Grimaud and Becker Supplementary File
1
Supplementary File
The dosage compensation complex shapes the conformation of the X chromosome in Drosophila
Charlotte Grimaud and Peter B. Becker
Adolf-Butenandt-Institute, Molecular Biology Unit and Centre for Integrated Protein Science
(CiPSM), Ludwig-Maximilians-University, 80336 Munich, Germany.
Page 2-4: Material and Methods
Page 5-24: Supplementary figures and figures legends
Page 25-26: Supplementary data composed of different tables
Page 27: Primer list used to produce FISH probes
Page 28: Primer list used for RNAi experiments in Drosophila cells
Page 29: References
Grimaud and Becker Supplementary File
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Material and Methods
Fly stocks and handling
Flies were raised on standard corn meal/yeast extract medium. OregonR yw was mainly
used and considered wild type with respect to dosage compensation. Transgenic lines for
expression of double stranded RNA for RNA interference were obtained from VDRC
(Vienna). The construct identification numbers are: 10636 (Nup153), 13871 (Mtor), 1242
(MSL1), 14745 (MSL2) and 1492 (MSL3). These homozygous lines were crossed with the
following Gal4 driver lines in order for specific RNA interference: ey-GAL4 (Bloomington
stock # 5248), SGS3-GAL4 (yw; {w+,GAL42314}) and, 69B-GAL4 (Bloomington stock # 1774).
These lines express GAL4 in the eye imaginal discs, in the salivary glands or predominantly
in the wing imaginal discs of 3rd instar larvae, respectively. All crosses were carried out at
25°C.
FISH-I on whole-mount tissues
Locus-specific FISH probes for consisted of pools of 6-7 PCR fragments each 1.2 to 1.5 kb
long. The amplicons were separated by approximately 2 kb in order to cover around 20 kb for
each locus. See table S4 for a list of amplification primers. Probe preparation and two-color
FISH on whole-mount tissues were performed as described ((Bantignies et al. 2003). A
detailed protocol is available at http://www.epigenome-
noe.net/researchtools/protocol.php?protid=5. Immunostaining after FISH was done as
follows. Whole-mount tissues were blocked in PBS, 0.3% Triton (PBS-Tr), 10% normal goat
serum (NGS) for 2 hours (h) at room temperature (RT), and incubated overnight at 4°C with
rabbit polyclonal sera specific for MSL1, MSL2, or MSL3 or with an Mtor rat monoclonal
antibody (A. Akhtar) at a dilution of 1/200 in PBS-Tr-10%NGS. The samples were washed
several times in PBS-Tr and blocked in PBS-Tr, 10% NGS, 1% BSA for 1 h at RT and
incubated sequentially each for 1 h with an anti-DIG-rhodamine (Roche Diagnosis) diluted
1/50, with an anti-biotin-FITC (Sigma Aldrich) diluted 1/200 and with a donkey-anti-rabbit or
donkey-anti-rat Cy5 (Jackson Laboratories) in PBS-Tr, 10% NGS diluted 1/100. After several
washes in PBS-Tr, DNA was counterstained with DAPI and tissues were mounted in Prolong
Antifade Medium (Molecular Probes).
Double Immunostaining on imaginal discs of third instar larvae
The anterior parts of 5-10 third instar larvae, which contains the majority of larval imaginal
discs were dissected, pooled in Eppendorf tubes and fixed in 4% formaldehyde, in PBT
Grimaud and Becker Supplementary File
3
(PBS, 0.1% Tween20) for 20 min at RT. After several washes in PBT, larvae were blocked in
PBS-Tr-10% NGS for 2 h at RT. They were incubated overnight at 4°C with the different
combination of primary antibodies. The MSL2 rabbit serum and the Mtor rat monoclonal
antibody were diluted to 1/200 in blocking solution. A monoclonal MSL2 antibody diluted to
1/200 was combined with a Rabbit Nup153 antibody diluted to 1/50 in blocking solution.
Larval tissues were washed in PBS-Tr 0.3%, blocked 1 h in PBS-Tr-0.3%, 10% NGS and
incubated sequentially with an anti-rabbit Cy3 (Jackson Laboratories) diluted 1/300 in PBS-
Tr-0,3%, 10% NGS for 1 h at RT and with an anti-rat Alexa 488 (Molecular Probes) diluted
1/500 in PBS-Tr-0.3%, 10% NGS for 1 h at RT. After several washes in PBS-Tr-0.3% DNA
was counterstained with DAPI and separated imaginal discs were mounted on slides in
Prolong Antifade Medium (Molecular Probes).
Microscopy and Image Analysis
Series of confocal sections through whole-mount embryos or imaginal discs were collected
with a Leica SP5 microscope equipped with a Plan/Apo 63X 1.4 NA oil immersion objective.
For each optical section, images (512*512 pixels) were collected sequentially for 3-4
fluorochromes. Stacks for each color were recorded as separated eight bit grayscale Z-
planes with voxel size 120x120x200 nm (XxYxZ). Chromatic shift was corrected using the z-
shift correction plugin of the ImageJ software corresponding to the measured chromatic shift
with the Leica SP5 microscope. RGB stacks were reconstructed with the 3 channels function
in ImageJ and used as raw data files for the FISH distance analysis. The 3D coordinates of
the visually determined center of mass of each FISH signal were recorded using the ImageJ
software and 3D distances were calculated in Excel with this formula: d=SQRT
(dxXVx)2+(dyXVy)2+(dzXVz)2 where dx, dy and dz correspond to the difference of the X, Y or
Z coordinates between the two FISH signals and, Vx,Vy and Vz correspond of the voxel size
in every direction. A similar average nuclear radius of 4.7+/- 0.3µm in the epidermal layer of
whole-mount embryos and of 3.5+/-0.4µm in interphase nuclei of imaginal discs in larvae
were measured using DAPI (in the FISH-I experiment) and using a lamin staining in the
different sexes, indicating that the embryonic and the larval nuclei were homogeneous in
size. Distances between the different loci were therefore directly expressed in micrometers.
3D distance values were sorted into the corresponding distance categories representing 0.5
µm intervals. Statistical analysis was performed using stacks collected for 6-8 whole-mount
embryos or larval imaginal discs and FISH coordinates were taken in approximately 50 nuclei
per stack. Mann-Whitney U-tests were applied on each set of data in order to determine
significance of the 3D distance difference observed between male and female nuclei.
Grimaud and Becker Supplementary File
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Immunostaining of polytene chromosomes
Immunostaining was carried out as described previously on the epigenome protocol:
http://www.epigenome-noe.net/researchtools/protocol.php?protid=4. Rabbit MSL2 polyclonal
serum was used at the dilution of 1/200 in blocking solution and revealed with a Donkey anti-
Rabbit Alexa 488 (Molecular probes) diluted at 1/500.
Drosophila SL2 cells culture and RNAi interference and Immunostaining
Culture of Drosophila male SL2 cells and RNA interference of specific target genes were
done with the same previously described protocol (Straub T 2005). Briefly, 1.5X106 cells
were deposited in 6-well plate and incubated with 10µg of specific dsRNA in a serum free
medium during 50 minutes. After the addition of serum-containing medium, cells were
incubated at 26°C during 6 days. SL2 cells were centrifuged 5 minutes at 1000 rpm and the
cell pellet was resuspended in Urea Buffer (9M Urea, 1% SDS, 25mM Tris pH 6.8, 1mM
EDTA) with a dilution of 0.5X106 cells per microliter. 10µL were loaded on SDS-Page for
each sample. Western blot was performed as previously described.
For immunostaining, 1X106 or 2X106 of cells were deposited on glass slides and incubated
during 2 hours at room temperature. Fixation was performed on ice with 2% formaldehyde
during 10 minutes. A second fixation with 1% formaldehyde diluted with 0,25% Triton in PBS
was applied to the cells during 7.5 minutes. Cells were washed and blocked with PBS-0.1%
Tween (PBT) -10% Normal Goat Serum (NGS) during 1 hour. Primary antibodies were
diluted in PBS-0.1% Tween 10%Normal Goat Serum (NGS), added on each slide and
incubated in a humid chamber overnight at 4°C. Several washes with PBT were performed at
room temperature and slides were incubated during 45 minutes with the secondary
fluorescent antibodies (Anti Rabbit Cy3 and Anti Rat Alexa 488 for Mtor and MSL, and Anti
Rabbit Alexa 488 and Anti Rat Cy3 for Nup153 and MSL2) diluted to 1/300 for Cy3 and 1/500
for Alexa 488. Slides were washed several times with PBT and DNA was counterstained with
DAPI. After a wash in PBT, slides were mounted with Prolong Antifade Medium. SL2 cells
immunostaining were analysed with SP5 confocal microscope.
Western blot on salivary glands of third instar larvae
4-5 pairs of salivary glands were dissected and dissolved in 30µL of Urea Buffer. Dissolved
salivary glands were heated at 65°C during 15 minutes with a 900 rpm agitation and several
times boiled at 95°C and frozen in liquid nitrogen. 15-20µL were loaded on a 7% SDS-PAGE
gels. Western blot was performed as previously described.
Grimaud-Suppl.-Fig.1
A
C
D
E
5
B
Grimaud and Becker Supplementary File
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Supplementary Figure 1: DCC interactions at loci probed by FISH.
The panels show genome browser screen shots visualizing the in vivo interactions of MSL1
and MSL2 in SL2 cells around the four loci selected for FISH localization in nuclei. The third
track in each panel shows the High Affinity Sites (HAS) for DCC interaction recently
described by (Straub et al. 2008) (red boxes). The FISH probes hybridize within an area of
20 kb around these HAS. The loci are named according to the gene (highlighted yellow in the
gene span) that is closest to the HAS, or central to the probe: usp (A), CG9650 (B), roX2 (C),
dpr8 (D), and Nup153 (E). The ruler on top of each panel reveals the coordinates on the X
chromosome, in kb.
DAPI Nup153 usp MSL2 merge
DAPI Nup153 roX2 MSL2 merge
DAPI rox2 usp MSL2 merge
DAPI Nup153 usp MSL2 merge
DAPI Nup153 roX2 MSL2 merge
usp dpr8DAPI MSL2 merge
roX2 dpr8DAPI MSL2 merge
Nup153dpr8DAPI MSL2 merge
A
B C
1µm
1µm
Grimaud‐Suppl.‐Fig.2
7
Dusp CG9650DAPI MSL2 merge
roX2 CG9650DAPI MSL2 merge
Nup153 CG9650DAPI MSL2 merge
1µm
1µm
1µm
1µm
1µm
1µm
1µm
1µm
1µm
1µm
1µm
Grimaud and Becker Supplementary File
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Supplementary Figure 2: A male-specific conformation of the dosage-compensated X
chromosome.
(A) Long-distance associations between high affinity sites (HAS) for DCC binding in
nuclei of whole-mount embryos. Pairs of chromosomal sites were visualized by two-
color fluorescence in situ hybridization (FISH) in female or male embryos of yw line
(wild type), respectively. DNA was stained with DAPI and the X chromosome territory
was immunostained with an antibody against MSL2 in males nuclei (there is not
MSL2 in females nuclei). A merge of all channels reveals the proximity of the sites
and their residence relative to the MSL2 territory (merge). The figure shows single
optical slices of representative images obtained with the Nup153 and usp probes
(top) or with Nup153 and roX2 probes (bottom).
(B) Long-distance proximity between high affinity sites (HAS) for DCC binding in nuclei of
imaginal discs of third instar larvae from the yw line. The analysis was as in (A). The
figure shows representative images obtained with roX2 and usp probes (top), with
Nup153 and usp probes (center) or with Nup153 and roX2 probes (bottom).
(C) Representative analysis in male nuclei obtained with the dpr8 and usp probes (top),
with the dpr8 and roX2 probes (center) or with the dpr8 and Nup153 probes (bottom).
(D) Representative analysis in male nuclei obtained with the CG9650 and usp probes
(top), with the CG9650 and roX2 probes (center) or with theCG9650 and Nup153
probes (bottom).
Female Male
0
10
20
30
40
50
60
0-0.5 0.5-1 1-1.5 1.5-2 2-2.5
dpr8-usp embryos
0
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20
30
40
50
60
0-0.5 0.5-1 1-1.5 1.5-2 2-2.5
dpr8-roX2 embryos
0
10
20
30
40
50
60
0-0.5 0.5-1 1-1.5 1.5-2 2-2.5
dpr8-Nup153 embryos
centromere Nup153
dpr8
roX2 usp 5 Mb 3 Mb 2 Mb
Perc
enta
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Pe
rcen
tage
of
nucl
ei
Perc
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Distance (µm) Distance (µm)
Distance (µm)
0 10 20 30 40 50 60 70
0-0.5 0.5-1 1-1.5 1.5-2
CG9650-dpr8 larvae
Perc
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Distance (µm)
B A
C D
Grimaud-Suppl.-Fig.3
4.5 Mb
CG9650
9
Grimaud and Becker Supplementary File
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Supplementary Figure 3: Sequences unbound by the DCC do not show sex-specific
proximity.
A schematic representation of the position of the different probes is presented at the top of
the figure. Quantitative analysis of the distribution of 3D distances between the dpr8 and the
usp loci (A), between the dpr8 and the roX2 (B), between the dpr8 and the Nup153 (C)
obtained in whole-mount embryos and between the CG9650 and the dpr8 (D) obtained in
larvae. Distribution of the distances in female nuclei is represented in light grey and in dark
grey for male nuclei from the yw line. The bars show the percentage of nuclei with distances
within the ranges indicated on the abscissa.
Grimaud‐Suppl.‐Fig.4
MtorRNAiSGS3‐GAL4
MtorRNAiey‐GAL4control
NupRNAiey‐GAL4control
NupRNAiSGS3‐GAL4
DAPI
DAPI
Mtor MSL2 merge
Nup153 MSL2 merge
20µm
20µm
B
C
D
MtorRNAiSGS3‐GAL4
Nup153RNAiSGS3‐GAL4
DAPI MSL2
11
Nup153
Lamin
Mtor
Lamin
A
Grimaud and Becker Supplementary File
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Supplementary Figure 4: The nuclear pore components Mtor and Nup153 are not
required for formation of the dosage-compensated X chromosome territory in the
larval salivary gland.
(A) Western blot analysis on salivary glands dissected from different lines. WT constitutes
a control line where each of the nucleoporins is present. Downregulation by RNAi
of Mtor (line MtorRNAi-SGS3GAL4) or Nup153 (line Nup153RNAi-SGS3GAL4),
using the salivary gland-specific GAL4 line (SGS3-GAL4) lead to a reduction of
each of the nucleoporins in this tissue. An arrow points at the expected size of
Mtor protein (260 KDa). Lamin was used as a loading control.
(B) Mtor expression was ablated by RNA interference in eyeless expressing cells (using
the driver line MtorRNAi-eyGAL4, upper panel), or in the salivary gland of third
instar larvae (using driver line MtorRNAi-SGS3GAL4, lower panel). Whole mount
salivary glands were stained for DNA (DAPI, grey), Mtor (green) and MSL2 (red).
The RNAi driven by eyeless does not affect Mtor levels in salivary glands. Each
panel represents Z-projection of 10 slices spaced by 0.5 µm.
(C) Nup153 expression was ablated by RNA interference in eyeless-expressing cells
(using the driver line Nup153NAi-eyGAL4, upper panel), or in the salivary gland
of third instar larvae (using driver line Nup153RNAi-SGS3GAL4, lower panel).
Whole mount salivary glands were stained for DNA (DAPI, grey), Mtor (green)
and MSL2 (red). The RNAi driven by eyeless does not affect Mtor levels in
salivary glands. Each panel represents Z-projection of 10 slices spaced by 0.5
µm.
(D) The expression of the nucleoporins Mtor and Nup153 was reduced by RNA
interference in the salivary glands in the transgenic driver lines MtorRNAi-
SGS3GAL4 and Nup153RNAi-SGS3GAL4 by transcription of interfering RNA
from an Sgs3 promoter. Polytene chromosome squashes were stained with
DAPI (left) or with an MSL2 antibody (right panel).
Mtor RNAi ey-GAL4 female
Mtor RNAi ey-GAL4
male
Nup153 RNAi ey-GAL4 female
Nup153 RNAi ey-GAL4
male
Grimaud-Suppl.-Fig.5
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Grimaud and Becker Supplementary File
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Supplementary Figure 5: Depletion of the nucleoporins Mtor or Nup153 in the larval
eye discs generates phenotypes in the adult eyes at 25°C.
(A) Representative examples of eye phenotypes obtained through RNA interference of
Mtor by an eyeless GAL4 driver (ey-GAL4) (MtorRNAi-eyGAL4 line). These
phenotypes manifest in 50 to 55% of the flies and in equal proportion in male or
female flies.
(B) Representative examples of eye phenotypes obtained using an eyeless GAL4 driver
(ey-GAL4) in the presence of Nup153RNAi transgene (Nup153RNAi-eyGAL4 line).
These phenotypes manifest in 15-20% of the flies and in equal proportion in every
sex.
MtorRNAiSGS3‐GAL4control
MtorRNAiey‐GAL4
DAPI Mtor MSL2 merge
MtorRNAiSGS3‐GAL4control
MtorRNAiey‐GAL4
DAPI Mtor MSL2 merge
Nup153RNAiSGS3‐GAL4control
Nup153RNAiey‐GAL4
DAPI Nup153 MSL2 merge
20µm
20µm
Grimaud‐Suppl.‐Fig.6
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Grimaud and Becker Supplementary File
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Supplementary Figure 6: The nuclear pore components Mtor and Nup153 are not
required for formation of the dosage-compensated X chromosome territory in the eye
disc.
(A) Mtor expression was ablated by RNA interference (MtorRNAi line) as a control in the
salivary gland (using the driver line SGS3-GAL4, top panel) or in the differentiated
part of the eye imaginal disc of third instar larvae (using the driver line eyGAL4, lower
panel). Eye imaginal discs were stained for DNA (DAPI, grey), Mtor (green) and
MSL2 (red). Each panel represents Z-projection of 10 slices spaced by 0.5 µm.
(B) Same analysis as in (A). Z-Sections zoomed in the differentiated part of the eye
imaginal disc of third instar larvae using driver line MtorRNAi-SGS3GAL4 (upper
panel), or using the driver line MtorRNAi-eyGAL4 (lower panel). Eye imaginal discs
were stained for DNA (DAPI, grey), Mtor (green) and MSL2 (red). Each panel
represents Z-projection of 10 slices spaced by 0.5 µm.
(C) Analysis as in (A) except for that Nup153 was reduced in the control line
Nup153RNAi-SGS3GAL4 (upper panel) and in the eye-specific line Nup153RNAi-
eyGAL4 driver lines an eye imaginal discs were stained for Nup153 (green) and
MSL2 (red).
control
Mtor‐1
Mtor‐2
DAPI Mtor MSL1 merge
control
Mtor‐1
Mtor‐2
DAPI Mtor MSL2 merge
DAPI Mtor MOF merge
DAPI Nup153 MSL2 merge
control
Mtor‐1
Mtor‐2
GST
Nup‐1
Nup‐2
DAPI
MSL2
MSL1
MOF
Lamin
Mtor
Nup153
MSL2
MSL1
MOF
Lamin
MtordsRNA12 Nup153
dsRNAGST
dsRNA
nodsRNA
12
Grimaud‐Suppl.‐Fig.7
2µm
2µm 2µm
2µm
A
B
17
Grimaud and Becker Supplementary File
18
Supplementary Figure 7: The nuclear pore components Mtor and Nup153 are not
required for formation of the dosage-compensated X chromosome territory in SL2
cells.
(A) Western blot analysis of total protein extracts of SL2 cells after 6 days of treatment
with two different types of double-stranded RNA (dsRNA) for each, Mtor and
Nup153. For reference and controls extracts from untreated cells (no dsRNA) or
treated with dsRNA directed against GST (GST dsRNA) were used. The specific
RNA interference is obvious from the upper panel. Levels of lamin, MSL1, MSL2 or
MOF in these extracts were not affected by the treatments.
(B) Double immunostaining of SL2 cells after 6 days of RNA interference. Staining was
for Mtor, Nup153, MSL1, MSL2 and MOF as indicated above the panels. The
sequence targeted by dsRNAs is given to the left of each panel.
Grimaud-Suppl.-Fig.8
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70
MSL1RNAi MSL2-RNAi MSL3-RNAi MOF-RNAi
Nup153-usp larvae
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0-0.5 µm 0.5-1 µm 1-1.5 µm 1.5-2 µm
19
Grimaud and Becker Supplementary File
20
Supplementary Figure 8: The male-specific X chromosome conformation depends on
MSL1 and MSL2, but not on MSL3 or MOF.
Quantitative analysis of 3D distances between the Nup153 and the usp loci in wing discs
male nuclei where MSL1, MSL2, MSL3 or MOF were specifically reduced by RNA
interference using the 69B-GAL4 driver line. The average total number of nuclei analysed per
condition was 400. The absence of MSL1 or MSL2 strongly affect the degree of associations
between these HAS (compare the distribution of 3D distances with the one obtained in yw
line for these two probes, Fig. 2), whereas neither the absence of the spreading factor MSL3
nor of the H4K16 acetyltransferase MOF disturb the nuclear connections established
between the HAS Nup153 and usp.
MOF‐RNAiSGS3‐GAL4
MSL3‐RNAiSGS3‐GAL4
MSL2
MSL2
MSL3‐RNAi69B‐GAL4
MOF‐RNAi69B‐GAL4
MOF
MSL3
DAPI
DAPI
merge
merge
Grimaud‐Suppl.‐Fig.9
21
A
B
Grimaud and Becker Supplementary File
22
Supplementary Figure 9: MSL3 and MOF downregulation in different tissues
(A) MOF or MSL3 expression was reduced by RNA interference in the salivary gland of
third instar larvae using the driver line SGS3GAL4 in the presence of the MOF-RNAi
transgene (top panel) or of the MSL3-RNAi transgene (bottom panel). Polytene
chromosome squashes were stained with DAPI (blue) and an MSL2 antibody
(green). MSL2 recruitment is only maintained on the highest binding sites for the
DCC, suggesting that a specific donwregulation of MOF or MSL3 by RNA
interference can phenocopy mutant alleles for these different genes.
(B) MOF or MSL3 expressions were ablated by RNA interference in the wing discs of
third instar larvae (using the driver lines MOF-RNAi-69BGAL4 or MSL3-RNAi-
69BGAL4). Whole-mount wing discs were stained for DNA (DAPI, grey), and MOF or
MSL3 (green). Each panel represents Z-projection of 10 slices spaced by 0.5 µm. A
specific downregulation of MOF (top) or MSL3 (bottom) is observed in the majority of
the diploid nuclei in wing imaginal discs (pointed by an arrow).
0
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0‐0.5 0.5‐1 1‐1.5 1.5‐2
autosomalsitesinlarvae
Percentageofn
uclei
Distance(μm)
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40
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60
0‐0.5 0.5‐1 1‐1.5 1.5‐2
autosomalsitesinembryos
Percentageofn
uclei
Distance(μm)
auto1 auto2
96C 88E3R 10Mb
A
B
C
Grimaud‐Suppl.‐Fig.10
Female Male
23
Grimaud and Becker Supplementary File
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Supplementary Figure 10: 3D distances between two autosomal loci do not differ in
male and female nuclei.
(A) Schematic representation of the localization of the two autosomal probes. The two
probes are located on the right arm of chromosome 3. The probe called auto1 is localized in
the cytological position 96C and the probe auto2 is localized around 10Mb away from the first
probe in cytological position 88E.
(B) Quantitative analysis of the distribution of 3D distances between the two autosomal sites
obtained in whole-mount embryos. 3D distances are similarly distributed in male and female
nuclei (p-value is 0.06). The average number of nuclei analysed was 200.
(C) Quantitative analysis of the distribution of 3D distances between the two autosomal sites
obtained in imaginal dicsc of third instar larvae. 3D distances distribution do not differ in male
and female nuclei (p-value 0.24). The average number of nuclei analysed was 300.
Grimaud and Becker Supplementary File
25
Supplementary Data
Supplementary Table S1: Percentage of nuclei where a maximal distance of 0.5 µm was
measured between different FISH probes in embryos (A) and in larvae (B)
A
EMBRYOS 0-0.5-female 0-0.5-male
roX2-usp 4 27.5
usp-Nup153 9.9 29.4
roX2-Nup153 24.2 44
dpr8-usp 6.6 6.6
dpr8-roX2 18.2 6
dpr8-Nup153 38 15.3
B
LARVAE 0-0.5-female 0-0.5-male
roX2-usp 9.5 44
usp-Nup153 10 38
roX2-Nup153 17.1 54.2
dpr8-usp 6 9
dpr8-roX2 39.3 10
dpr8-Nup153 49 14
usp-CG9650 24.4 16.1
roX2-CG9650 6.4 8.5
Nup153-CG9650 13.1 8.6
dpr8-CG9650 11 10
Grimaud and Becker Supplementary File
26
Supplementary Table S2: p-values obtained for each two-color FISH experiments done in
third instar larvae. p-value evaluates the significance of the difference in 3D-distances
obtained with different probes, between male and female nuclei.
Supplementary Table S3: Numbers of flies obtained upon MSLs RNAi using the 69B-GAL4
driver
FISH experiments p-value
roX2-usp <4.9*10-6
Nup153-usp <2.4*10-6
Nup153-roX2 <2.75*10-6
dpr8-usp 0.13
dpr8-roX2 <2*10-6
dpr8-Nup153 <2.86*10-6
usp-CG9650 2.5*10-4
roX2-CG9650 0.02
Nup153-CG9650 0.16
dpr8-CG9650 0.146
25°C Female Male
MSL1RNAi 224 4
MSL2RNAi 242 29
MSL3RNAi 300 46
MOFRNAi 195 33
Grimaud and Becker Supplementary File
27
FISH
Probes
Primer coordinates* forward reverse
usp-1 1928196-1929511 TTGTTGGAGCTGGCGGAGTT CTGGTTCCGTTTGGTCTTGG
usp-2 1931542-1932747 CACACACGAAATCGCAAATC GTGCGTTTCAGGACTGTAAG
usp-3 1934756-1936046 CAGACAACAGTGGTTGGTAG TTTGCGCCATCGATCGGTCAA
usp-4 1938180-1939419 AGCTCCGCAGTTTGAGAAC TGTCCACGGACTCATCAACA
usp-5 1941538-1942841 TCTCACTTGGCACCCACTTG CTCGAGCGGGAACAGAGAAT
usp-6 1926021-1927387 ACGTAACGATCACAACGGTT GACATGTATGCGTGGCATCT
usp-7 1922902-1924350 GAGCGCGCACACAGAGATAA GACTACGCCAAGCTGTCCAA
roX2-1 11473695-11474993 TCGTTTAGGTAGCTCGGAT CGCAATCCGAACTGCGCTTA
roX2-2 11470506-11471838 GCTCATCCTTGTGTCGCGTA ATCGTTGTGCATGTGCAGGT
roX2-3 11467508-11468801 CATCATCAAGCAACCAGCAA GCACCTCCGAGGTATCATCA
roX2-4 11476692-11477935 CGCATCAGTTGCCAGTCGAT GTGCTGAGGTGGAGCACATT
roX2-6 11464530-11466294 AAAGGCCAGTACCTGTTGAAT CCAACTGGAGGCCACTTTGA
roX2-7 11460769-11462705 AAGACGACGCTGGATGTAAC CAAGTATGCGCGTCCCGATT
roX2-8 11458321-11459738 CCGTCGACTGTGGATTGAAA CGCCCTGGTTGCTACGTATT
dpr8-1 14216390-14217720 CGAATCCTCGACAACTGAAT AAAGTTCGCAATTCGCCTTA
dpr8-2 14219872-14221394 CGGCCATATGGGCAATAACC GTGCCTGGATGTAAGAGTCA
dpr8-3 14223268-14224703 GCAACAGAAATTGTTGCCAT AGCTGCCGTCTGTAAGACAA
dpr8-4 14226886-14228490 AAGCCAAAGCCAAAGCCTCT GGGGCATCTTTGGAAAACAA
dpr8-5 14230742-14232063 GCCAGTAGTCCGTGTTGTTT CCCGAATTTATTGCCAACGA
dpr8-6 14233875-14235437 CATGAATCCTTGGCCGAATC GCTGCCTGGCAGGTATTAAG
dpr8-7 1423790214239249 CGATAGGGTGTGTTGTGATT CGGATACCGTAAAGCAGAGT
Nup153-1 16490075-16491255 GCTGGTTGGTTTAGAATCATG ATCAAGCTCTACCCGAATGT
Nup153-2 16493504-16494684 TGAGCAGAGCGTGAAGAAGA AGGGCCACCTCCTCATAGTT
Nup153-3 16496674-16497795 AGTCGGCTAACCATACTCAG TCGCTATTGGAGTCGAAGAT
Nup153-4 16499849-16501046 CACGAATTCCAATCCGCTTT TGACCTCGCATTACCTGCAT
Nup153-5 16503305-16504726 GGTTCGGTCCAGAGCCAATA ATTAGTGCTGGAACGGCTAT
Nup153-6 16510032-16511083 CTTCAGCGAGTTGAGGAAGTC ATTGAATAACCCGTCCAACCA
Nup153-7 16513197-16514290 CGACTTAATGGGCTGACGTC CGGCATGGTGTATTATGCAA
CG9650-1 7087131-7088418 AGGCGTCCATCGATAGCTGA CCATAGAGTTGATCCCTCAT
CG9650-2 7091057-7092227 AGCTGAAGCTGTCTGATTAG GCTCTGATTTCCCTTTGAAC
CG9650-3 7094341-7095530 CAAGGACATGGCGGATATAA AACTATGACAAAGGTGCCAA
CG9650-4 7098505-7100013 GACGCGCTCTGATCTTTATT CCAAAAGGATCCACCACCTT
CG9650-5 7102346-7104380 GCGATTGAGTTGAGTTTCAG GACCAAGAGCCGAGTGAGTT
CG9650-6 7105392-7106692 GTGTCAATGCAGGGCGAGAA TTGGATGGCGTCGTGTTAGT
CG9650-7 7108704-7109872 CCTCACACTTTCGGCTGACA GCGTTGGTGGAGAGTTCGTT
Grimaud and Becker Supplementary File
28
Supplementary Table S4: List of the different primers used for each FISH probe
*Each primer has been blasted on the more recent version of the Drosophila melanogaster
genome release (FB2009_03, released March 20, 2009): http://flybase.org/blast/
Supplementary Table S5: Description of the primers used for the production of dsRNA in SL2
cells
Forward primer Reverse primer
Mtor1
(Mendjan et al.
2006)
TTAATACGACTCACTATAGGGA
GACCTCCTCACCCACTTCCTC
ACA
TTAATACGACTCACTATAGGG
AGACCTCCTCACCCACTTCCT
CACA
Mtor2
(Dietzl et al.
2007)
TTAATACGACTCACTATAGGGA
GAGGACGAGGCGCGCAAGAA
GCAAGTGGACG
TTAATACGACTCACTATAGGG
AGACTCACGGGTGAGTCGCT
GGTTGATGTCC
Nup1
(Mendjan et al.
2006)
TTAATACGACTCACTATAGGGA
GACCTCCTCACCCACTTCCTC
ACA
TTAATACGACTCACTATAGGG
AGACCTCCTCACCCACTTCCT
CACA
Nup2
(Dietzl et al.
2007)
TTAATACGACTCACTATAGGGA
GAGCGAACAGTAGTTACCCGT
CGATCAAC
TTAATACGACTCACTATAGGG
AGAGTTGCGGCAGGGTTAAA
TGCTTCCGGC
FISH
Probes
Primer coordinates* forward reverse
Auto1-1 21145960-21147093 GGCGTAAACCAGCCCATAAA AGATGCAGCTCTACAACAGT
Auto1-2 21140854-21141982 GCAGTTGGAGATCCACGAAA ACTCTCCATATCCAGCACTC
Auto1-3 21142502-21143765 CAATATAGTTGCGGTGCAGA GCCAAAAACAGGTCACCATT
Auto1-4 21149067-21150124 AGGATAGCCTGCATGTGCTT CGAAATCGAACGCACCATGT
Auto1-5 21154905-21156085 ACATCTTCGAAACGTTCAGC ACATCTCAACTGACGGATCT
Auto2-1 11024551-11025727 CGAGATTCTCAACGGGTCTA AAACCAAAGCGTGGCATATG
Auto2-2 11028277-11029457 AGGCAAGGATCGTGTTGAAC CCGGATGAGAACTCGGACAT
Auto2-3 11031682-11032861 GCAGAGCTATGTGTCACTCA CCTGGAACTGACCCCTGTAA
Auto2-4 11034917-11036377 ATTCCGCACTTGACAACACT CATCAGCGCTTGACTGCTAT
Auto2-5 11037588-11039099 TGAATCTCATGGGGCAGAGAT CGAGAAGGTGTGCCTAAGCT
Auto2-6 11040202-11041428 GTACTTCTTCGTCGAGGACA CTGATCTCCTGAGCTCGTCT
Grimaud and Becker Supplementary File
29
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