Telomere-associated proteins in Arabidopsis thaliana

Telomeres comprise the physical ends of chromosomes. Essential functions of telomeres include protecting the terminus from being recognized as a DNA doublestrand break and facilitating the complete replication of the physical end of the DNA. Telomere functions are mediated by a large array of telomere-associated proteins. Mutations in telomere-related genes cause immediate telomere dysfunction, activation of DNA damage response, and accumulation of end-to-end chromosome fusions. In addition, changes in telomere complex composition may affect the ability of the telomerase enzyme to maintain telomeres in vivo. Here, we describe the characterization of telomere-associated proteins in the flowering plant, Arabidopsis thaliana. Using a bioinformatics approach, we identified twelve proteins with sequence similarity to vertebrate duplex telomere DNA binding proteins TRF1 and TRF2. We showed that, like their vertebrate counterparts, some of the Arabidopsis TRFL (TRF-LIKE) proteins can homodimerize and bind telomeric DNA in vitro, indicating that Arabidopsis encodes a large family of double-strand telomeric DNA binding proteins. We have also characterized three Arabidopsis POT1 proteins whose homologs in yeast and vertebrates associate with the single-stranded portion of telomeric DNA. Unexpectedly, we found that unlike POT1 protein in other organisms, Arabidopsis AtPOT1a protein associates with telomeres only in the S phase of the cell cycle and is a physical component of the active telomerase RNP complex, providing positive telomere length regulation. Our data implicated AtPOT1b, another Arabidopsis POT1 protein, in chromosome end protection. Finally, we showed that Arabidopsis thaliana has evolved a third POT1 protein, AtPOT1c, which contributes to both telomere length regulation and telomerase activity, and maintenance of the structure of the chromosome terminus. Thus, Arabidopsis has evolved a set of POT1 proteins that make distinct and novel contributions to telomere biology. Finally, we describe the identification and characterization of a novel Arabidopsis protein CIT1 (Critical for Integrity of Telomeres 1), and show that CIT1 deficiency leads to an immediate and profound telomere dysfunction and chromosome end deprotection. Altogether, these data provide new insight into plant telomereassociated factors and significantly improve our understanding of the overall architecture and evolution of telomeric complex in Arabidopsis

STN1 protects chromosome ends in Arabidopsis thaliana

Telomeres shield the natural ends of chromosomes from nucleolytic attack, recognition as double-strand breaks, and inappropriate processing by DNA repair machinery. The trimeric Stn1/Ten1/Cdc13 complex is critical for chromosome end protection in Saccharomyces cerevisiae, while vertebrate telomeres are protected by shelterin, a complex of six proteins that does not include STN1 or TEN1. Recent studies demonstrate that Stn1 and Ten1 orthologs in Schizosaccharomyces pombe contribute to telomere integrity in a complex that is distinct from the shelterin components, Pot1 and Tpp1. Thus, chromosome-end protection may be mediated by distinct subcomplexes of telomere proteins. Here we report the identification of a STN1 gene in Arabidopsis that is essential for chromosome-end protection. AtSTN1 encodes an 18-kDa protein bearing a single oligonucleotide/oligosaccharide binding fold with significant sequence similarity to the yeast Stn1 proteins. Plants null for AtSTN1 display an immediate onset of growth and developmental defects and reduced fertility. These outward phenotypes are accompanied by catastrophic loss of telomeric and subtelomeric DNA, high levels of end-to-end chromosome fusions, increased G-overhang signals, and elevated telomere recombination. Thus, AtSTN1 is a crucial component of the protective telomere cap in Arabidopsis, and likely in other multicellular eukaryotes

The Arabidopsis Pot1 and Pot2 Proteins Function in Telomere Length Homeostasis and Chromosome End Protection

Pot1 (protection of telomeres 1) is a single-stranded telomere binding protein that is essential for chromosome end protection and telomere length homeostasis. Arabidopsis encodes two Pot1-like proteins, dubbed AtPot1 and AtPot2. Here we show that telomeres in transgenic plants expressing a truncated AtPot1 allele lacking the N-terminal oligonucleotide/oligosaccharide binding fold (P1ΔN) are 1 to 1.5 kb shorter than in the wild type, suggesting that AtPot1 contributes to the positive regulation of telomere length control. In contrast, telomere length is unperturbed in plants expressing the analogous region of AtPot2. A strikingly different phenotype is observed in plants overexpressing the AtPot2 N terminus (P2ΔC) but not the corresponding region in AtPot1. Although bulk telomeres in P2ΔC mutants are 1 to 2 kb shorter than in the wild type, these plants resemble late-generation telomerase-deficient mutants with severe growth defects, sterility, and massive genome instability, including bridged chromosomes and aneuploidy. The genome instability associated with P2ΔC mutants implies that AtPot2 contributes to chromosome end protection. Thus, Arabidopsis has evolved two Pot genes that function differently in telomere biology. These findings provide unanticipated information about the evolution of single-stranded telomere binding proteins

A C-terminal Myb extension domain defines a novel family of double-strand telomeric DNA-binding proteins in Arabidopsis

Little is known about the protein composition of plant telomeres. We queried the Arabidopsis thaliana genome data base in search of genes with similarity to the human telomere proteins hTRF1 and hTRF2. hTRF1/hTRF2 are distinguished by the presence of a single Myb-like domain in their C terminus that is required for telomeric DNA binding in vitro. Twelve Arabidopsis genes fitting this criterion, dubbed TRF-like (TRFL), fell into two distinct gene families. Notably, TRFL family 1 possessed a highly conserved region C-terminal to the Myb domain called Myb-extension (Myb-ext) that is absent in TRFL family 2 and hTRF1/hTRF2. Immunoprecipitation experiments revealed that recombinant proteins from TRFL family 1, but not those from family 2, formed homodimers and heterodimers in vitro. DNA binding studies with isolated C-terminal fragments from TRFL family 1 proteins, but not family 2, showed specific binding to double-stranded plant telomeric DNA in vitro. Removal of the Myb-ext domain from TRFL1, a family 1 member, abolished DNA binding. However, when the Myb-ext domain was introduced into the corresponding region in TRFL3, a family 2 member, telomeric DNA binding was observed. Thus, Myb-ext is required for binding plant telomeric DNA and defines a novel class of proteins in Arabidopsis

Arabidopsis POT1 associates with the telomerase RNP and is required for telomere maintenance

POT1 is a single-copy gene in yeast and humans that encodes a single-strand telomere binding protein required for chromosome end protection and telomere length regulation. In contrast, Arabidopsis harbors multiple, divergent POT-like genes that bear signature N-terminal OB-fold motifs, but otherwise share limited sequence similarity. Here, we report that plants null for AtPOT1 show no telomere deprotection phenotype, but rather exhibit progressive loss of telomeric DNA. Genetic analysis indicates that AtPOT1 acts in the same pathway as telomerase. In vitro levels of telomerase activity in pot1 mutants are significantly reduced and are more variable than wild-type. Consistent with this observation, AtPOT1 physically associates with active telomerase particles. Although low levels of AtPOT1 can be detected at telomeres in unsynchronized cells and in cells arrested in G2, AtPOT1 binding is significantly enhanced during S-phase, when telomerase is thought to act at telomeres. Our findings indicate that AtPOT1 is a novel accessory factor for telomerase required for positive telomere length regulation, and they underscore the coordinate and extraordinarily rapid evolution of telomere proteins and the telomerase enzyme

A high-throughput assay for directly monitoring nucleolar rRNA biogenesis

Studies of the regulation of nucleolar function are critical for ascertaining clearer insights into the basic biological underpinnings of ribosome biogenesis (RB), and for future development of therapeutics to treat cancer and ribosomopathies. A number of high-throughput primary assays based on morphological alterations of the nucleolus can indirectly identify hits affecting RB. However, there is a need for a more direct high-throughput assay for a nucleolar function to further evaluate hits. Previous reports have monitored nucleolar rRNA biogenesis using 5-ethynyl uridine (5-EU) in low-throughput. We report a miniaturized, high-throughput 5-EU assay that enables specific calculation of nucleolar rRNA biogenesis inhibition, based on co-staining of the nucleolar protein fibrillarin (FBL). The assay uses two siRNA controls: a negative non-targeting siRNA control and a positive siRNA control targeting RNA Polymerase 1 (RNAP1; POLR1A), and specifically quantifies median 5-EU signal within nucleoli. Maximum nuclear 5-EU signal can also be used to monitor the effects of putative small-molecule inhibitors of RNAP1, like BMH-21, or other treatment conditions that cause FBL dispersion. We validate the 5-EU assay on 68 predominately nucleolar hits from a high-throughput primary screen, showing that 58/68 hits significantly inhibit nucleolar rRNA biogenesis. Our new method establishes direct quantification of nucleolar function in high-throughput, facilitating closer study of RB in health and disease

Diverse Regulators of Human Ribosome Biogenesis Discovered by Changes in Nucleolar Number

Summary: Ribosome biogenesis is a highly regulated, essential cellular process. Although studies in yeast have established some of the biological principles of ribosome biogenesis, many of the intricacies of its regulation in higher eukaryotes remain unknown. To understand how ribosome biogenesis is globally integrated in human cells, we conducted a genome-wide siRNA screen for regulators of nucleolar number. We found 139 proteins whose depletion changed the number of nucleoli per nucleus from 2–3 to only 1 in human MCF10A cells. Follow-up analyses on 20 hits found many (90%) to be essential for the nucleolar functions of rDNA transcription (7), pre-ribosomal RNA (pre-rRNA) processing (16), and/or global protein synthesis (14). This genome-wide analysis exploits the relationship between nucleolar number and function to discover diverse cellular pathways that regulate the making of ribosomes and paves the way for further exploration of the links between ribosome biogenesis and human disease

Phenotype of bone-marrow mononuclear cells before and after short-time precondition with erythropoietin from patients with ischemic heart failure

Aim ― To reveal the results of the condition of the bone marrow mononuclear cells (BM-MNCs) with erythropoietin (Epo). Material and Methods ― There were 30-ty patients with coronary artery disease (CAD) enrolled in this study during 2016-2017 years. 95% were men. All were in angina NYHA class II-III. Hypertension presented in 90%, peripheral atherosclerosis in 60%. BM-MNCs were obtained by centrifugation of bone marrow aspirate on Ficoll-Paque, washed, and then preconditioned with Epo. Phenotype, cell cycle, cell death, proliferation, migration, and tube formation before, and after precondition BM-MNCs with Epo were done. Results ― In this study, we observed the presence in cellular graft of the hematopoietic stem cells (HSCs), and endothelial progenitor cells (EPCs) at the different stage of maturation/differentiation, and mesenchymal stem cells (MSCs). Precondition BM-MNCs with Epo increased number of HSCs carrying erythropoietin receptor (EpoR), and EPCs carrying CD184. Also, Epo detained СВ34+ cells in a rest phase of cell cycle (G0G1). Condition media from BM-MNCs treated with Epo augment tube formation and wound healing by EA.hy 929. Conclusion ― Epo in vitro increased number of the stem cells carrying EpoR and CD184, and increased accumulation of CD34+ cell in G0G1 phase of cell cycle, and induced production of proangiogenic factor by BM-MNCs. Further investigation is needed to assess the type of cytokines produced by BM-MNCs after condition with Epo

Characterization of Cardiac Glycoside Natural Products as Potent Inhibitors of DNA Double-Strand Break Repair by a Whole-Cell Double Immunofluorescence Assay

Small-molecule inhibitors of DNA repair pathways are being intensively investigated as primary and adjuvant chemotherapies. We report the discovery that cardiac glycosides, natural products in clinical use for the treatment of heart failure and atrial arrhythmia, are potent inhibitors of DNA double-strand break (DSB) repair. Our data suggest that cardiac glycosides interact with phosphorylated mediator of DNA damage checkpoint protein 1 (phospho-MDC1) or E3 ubiquitin–protein ligase ring finger protein 8 (RNF8), two factors involved in DSB repair, and inhibit the retention of p53 binding protein 1 (53BP1) at the site of DSBs. These observations provide an explanation for the anticancer activity of this class of compounds, which has remained poorly understood for decades, and provide guidance for their clinical applications. This discovery was enabled by the development of the first high-throughput unbiased cellular assay to identify new small-molecule inhibitors of DSB repair. Our assay is based on the fully automated, time-resolved quantification of phospho-SER139-H2AX (γH2AX) and 53BP1 foci, two factors involved in the DNA damage response network, in cells treated with small molecules and ionizing radiation (IR). This primary assay is supplemented by robust secondary assays that establish lead compound potencies and provide further insights into their mechanisms of action. Although the cardiac glycosides were identified in an evaluation of 2366 small molecules, the assay is envisioned to be adaptable to larger compound libraries. The assay is shown to be compatible with small-molecule DNA cleaving agents, such as bleomycin, neocarzinostatin chromophore, and lomaiviticin A, in place of IR