Spindle-independent condensation-mediated segregation of yeast ribosomal DNA in late anaphase

Mitotic cell division involves the equal segregation of all chromosomes during anaphase. The presence of ribosomal DNA (rDNA) repeats on the right arm of chromosome XII makes it the longest in the budding yeast genome. Previously, we identified a stage during yeast anaphase when rDNA is stretched across the mother and daughter cells. Here, we show that resolution of sister rDNAs is achieved by unzipping of the locus from its centromere-proximal to centromere-distal regions. We then demonstrate that during this stretched stage sister rDNA arrays are neither compacted nor segregated despite being largely resolved from each other. Surprisingly, we find that rDNA segregation after this period no longer requires spindles but instead involves Cdc14-dependent rDNA axial compaction. These results demonstrate that chromosome resolution is not simply a consequence of compacting chromosome arms and that overall rDNA compaction is necessary to mediate the segregation of the long arm of chromosome XII

Evolutionary origins of apoB mRNA editing: Catalysis by a cytidine deaminase that has acquired a novel RNA-binding motif at its active site

AbstractThe site-specific C to U editing of apolipoprotein B100 (apoB100) mRNA requires a 27 kDa protein (p27) with homology to cytidine deaminase. Here, we show that p27 is a zinc-containing deaminase, which operates catalytically like the E. coli enzyme that acts on monomeric substrate. In contrast with the bacterial enzyme that does not bind RNA, p27 interacts with its polymeric apoB m RNA substrate at AU sequences adjacent to the editing site. This interaction is necessary for editing. RNA binding is mediated through amino acid residues involved in zinc coordination, in proton shuttling, and in forming the αβα structure that encompasses the active site. However, certain mutations that inactivate the enzyme do not affect RNA binding. Thus, RNA binding does not require a catalytically active site. The acquisition of polymeric substrate binding provides a route for the evolution of this editing enzyme from one that acts on monomeric substrates

Sgs1’s roles in DNA end resection, HJ dissolution, and crossover suppression require a two-step SUMO regulation dependent on Smc5/6

The RecQ helicase Sgs1 plays critical roles during DNA repair by homologous recombination, fromend resection to Holliday junction (HJ) dissolution. Sgs1 has both pro- and anti-recombinogenic roles, and therefore its activity must be tightly regulated. However, the controls involved in recruitment and activation of Sgs1 at damaged sites are unknown. Here we show a two-step role for Smc5/6 in recruiting and activating Sgs1 through SUMOylation. First, auto-SUMOylation of Smc5/6 subunits leads to recruitment of Sgs1 as part of the STR (Sgs1–Top3–Rmi1) complex, mediated by two SUMO-interacting motifs (SIMs) on Sgs1 that specifically recognize SUMOylated Smc5/6. Second, Smc5/6-dependent SUMOylation of Sgs1 and Top3 is required for the efficient function of STR. Sgs1 mutants impaired in recognition of SUMOylated Smc5/6 (sgs1-SIMΔ) or SUMO-dead alleles (sgs1-KR) exhibit unprocessed HJs at damaged replication forks, increased crossover frequencies during double-strand break repair, and severe impairment in DNA end resection. Smc5/6 is a key regulator of Sgs1’s recombination functions.We thank the Aragon laboratory for discussions and critical reading of the manuscript.We thank the Clinical Sciences Centre Proteomics Facility (P. Cutillas and P. Faull) for help and advice on our proteomic analysis. Work in J.T.-R.’s laboratory is supported by grants BFU2015-71308-P and BFU2013-50245-EXP from the Spanish Ministry of Economy and Competitivity.Work in the Aragon laboratory was supported by the intramural programme of the Medical Research Council UK and the Wellcome Trust (100955)

Transcription of ribosomal genes can cause nondisjunction

Mitotic disjunction of the repetitive ribosomal DNA (rDNA) involves specialized segregation mechanisms dependent on the conserved phosphatase Cdc14. The reason behind this requirement is unknown. We show that rDNA segregation requires Cdc14 partly because of its physical length but most importantly because a fraction of ribosomal RNA (rRNA) genes are transcribed at very high rates. We show that cells cannot segregate rDNA without Cdc14 unless they undergo genetic rearrangements that reduce rDNA copy number. We then demonstrate that cells with normal length rDNA arrays can segregate rDNA in the absence of Cdc14 as long as rRNA genes are not transcribed. In addition, our study uncovers an unexpected role for the replication barrier protein Fob1 in rDNA segregation that is independent of Cdc14. These findings demonstrate that highly transcribed loci can cause chromosome nondisjunction

Assessing Various Control Samples for Microarray Gene Expression Profiling of Laryngeal Squamous Cell Carcinoma

Selection of optimal control samples is crucial in expression profiling tumor samples. To address this issue, we performed microarray expression profiling of control samples routinely used in head and neck squamous cell carcinoma studies: human bronchial and tracheal epithelial cells, squamous cells obtained by laser uvulopalatoplasty and tumor surgical margins. We compared the results using multidimensional scaling and hierarchical clustering versus tumor samples and laryngeal squamous cell carcinoma cell lines. A general observation from our study is that the analyzed cohorts separated according to two dominant factors: "malignancy", which separated controls from malignant samples and "cell culture-microenvironment" which reflected the differences between cultured and non-cultured samples. In conclusion, we advocate the use of cultured epithelial cells as controls for gene expression profiling of cancer cell lines. In contrast, comparisons of gene expression profiles of cancer cell lines versus surgical margin controls should be treated with caution, whereas fresh frozen surgical margins seem to be appropriate for gene expression profiling of tumor samples.</p

Laryngeal squamous cell carcinoma cell lines show high tolerance for siRNA-mediated CDK1 knockdown

Alterations of the cell cycle checkpoints lead to uncontrolled cell growth and result in tumorigenesis. One of the genes essential for cell proliferation and cell cycle regulation is CDK1. This makes it a potential target in cancer therapy. In our previous study we have shown upregulation of this gene in laryngeal squamous cell carcinoma (LSCC). Here we analyze the impact of siRNA-mediated CDK1 knockdown on cell proliferation and viability, measured with cell growth monitoring and colorimetric test (CCK8 assay), respectively. We proved that a reduction of CDK1 expression by more than 50% has no effect on these cellular processes in LSCC cell lines (n=2). Moreover, using microarrays, we analyzed global gene expression deregulation in these cell lines after CDK1 knockdown. We searched for enriched ontologies in the group of identified 137 differentially expressed genes (>2-fold change). Within this group we found 3 enriched pathways: protein binding (GO:0005515), mitotic nuclear division (GO:0007067) and transmembrane receptor protein tyrosine kinase signaling pathway (GO:0007169) and a group of 11 genes encoding proteins for which interaction with CDK1 was indicated with the use of bioinformatic tools. Among these genes we propose three: CDK6, CALD1 and FYN as potentially dependent on CDK1

Loss of the MAF Transcription Factor in Laryngeal Squamous Cell Carcinoma

MAF is a transcription factor that may act either as a tumor suppressor or as an oncogene, depending on cell type. We have shown previously that the overexpressed miR-1290 influences MAF protein levels in LSCC (laryngeal squamous cell carcinoma) cell lines. In this study, we shed further light on the interaction between miR-1290 and MAF, as well as on cellular MAF protein localization in LSCC. We confirmed the direct interaction between miR-1290 and MAF 3'UTR by a dual-luciferase reporter assay. In addition, we used immunohistochemistry staining to analyze MAF protein distribution and observed loss of MAF nuclear expression in 58% LSCC samples, of which 10% showed complete absence of MAF, compared to nuclear and cytoplasmatic expression in 100% normal mucosa. Using TCGA data, bisulfite pyrosequencing and CNV analysis, we excluded the possibility that loss-of-function mutations, promoter region DNA methylation or CNV are responsible for MAF loss in LSCC. Finally, we identified genes involved in the regulation of apoptosis harboring the MAF binding motif in their promoter region by applied FIMO and DAVID GO analysis. Our results highlight the role of miR-1290 in suppressing MAF expression in LSCC. Furthermore, MAF loss or mislocalization in FFPE LSCC tumor samples might suggest that MAF acts as a LSCC tumor suppressor by regulating apoptosis.</p

Rtt107 Phosphorylation Promotes Localisation to DNA Double-Stranded Breaks (DSBs) and Recombinational Repair between Sister Chromatids

Efficient repair of DNA double-stranded breaks (DSB) requires a coordinated response at the site of lesion. Nucleolytic resection commits repair towards homologous recombination, which preferentially occurs between sister chromatids. DSB resection promotes recruitment of the Mec1 checkpoint kinase to the break. Rtt107 is a target of Mec1 and serves as a scaffold during repair. Rtt107 plays an important role during rescue of damaged replication forks, however whether Rtt107 contributes to the repair of DSBs is unknown. Here we show that Rtt107 is recruited to DSBs induced by the HO endonuclease. Rtt107 phosphorylation by Mec1 and its interaction with the Smc5–Smc6 complex are both required for Rtt107 loading to breaks, while Rtt107 regulators Slx4 and Rtt101 are not. We demonstrate that Rtt107 has an effect on the efficiency of sister chromatid recombination (SCR) and propose that its recruitment to DSBs, together with the Smc5–Smc6 complex is important for repair through the SCR pathway

Cdc14 phosphatase promotes segregation of telomeres through repression of RNA polymerase II transcription

Kinases and phosphatases regulate messenger RNA synthesis through post-translational modification of the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (ref. 1). In yeast, the phosphatase Cdc14 is required for mitotic exit2,3 and for segregation of repetitive regions4. Cdc14 is also a subunit of the silencing complex RENT (refs 5, 6), but no roles in transcriptional repression have been described. Here we report that inactivation of Cdc14 causes silencing defects at the intergenic spacer sequences of ribosomal genes during interphase and at Y′ repeats in subtelomeric regions during mitosis. We show that the role of Cdc14 in silencing is independent of the RENT deacetylase subunit Sir2. Instead, Cdc14 acts directly on RNA polymerase II by targeting CTD phosphorylation at Ser 2 and Ser 5. We also find that the role of Cdc14 as a CTD phosphatase is conserved in humans. Finally, telomere segregation defects in cdc14 mutants4 correlate with the presence of subtelomeric Y′ elements and can be rescued by transcriptional inhibition of RNA polymerase II