Evolution of Telomeres in Schizosaccharomyces pombe and Its Possible Relationship to the Diversification of Telomere Binding Proteins

Telomeres of nuclear chromosomes are usually composed of an array of tandemly repeated sequences that are recognized by specific Myb domain containing DNA-binding proteins (telomere-binding proteins, TBPs). Whereas in many eukaryotes the length and sequence of the telomeric repeat is relatively conserved, telomeric sequences in various yeasts are highly variable. Schizosaccharomyces pombe provides an excellent model for investigation of co-evolution of telomeres and TBPs. First, telomeric repeats of S. pombe differ from the canonical mammalian type TTAGGG sequence. Second, S. pombe telomeres exhibit a high degree of intratelomeric heterogeneity. Third, S. pombe contains all types of known TBPs (Rap1p [a version unable to bind DNA], Tay1p/Teb1p, and Taz1p) that are employed by various yeast species to protect their telomeres. With the aim of reconstructing evolutionary paths leading to a separation of roles between Teb1p and Taz1p, we performed a comparative analysis of the DNA-binding properties of both proteins using combined qualitative and quantitative biochemical approaches. Visualization of DNA-protein complexes by electron microscopy revealed qualitative differences of binding of Teb1p and Taz1p to mammalian type and fission yeast telomeres. Fluorescence anisotropy analysis quantified the binding affinity of Teb1p and Taz1p to three different DNA substrates. Additionally, we carried out electrophoretic mobility shift assays using mammalian type telomeres and native substrates (telomeric repeats, histone-box sequences) as well as their mutated versions. We observed relative DNA sequence binding flexibility of Taz1p and higher binding stringency of Teb1p when both proteins were compared directly to each other. These properties may have driven replacement of Teb1p by Taz1p as the TBP in fission yeast

Evolution of Telomeres in Schizosaccharomyces pombe and Its Possible Relationship to the Diversification of Telomere Binding Proteins.

Telomeres of nuclear chromosomes are usually composed of an array of tandemly repeated sequences that are recognized by specific Myb domain containing DNA-binding proteins (telomere-binding proteins, TBPs). Whereas in many eukaryotes the length and sequence of the telomeric repeat is relatively conserved, telomeric sequences in various yeasts are highly variable. Schizosaccharomyces pombe provides an excellent model for investigation of co-evolution of telomeres and TBPs. First, telomeric repeats of S. pombe differ from the canonical mammalian type TTAGGG sequence. Second, S. pombe telomeres exhibit a high degree of intratelomeric heterogeneity. Third, S. pombe contains all types of known TBPs (Rap1p [a version unable to bind DNA], Tay1p/Teb1p, and Taz1p) that are employed by various yeast species to protect their telomeres. With the aim of reconstructing evolutionary paths leading to a separation of roles between Teb1p and Taz1p, we performed a comparative analysis of the DNA-binding properties of both proteins using combined qualitative and quantitative biochemical approaches. Visualization of DNA-protein complexes by electron microscopy revealed qualitative differences of binding of Teb1p and Taz1p to mammalian type and fission yeast telomeres. Fluorescence anisotropy analysis quantified the binding affinity of Teb1p and Taz1p to three different DNA substrates. Additionally, we carried out electrophoretic mobility shift assays using mammalian type telomeres and native substrates (telomeric repeats, histone-box sequences) as well as their mutated versions. We observed relative DNA sequence binding flexibility of Taz1p and higher binding stringency of Teb1p when both proteins were compared directly to each other. These properties may have driven replacement of Teb1p by Taz1p as the TBP in fission yeast

NF449 Is a Novel Inhibitor of Fibroblast Growth Factor Receptor 3 (FGFR3) Signaling Active in Chondrocytes and Multiple Myeloma Cells*

The FGFR3 receptor tyrosine kinase represents an attractive target for therapy due to its role in several human disorders, including skeletal dysplasias, multiple myeloma, and cervical and bladder carcinomas. By using molecular library screening, we identified a compound named NF449 with inhibitory activity toward FGFR3 signaling. In cultured chondrocytes and murine limb organ culture, NF449 rescued FGFR3-mediated extracellular matrix loss and growth inhibition, which represent two major cellular phenotypes of aberrant FGFR3 signaling in cartilage. Similarly, NF449 antagonized FGFR3 action in the multiple myeloma cell lines OPM2 and KMS11, as evidenced by NF449-mediated reversal of ERK MAPK activation and transcript accumulation of CCL3 and CCL4 chemokines, both of which are induced by FGFR3 activation. In cell-free kinase assays, NF449 inhibited the kinase activity of both wild type and a disease-associated FGFR3 mutant (K650E) in a fashion that appeared non-competitive with ATP. Our data identify NF449 as a novel antagonist of FGFR3 signaling, useful for FGFR3 inhibition alone or in combination with inhibitors that target the ATP binding site

Parameters of binding of Teb1 and Taz1 proteins to <i>S</i>. <i>pombe</i> telomere (SpTEL), mammalian telomeres (HsTEL) and <i>S</i>. <i>pombe</i> histone box (HisBox).

<p>Parameters of binding of Teb1 and Taz1 proteins to <i>S</i>. <i>pombe</i> telomere (SpTEL), mammalian telomeres (HsTEL) and <i>S</i>. <i>pombe</i> histone box (HisBox).</p

Electron microscopic analysis of the binding of Teb1p and Taz1p to model mammalian and fission yeast telomeres.

<p>(A) Schematic representation of the proteins used in all experiments. 6HN-Teb1 was produced in a fusion with GST, purified on glutathione-agarose and GST was removed by cleavage by <i>PreScission</i> Protease (the arrow indicates a position of the protease recognition site). Both proteins are represented by their full-length sequences (lacking the first methionine) fused with a 6HN tag at the N-terminus. The numbers below the rectangles indicate the range of amino acids from Teb1p and Taz1p present in the recombinant proteins. C, two cysteine residues located in Myb-1 (M1) and Myb-2 (M2) domain of Teb1p, respectively, that may be involved in mediating inhibitory effect of reducing agents on binding of Teb1p to SpTEL (see below). Note that Taz1p contains a single Myb domain (M) a it carries nine cysteine residues (not shown) and in contrast to Teb1p reducing agent is required for efficient DNA-binding. (B) Linearized plasmids carrying either mammalian (HsTEL) or <i>S</i>. <i>pombe</i> (SpTEL) telomeres at one end were incubated with purified Teb1p or Taz1p and the DNA-protein complexes were visualized by EM as described in Materials and Methods.</p

Fluorescence anisotropy analysis of the DNA binding properties of Teb1p and Taz1p.

<p>Fluorescence anisotropy measurements of binding of Teb1p (A) and Taz1p (B) to FAM labeled DNA probes were performed as described in Materials and Methods. SpTEL (closed circle), HsTEL (open square), HisBox (closed triangle).</p

List of putative telomere-binding proteins identified by <i>in silico</i> analysis of the corresponding fungal genomes (see also Fig 1).

<p>List of putative telomere-binding proteins identified by <i>in silico</i> analysis of the corresponding fungal genomes (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154225#pone.0154225.g001" target="_blank">Fig 1</a>).</p

Comparison of the binding of Teb1p and Taz1p to mammalian telomeres, fission yeast telomeres and HisBox using EMSA.

<p>The indicated radioactively labeled DNA probes were incubated with increasing concentrations (indicated above the lanes) of Teb1p or Taz1p and DNA (15 nM) and DNA-protein complexes were separated by electrophoresis in 5% polyacrylamide gels as described in Materials and Methods.</p