1. Taz1 Enforces Cell-Cycle Regulation of Telomere Synthesis
Pierre-Marie Dehé, Ofer Rog, Miguel Godinho Ferreira, Jessica Greenwood, Julia Promisel Cooper 
Molecular Cell 
Volume 46, Issue 6, Pages (June 2012)
DOI: /j.molcel
Copyright © 2012 Elsevier Inc. Terms and Conditions

2. Figure 1 taz1Δ and rap1Δ Telomeres Recruit High Levels of Trt1
(A) Asynchronous cultures of WT, taz1Δ and rap1Δ cells carrying endogenously tagged Trt1-V5 were fixed and viewed by IF using anti-V5 and anti-Taz1 antibodies. DNA was stained with DAPI (blue in Merge). Quantitation is shown in Figure S1.
(B) Schematic representation of telomere showing relative positions of PCR primers and dot blot probe (TELO) for ChIP.
(C) Levels of telomeric enrichment in Trt1-V5 IPs derived from asynchronously growing cells analyzed by ChIP followed by dot blot Southern analysis with the TELO probe. Dot blots are quantified above as the ratio of signal intensity in immunoprecipitated fragments (IP) to that in the whole cell extract (WCE). Error bars here and in (F) represent standard deviations of at least two biological repeats.
(D) Levels of telomeric enrichment in Trt1-V5 IPs derived from synchronized cdc25-22 cultures quantified by PCR using the subTELO primers as described in Experimental Procedures. A repeat of this experiment (with fewer time points) yielded identical results.
(E) Dot blot analysis of telomeric enrichment in Trt1-V5 IPs derived from cdc25-22 synchronized cells.
(F) Quantitation of (E). Figure S5 shows the WT and rap1Δ data on an expanded scale.
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3. Figure 2
Telomerase Is Present and Active at Telomeres in G1 Arrested Cells Lacking Taz1
(A) G1 arrest induced by nitrogen starvation was confirmed by FACS analysis.
(B) Levels of telomeric DNA in Trt1-V5 IPs of G1-arrested cells were analyzed by ChIP followed by dot blot analysis using a telomeric probe. Error bars correspond to the standard deviations of two to three biological repeats.
(C) Southern blot analysis of ApaI-digested genomic DNA from asynchronous log-phase cultures (Log lanes A) and G1 arrested cells (-N lanes B–D), probed with TELO. Quantitation of this data is shown in Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 Est1 Recruitment Increases at taz1Δ and rap1Δ Telomeres
(A) Levels of telomeric DNA in Est1 IPs analyzed by ChIP and dot blot analysis as for Trt1 IPs in Figure 1C. Error bars correspond to the standard deviations of two to three biological repeats.
(B) Levels of telomeric DNA in Est1 IPs of cdc25-22 synchronized cells analyzed by ChIP followed by qPCR using subTELO primers (1B). Error bars here and in (D) represent standard deviations of at least two biological repeats.
(C) Levels of telomere bound Trt1 were analyzed by ChIP and dot blot in cdc25-22 synchronized Trt1-V5 cells.
(D) Quantitation of the data shown in (C), expressed as percentage of the TELO signal in ChIP versus whole cell extract (WCE). Figure S5 shows this data on an expanded scale that allows comparison of WT and rap1Δ cells.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Synchronous C−Strand Resection Occurs in WT and rap1Δ Cells while It Is Deregulated in taz1Δ Cells
(A) Southern blot analysis of ApaI digested genomic DNA from cdc25-22 synchronized cells. An internal genomic 1.5kb ApaI fragment is used as a loading control (bottom panels). The dark gray vertical bar (top right) indicates slowly migrating species representing replication intermediates. Quantitation of these data is shown in Figure S8A.
(B) In-gel hybridization of EcoRV digested genomic DNA from cdc25-22 synchronized taz1Δ and rap1Δ cells to a C-rich telomeric oligonucleotide in native and denaturing conditions. The red vertical bars (left) indicate ssDNA overhangs and dark gray vertical bar (right) indicates replication intermediates. Quantitation of these data is shown in Figure S8B.
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6. Figure 5
taz1Δ Telomeres Replicate throughout S Phase while WT and rap1Δ Telomeres Replicate Late
(A) Representative cell-cycle profile for cdc25-22 arrest and release. Cells were grown to log phase at permissive temperature (25°C) and shifted to restrictive temperature (36°C) for 3 hr, inducing G2/M arrest. Cells were released in rich medium (YES) for the experiments shown in Figures 1, 3, and 4, and in YES supplemented with BrdU for the experiments shown in Figure 5. Anaphase timing and septation indeces were measured using DAPI and calcofluor staining, respectively. The schematic above indicates cell-cycle phase.
(B) Western blot analysis of BrdU substitution in newly replicated DNA using an anti-BrdU antibody.
(C) Schematic representation of chromosome and relative positions of primers used to analyze replication in BrdU ChIPs at the early origin ars2004 (gray) and subtelomeres (black).
(D) BrdU incorporation at subtelomeres and ars2004 was quantified by qPCR of BrdU ChIP samples. The ratio of ChIP signal in the subtelomere versus ars2004 (in the same sample) is plotted over time.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Telomerase Approaches the Telomere-Subtelomere Boundary at taz1Δ Telomeres, despite Their Excessive Lengths, and Taz1 Overexpression Suppresses rap1Δ Telomere Overelongation
(A) Schematic showing how the sonication step of ChIP protocols will separate telomere end-binding proteins from the subtelomere at long, but not short, telomeres.
(B) Southern blot of genomic DNA digested with ApaI or sonicated and probed with a telomeric probe.
(C) Level of telomere bound Trt1-V5 analyzed by ChIP in asynchronously growing strains lacking (WT) or harbouring the Trt1-V5 tag. p values derived from a Student's t test based on four independent experiments are shown above the corresponding bars. While the distribution of Trt1-V5 enrichment values is statically different from the untagged (p < 0.05), there is no significant difference between WT and rap1Δ strains (p > 0.05). Error bars correspond to the standard deviations of two to three biological repeats.
(D) rap1Δ cells harboring the taz1+ gene under control of an inducible promoter (nmt1 or nmt41, see text; both are repressed by thiamine and induced in its absence). DNA was harvested from cells grown in the presence or absence of thiamine, digested with ApaI and subjected to Southern blot analysis using a telomere probe.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
Different Mechanisms of taz1Δ and rap1Δ Telomere Overelongation
Top panel: WT telomeres replicate late in S phase (a) and undergo synchronous C-strand resection (b). Telomerase is recruited specifically to telomeres requiring elongation, and its processivity is controlled by Taz1/Rap1 (c). Middle panel: taz1Δ cells lose temporal and spatial control of telomere replication. taz1Δ telomeres undergo early S phase replication onset and suffer replication fork stalling (a). A speculative model (b) for processing of the stalled fork (see text): Isomerization of telomeric replication intermediate to form chicken foot structure, which is in turn subjected to nucleolytic digestion, resulting in 3′ ss overhang generation. This processing creates a substrate for telomerase (c). This structure can also be acted on by the recombination machinery (Rog et al., 2009). Lower panel: rap1Δ telomeres cannot be fully covered by Taz1; therefore, they lose telomere length homeostasis. rap1Δ telomeres contain an excess of Taz1 binding sites, resulting in a partially Taz1-deficient status. Hence, the ends of rap1Δ telomeres behave in a taz1Δ-like manner.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions