintenance.

The insulator protein Suppressor of Hairy wing is required for proper ring canal development during oogenesis in Drosophila.

Chromatin insulators orchestrate gene transcription during embryo development and cell differentiation by stabilizing interactions between distant genomic sites. Mutations in genes encoding insulator proteins are generally lethal, making in vivo functional analyses of insulator proteins difficult. In Drosophila, however, mutations in the gene encoding the Suppressor of Hairy wing insulator protein [Su(Hw)] are viable and female sterile, providing an opportunity to study insulator function during oocyte development. Whereas previous reports suggest that the function of Su(Hw) in oogenesis is independent of its insulator activity, many aspects of the role of Su(Hw) in Drosophila oogenesis remain unexplored. Here we show that mutations in su(Hw) result in smaller ring canal lumens and smaller outer ring diameters, which likely obstruct molecular and vesicle passage from nurse cells to the oocyte. Fluorescence microscopy reveals that lack of Su(Hw) leads to excess accumulation of Kelch (Kel) and Filament-actin (F-actin) proteins in the ring canal structures of developing egg chambers. Furthermore, we found that misexpression of the Src oncogene at 64B (Src64B) may cause ring canal development defects as microarray analysis and real-time RT-PCR revealed there is a three fold decrease in Src64B expression in su(Hw) mutant ovaries. Restoration of Src64B expression in su(Hw) mutant female germ cells rescued the ring phenotype but did not restore fertility. We conclude that loss of su(Hw) affects expression of many oogenesis related genes and down-regulates Src64B, resulting in ring canal defects potentially contributing to obstruction of molecular flow and an eventual failure of egg chamber organization.

Editing of accD and ndhF chloroplast transcripts is partially affected in the Arabidopsis vanilla cream1 mutant.

The vanilla cream1 (vac1) albino mutant is defective in a gene encoding a chloroplast-localized pentatricopeptide repeat protein of the DYW subgroup. However, the carboxyl-terminal DYW motif is truncated in VAC1. To identify vac1-specific phenotypes, we compared 34 chloroplast RNA editing sites and approximately 90 chloroplast gene expression patterns among wild type, vac1 and another albino mutant ispH, which is defective in the plastid isoprenoid biosynthesis pathway. We found that the editing of accD and ndhF transcripts is partially affected in vac1. In addition, steady-state levels of chloroplast rRNAs are significantly decreased in vac1. The expression of plastid-encoded RNA polymerase transcribed genes is down-regulated, whereas the expression of nucleus-encoded RNA polymerase transcribed genes is up-regulated in vac1. Although the development and function of mutant chloroplasts are severely impaired, steady-state mRNA levels of nucleus-encoded photosynthetic genes are not affected or are only slightly decreased in vac1. The ZAT10 gene encodes a transcription factor and its expression is down-regulated by norflurazon treatment in wild type. This norflurazon effect was not observed in vac1. These results suggest that the VAC1 protein may be involved in plastid-to-nucleus retrograde signaling in addition to its role in chloroplast RNA editing and gene expression. A defect in a key biosynthetic pathway can have many indirect effects on chloroplast gene expression as is seen in the ispH mutant. Similarly, the vac1 mutant has pleiotropic molecular phenotypes and most of which may be indirect effects.

Changes in chromatin structure correlate with transcriptional activity of nucleolar rDNA in polytene chromosomes.

Ribosomal DNA genes (rDNA) are found in tandem arrays of hundreds of repeated genes, but only a fraction of these genes are actively transcribed. The regulatory mechanism controlling the transition between active and inactive rDNA in higher eukaryotes is vital for cell survival. Here, we show that the nucleolus from Drosophila salivary gland cells contains two levels of chromatin organization reflecting differences in transcriptional activity: Decondensed chromatin is highly occupied with TATA-box-binding protein (TBP), phosphorylated H3S10, and acetylated H3K14, suggesting that rDNA in decondensed nucleolar areas is actively transcribed. Condensed chromatin lacks TBP, phosphorylated H3S10, or acetylated H3K14 and is enriched in the rDNA retrotransposons R1 and R2. The data show that R1 and R2 retrotransposons are not actively transcribed in salivary glands and may lead to the epigenetic silencing of flanking rDNA genes and that the silencing mechanisms of these sequences might be partially independent of heterochromatin formation by methylation of histone H3 at lysine 9 and binding of heterochromatin protein 1.

Chloroplast localization of methylerythritol 4-phosphate pathway enzymes and regulation of mitochondrial genes in ispD and ispE albino mutants in Arabidopsis.

Plant isoprenoids are derived from two independent pathways, the cytosolic mevalonate pathway and the plastid methylerythritol 4-phosphate (MEP) pathway. We used green fluorescent fusion protein assays to demonstrate that the Arabidopsis MEP pathway enzymes are localized to the chloroplast. We have also characterized three Arabidopsis albino mutants, ispD-1, ispD-2 and ispE-1, which have T-DNA insertions in the IspD and IspE genes of the MEP pathway. Levels of photosynthetic pigments are almost undetectable in these albino mutants. Instead of thylakoids, the ispD and ispE mutant chloroplasts are filled with large vesicles. Impairments in chloroplast development and functions may signal changes in the expression of nuclear, chloroplast and mitochondrial genes. We used northern blot analysis to examine the expression of photosynthetic and respiratory genes in the ispD and ispE albino mutants. Steady-state mRNA levels of nucleus- and chloroplast-encoded photosynthetic genes are significantly decreased in the albino mutants. In contrast, transcript levels of nuclear and mitochondrial genes encoding subunits of the mitochondrial electron transport chain are increased or not affected in these mutants. Genomic Southern blot analysis revealed that the DNA amounts of mitochondrial genes are not enhanced in the ispD and ispE albino mutants. These results support the notion that the functional state of chloroplasts may affect the expression of nuclear and mitochondrial genes. The up-regulation of mitochondrial genes in the albino mutants is not caused by changes of mitochondrial DNA copy number in Arabidopsis.

Expression and regulation of retinoid-inducible gene 1 (RIG1) in breast cancer.

BACKGROUND: Retinoid-inducible gene I (RIG1) is a growth regulator protein that exhibits activities to suppress cellular growth and induce cellular differentiation and apoptosis. This study analyzed the expression and regulation of RIG1 in breast cancer cells in vitro and in vivo. MATERIALS AND METHODS: Expression of RIG1 RNA in breast cancer tissues was analyzed using RNA in situ hybridization. Regulation of RIG1 expression by 17beta-estradiol (E2) was analyzed by semi-quantitative reverse transcription polymerase chain reaction. RESULTS: RIG1 expression in 47 breast cancer tissues was detected mostly in the cytoplasm and in some nuclei. Levels of both cytoplasmic and nuclear RIG1 mRNA were significantly lower in 20 estrogen receptor-positive (ER+) than in 27 ER-negative (ER-) tissues (p < 0.05), in 20 progesterone receptor-positive (PR+) than in 27 PR-negative (PR-) tissues (p < 0.01), and in 14 ER+/PR+ than in 21 ER-/PR-tissues (p < 0.05). Basal levels of RIG1 and ER mRNA were inversely related between ER+ (MCF-7 WS8 and ZR75-1) and ER- (ZR-75-30) breast cancer cells. E2 (1 nM) treatment for two days suppressed RIG1 mRNA levels in MCF-7 WS8 and ZR-75-1 cells, but not in the ER- ZR-75-30 cells. The E2-mediated down-regulation of RIG1 expression was time- and concentration-dependent in ZR-75-1 cells. CONCLUSION: The negative association between RIG1 and ER expression in breast cancer tissues and down-regulation of RIG1 by E2 in breast cancer cells in vitro suggest that RIG1 expression is negatively regulated by E2 through activation of the ER in ER+ breast cancer cells.