Mireille Tittel-Elmer

The University of Calgary | HBI · Department of Biochemistry and Molecular Biology

Eukaryotic cells have evolved a replication stress response that helps to overcome stalled/collapsed replication forks and ensure proper DNA replication. The replication checkpoint protein Mrc1 plays important roles in these processes, although its functional interactions are not fully understood. Here, we show that MRC1 negatively interacts with C...

Mre11-Rad50-Xrs2 (MRX) is a highly conserved complex with key roles in various aspects of DNA repair. Here, we report a new function for MRX in limiting transcription in budding yeast. We show that MRX interacts physically and colocalizes on chromatin with the transcriptional co-regulator Mediator. MRX restricts transcription of coding and noncodin...

Eukaryotic chromosomes are replicated in interphase and the two newly duplicated sister chromatids are held together by the cohesin complex and several cohesin auxiliary factors. Sister chromatid cohesion is essential for accurate chromosome segregation during mitosis, yet has also been implicated in other processes, including DNA damage repair, tr...

SMC proteins constitute the core members of the Smc5/6, cohesin and condensin complexes. We demonstrate that Smc5/6 is present at telomeres throughout the cell cycle and its association with chromosome ends is dependent on Nse3, a subcomponent of the complex. Cells harboring a temperature sensitive mutant, nse3-1, are defective in Smc5/6 localizati...

Summary of mutant phenotypes. (PDF)

Sir4 Sumoylation in mutant backgrounds. Sumoylated proteins were isolated by Ni-NTA affinity purification of His-Smt3 as described previously [48, 57, 80, 85] followed by western blotting with αMyc antibodies to visualize sumoylated proteins in cells containing Myc-tagged Sir4 with un-tagged Smt3 wild type (JC3433), or His8-tagged Smt3 in wild type...

Strains used in this study. (PDF)

TERRA expression and telomeres length in smc6-9 mutants. (A and B) TERRA expression was determined by RT-qPCR for Tel1R and Tel6R, X only telomeres, at 28C (A) and 34C (B). Statistical significance with p values < .05 (*) or < .01(**) are reported from a two-tailed t-test. (C) Telomere length was determined as in Fig 1F by Southern blot analysis on...

qPCR primers used in this study. (PDF)

ChIP performed on Rap1Myc and Rif1Myc and in non-tagged (nt) strains. ChIP was perform with Chromatin immunoprecipitation (ChIP) was performed on (A) Rap1Myc in wild type (JC2381) and nse3-1 (JC3272), (B) Rif1Myc in wild type (JC3277) and nse3-1 (JC3295), (C) α Myc in non-tagged wild type (JC470) and nse3-1 (JC3607) cells and (D) α FLAG in non-tagg...

Rif1, Rif2 and Smc6 recruitment at native telomeres in various mutant cells. (A) Yeast-two Hybrid analysis was performed as previously described [48]. NSE3 full-length, nse3(1–150)—N-terminal end, nse3(150–300)—C-terminal end, or the nse3-1 mutant were cloned into bait plasmid (pEG202) and RIF2 into prey plasmid (pJG4-6) [86]. Plasmids containing b...

TERRA expression levels in rif2Δ and nse3-1 mutants. (A and B) TERRA expression was determined for Y’ at 28°C and 34°C in wild type (JC470), nse3-1 (JC3607), rif2Δ (JC2992), nse3-1 rif2Δ (JC3269), sir4Δ (JC3737), nse3-1 sir4Δ (JC3741), and sir4Δ rif2Δ (JC3738). Statistical significance with p values < .05 (*) or < .01 (**) are reported from a two-t...

ChIP of yKu70 at telomeres in nse3-1 mutant and wild type cells. Chromatin immunoprecipitation (ChIP) was performed on yKu70Myc in wild type (JC1352) and nse3-1 (JC3392). The enrichment at three native subtelomeres (Tel1L, Tel6R and Tel15L) normalized to the negative control region as described in Fig 1B. (TIFF)

The nse3-1 mutants do not synchronize properly, however components of the Smc5/6 complex still interact. (A) Flow cytometry was performed as described in Fig 1. (B) The fold enrichment levels are relative to the late-replicating control region on Chr V for n = 3 experiments with the mean ± SD at the silent mating type locus (HMR) and two regions in...

TPE measurements from the URA3 reporter at Telomere VII L. TPE was determined in strains with the URA3 reporter at the adh4 locus of Chromosome VIIL. Overnight cultures were spotted onto SC (complete medium) and SC + .1% 5-FOA plates and photographed after incubation at 25C and 34C in wild type (JC1991), sir4Δ (JC3818), nse3-1(JC3860), nse3-1 sir4Δ...

Transcription at sub-telomeric genes in smc6-9 mutants. Levels of transcription were compared at sub-telomeric genes CHA1, VAC17 and YR043C as described in Fig 4 in wild type (JC470), sir4Δ (JC3737), smc6-9 (JC3039), and sir4Δ smc6-9 (JC3925). Expression values are mRNA levels relative to ACT1 and normalization to wild type cells. Error bars repres...

The nse3-1 allele, but not the smc6-9 allele shortens the long telomeres in cells lacking RIF2. Telomere length is determined for the indicated strains by performing southern blot analysis using radiolabeled poly GT/CA probe as explained in Fig 1F and in the experimental procedures section for wild type (JC470), rif2Δ (JC2992), smc6-9 (JC3039), and...

Comparison of ChIP levels for Smc6 at telomeres in sir4Δ and nse3-1 mutants and wild type cells. (A) Chromatin immunoprecipitation (ChIP) on Smc6FLAG in wild type (JC1594) and nse3-1 (JC2630) at 25°C. (B) ChIP comparison of Smc6FLAG in wild type (JC1594), sir4Δ (JC3732), nse3-1 (JC2630). The enrichment at three native subtelomeres (Tel1L, Tel6R and...

The cohesin complex holds together newly replicated chromatids and is involved in diverse pathways that preserve genome integrity. We show that in budding yeast, cohesin is transiently recruited to active replication origins, and it spreads along DNA as forks progress. When DNA synthesis is impeded, cohesin accumulates at replication sites and is c...

List of genes significantly upregulated (greater than twofold, P<0.01) in ITS versus CTS. The comparison of transcript levels between ITS+2d and CTS+2d for the corresponding genes is also presented. (0.38 MB XLS)

Impact of ITS on histone post-translational modifications. (a) Input and mock controls of ChIP analysis of MULE F19G14, 106B repeats and 5S repeats using antibodies specific for (b) H3K4me3 and H3K9ac-K14ac , which are associated with active transcription, and for (c) H3K9me2, H3K27me3 and H3K27me2, which are associated with repressed transcription...

List of primers used for RT-PCR and/or chromatin immunoprecipitation analysis. (0.03 MB DOC)

Genome-wide analysis of ITS-induced transcriptional changes at transposons. The upper plots show the relative densities of repeats (blue lines) and DNA methylation (green lines) along the 5 chromosomes of Arabidopsis. Graphs show the chromosome-wide distribution and variation in transcript abundance (Log2 scale) of transposons (grouped by superfami...

List of transposable elements significantly upregulated (greater than fourfold, P<0.01) in ITS versus CTS. Transposons for which transcripts still over-accumulate in ITS+2d compared with CTS+2d, are listed at the end of the Table. (0.11 MB XLS)

Plants of the Zürich ecotype display a better fitness following ITS than plants of the Columbia ecotype. (a) Wild-type seedlings of the Columbia (WT-Col-0, left) and the Zürich ecotypes (WT-Zh, right) grown in vitro under the indicated conditions. (b) Enlargement of plates shown in (a). Unlike WT-Zh, some WT-Col-0 seedlings did not survive the ITS...

Tiling Array data and RT-PCR validation. (a) Relative accumulation transcripts from selected loci (MULE F19G14, ROS1, DML2 and SDC) comparing ITS and CTS from the tiling array data. (b) RT-PCR validation of the tiling data of the slightly differentially expressed targets, ROS1, DML2 and SDC, after ITS compared with CTS; amplification of 18S rRNA wa...

List of genes significantly downregulated (greater than twofold, P<0.01) in ITS versus CTS. The comparison of transcript levels between ITS+2d and CTS+2d for the corresponding genes is also presented. (0.50 MB XLS)

List of transposable elements significantly downregulated (greater than fourfold, P<0.01) in ITS versus CTS. (0.04 MB XLS)

Author Summary In eukaryotic cells, DNA is packaged into chromatin that is present in two different forms named euchromatin and heterochromatin. Gene-rich euchromatin is relaxed and permissive to transcription compared with heterochromatin that essentially contains transcriptionally inert non-coding repeated DNA. The silent state associated with he...

The Mre11-Rad50-Xrs2 (MRX) complex has an important function in the maintenance of genomic integrity by contributing to the detection and repair of chromosome breaks. Here we show that the complex is recruited to sites of paused forks where it stabilizes the association of essential replisome components. Interestingly, this function is not dependen...

All developmental transitions throughout the life cycle of a plant are influenced by light. In Arabidopsis, multiple photoreceptors including the UV-A/blue-sensing cryptochromes (cry1-2) and the red/far-red responsive phytochromes (phyA-E) monitor the ambient light conditions. Light-regulated protein stability is a major control point of photomorph...