Supplementary Material S1

Live Imaging of Telomeres: yKu and Sir Proteins

Define Redundant Telomere-Anchoring Pathways
in Yeast
Florence Hediger, Frank R. Neumann,
Griet Van Houwe, Karine Dubrana,
and Susan M. Gasser

Supplementary References

S1. Gotta, M., Palladino, F., and Gasser, S.M. (1998). Functional
characterization of the N terminus of Sir3p. Mol. Cell. Biol. 18,
6110–6120.
S2. Heun, P., Laroche, T., Shimada, K., Furrer, P., and Gasser, S.M.
(2001). Chromosome dynamics in the yeast interphase nucleus.
Science 294, 2181–2186.

Figure S1. Internal Locus ARS607 Localiza-

tion in wt and Mutant Strains
The strain used carries the lacop insertion 70
kb from the right telomere of Chr VI, at a site
near the early origin, ARS607. The positions
of the ARS607 GFP spot were classified into
three concentric zones of equal surface in
cells from wt, hdf1, and sir4 strains. For each
stage of the cell cycle, G1, early S (eS), and
mid-to-late S phase (mlS), data are repre-
sented in bar graphs as percentages of spot
(y axis) per zone (x axis). The horizontal bar
at 33% corresponds to a random distribution.
The number of cells analyzed and confidence
values (p) for the ␹2 analysis between random
and test distributions are indicated here in
parentheses for each cell cycle stage (G1; eS;
mlS): ARS607::lacop wt (322, p ⫽ 0.21; 61, p ⫽
0.30; 146, p ⫽ 0.18), hdf1 (192, p ⫽ 0.32; 75,
p ⫽ 0.01; 96, p ⫽ 0.38), sir4 (294, p ⫽ 0.53;
75, p ⫽ 0.14; 168, p ⫽ 0.07).
S2

Figure S2. Distribution of Bulk DNA in wt, sir4, and hdf1 Strains
Immunolocalization of nuclear pore (Mab414, green) and DNA (TOTO dye, red) was performed in wt (GA-1459), hdf1 (GA-1489), and sir4 (GA-
1867) strains. The intensity of red and green signals along the line crossing the nucleus is plotted to show the distribution of total DNA within
the nucleus.

Figure S3. Effects of Sir3 and Sir3C Overexpression on Tel VI-R Position
The strain GA-1459 was transformed with pADH-SIR3 (p2␮-ASir3) or pADH-SIR3C (expressing aa 568–978) based on the vector pAAH5 [S1].
Strains were grown on SD-leu-his and were scored for Tel VI-R location as in Figure 1. Wild-type values are from GA-1459 grown on SD-his.
The positions of the Tel VI-R GFP spots were classified into three concentric zones of equal surface for G1 and mid-to-late S phase (mlS).
Data are represented in bar graphs as percentages of spot (y axis) per zone (x axis). The horizontal bar at 33% corresponds to a random
distribution. Repression of a subtelomeric URA3 marker in the presence or absence of each plasmid was assayed by dilution series on 5-FOA
as described in [S1] and confirm a loss of TPE in the presence of pADH-SIR3C and slight enhancement with pADH-SIR3 (p2␮-ASir3). The
number of cells analyzed and the confidence values (p) for the ␹2 analysis between random and test distributions are indicated here in
parentheses for each cell cycle stage (G1; eS; mlS): Tel VI-R::lacop Sir3 o/e (204, p ⫽ 5.3 ⫻ 10⫺17; 60, p ⫽ 1.8 ⫻ 10⫺5; 76, p ⫽ 2.6 ⫻ 10⫺9), Tel
VI-R::lacop Sir3C o/e (188, p ⫽ 7.9 ⫻ 10⫺5; 63, p ⫽ 1.2 ⫻ 10⫺2; 86, p ⫽ 8.6 ⫻ 10⫺2).
S3

Table S1. Yeast Strains Used in This Study

Name Genotype Reference

GA-1320 MATa ade2-1 can1-100 his3-11,15::GFP-LacI-HIS3 trp1-1 ura3-1 leu2-3,112 [33]

nup49::NUP49-GFP-URA3
GA-1459 MATa ade2-1 can1-100 his3-11,15::GFP-LacI-HIS3 trp1-1 ura3-1 leu2-3,112 [35]
nup49::NUP49-GFP-URA3 TELVI-R::lacO-lexAop-TRP1
GA-1985 MATa ade2-1 can1-100 his3-11,15::GFP-LacI-HIS3 trp1-1 ura3-1 leu2-3,112 this study
nup49::NUP49-GFP-URA3 TELXIV-L::lacO-lexA-TRP1
GA-1986 MATa ade2-1 can1-100 his3-11,15::GFP-LacI-HIS3 trp1-1 ura3-1 leu2-3,112 this study
nup49::NUP49-GFP-URA3 TELVIII-L::lacO-lexA-TRP1
GA-1489 GA-1459 hdf1::URA3 this study
GA-1731 GA-1459 mlp1::kanMX6 mlp2::URA3 this study
GA-1867 GA-1459 sir4::kanMX6 this study
GA-1846 GA-1459 sir2::kanMX6 this study
GA-1842 GA-1459 rpd3::LEU2 this study
GA-1844 GA-1459 rpd3::LEU2 sir2::kanMX6 this study
GA-1793 GA-1459 rif1::kanMX6 this study
GA-1794 GA-1459 hdf1::URA3 rif1::kanMX6 this study
GA-1866 GA-1459 hdf1::URA3 rif1::kanMX6 sir3::LEU2 this study
GA-1841 GA-1459 TELVII-L::URA3 this study
GA-1842 GA-1841 rpd3::LEU2 this study
GA-1844 GA-1841 rpd3::LEU2; sir2::kanMX6 this study
GA-1846 GA-1841 sir2::kanMX6 this study
GA-1987 GA-1985 spc42::SPC42-CFP-URA3 this study
GA-1917 GA-1320 TELVI-R::lacO-lexAop-TRP1-//-ADE2-TG this study
GA-1918 GA-1917 hdf1::URA3 this study
GA-2067 GA-1917 sir4::kanMX6 this study
GA-2068 GA-1917 sir4:: kanMX6; hdf1::URA3 this study
GA-1983 GA-1985 hdf1::URA3 this study
GA-2113 GA-1985 sir4::kanMX6 this study
GA-2114 GA-1985 hdf1::URA3 sir4::kanMX6 this study
YG345 hdf1::LEU2 [41]
YG342 hdf1:: LEU2; rif1::HIS3 [41]
YG526 rif1::TRP1 [41]

Figure S4. Localization of Tel VIII-L in wt and hdf1 Strains

The positions of the GFP spots for tagged Tel VIII-L were classified into three concentric zones of equal surface in a wild-type background
and the same with a full hdf1 deletion. For each stage of the cell cycle, G1, early S (eS), and mid-to-late S phase (mlS), data are represented
in bar graphs as percentages of spot (y axis) per zone (x axis). The horizontal bar at 33% corresponds to a random distribution. The fold
enrichment or depletion in comparison to a random distribution is indicated at the bottom of each column. The strain number, number of
cells analyzed, and confidence values (p) for the ␹2 analysis between random and test distributions are indicated here in parentheses for each
cell cycle stage (G1; eS; mlS): Tel VIII-L::lacop wt (GA-1986, 218, p ⫽ 5.2 ⫻ 10⫺6; 35, p ⫽ 9.4 ⫻ 10⫺4; 87, p ⫽ 2.2 ⫻ 10⫺2), hdf1 (GA-1916, 207,
p ⫽ 0.54; 32, p ⫽ 0.22; 61, p ⫽ 0.29).
S4

Movie 1. Tel VI-R::lacop in Wild-Type Cells with Nup49-GFP: G1

Phase

This movie was run at 10 frames/s or 15⫻ real time and were taken
on a Zeiss LSM 510 at 1.5 s/frame by using a 100⫻ Planapo objective
and zoom 2 (90 nm/pixel; [S2] and the Experimental Procedures).

Movie 2. Tel VI-R::lacop in Wild-Type Cells with Nup49-GFP: S Phase

See legend for Movie 1.

Movie 3. Tel VI-R::lacop in Wild-Type Cells with Nup49-GFP: G2/M

Phase

See legend for Movie 1.

Movie 4. Tel VI-R::lacop in hdf1 Cells with Nup49-GFP: G1 Phase

See legend for Movie 1.

Movie 5. Tel VI-R::lacop in mlp1 mlp2 Cells with Nup49-GFP: G1

Phase

See legend for Movie 1.