Charlotte Grimaud and Peter B. Becker Adolf-Butenandt ...

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


2

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


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.


4

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


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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


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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

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30

40

50

60

0-0.5 0.5-1 1-1.5 1.5-2 2-2.5

dpr8-roX2 embryos

0

10

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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

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Pe

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tage

of

nucl

ei

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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

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Distance (µm)

B A

C D


4.5 Mb

CG9650

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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.


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


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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


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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


Nup153RNAiSGS3‐GAL4control

Nup153RNAiey‐GAL4

DAPI Nup153 MSL2 merge

20µm

20µm


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16


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


control

Mtor‐1

Mtor‐2


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


2µm

2µm 2µm

2µm

A

B

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18


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.


0

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50

60

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

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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


21

A

B


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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).

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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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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


Female Male

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24

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.


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


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


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


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)


GAGGACGAGGCGCGCAAGAA

GCAAGTGGACG


AGACTCACGGGTGAGTCGCT

GGTTGATGTCC

Nup1

(Mendjan et al.

2006)


GACCTCCTCACCCACTTCCTC

ACA


AGACCTCCTCACCCACTTCCT

CACA

Nup2

(Dietzl et al.

2007)


GAGCGAACAGTAGTTACCCGT

CGATCAAC


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


29

References:

Bantignies, F., Grimaud, C., Lavrov, S., Gabut, M., and Cavalli, G. 2003. Inheritance of

Polycomb-dependent chromosomal interactions in Drosophila. Genes Dev 17(19):

2406-2420.

Dietzl, G., Chen, D., Schnorrer, F., Su, K.C., Barinova, Y., Fellner, M., Gasser, B., Kinsey,

K., Oppel, S., Scheiblauer, S., Couto, A., Marra, V., Keleman, K., and Dickson, B.J.

2007. A genome-wide transgenic RNAi library for conditional gene inactivation in

Drosophila. Nature 448(7150): 151-156.

Mendjan, S., Taipale, M., Kind, J., Holz, H., Gebhardt, P., Schelder, M., Vermeulen, M.,

Buscaino, A., Duncan, K., Mueller, J., Wilm, M., Stunnenberg, H.G., Saumweber, H.,

and Akhtar, A. 2006. Nuclear pore components are involved in the transcriptional

regulation of dosage compensation in Drosophila. Mol Cell 21(6): 811-823.

Straub T, G.G., Maier VK, Becker PB. 2005 The Drosophila MSL complex activates the

transcription of target genes. Genes Dev 19(Oct 1): 2284-2288.

Straub, T., Grimaud, C., Gilfillan, G.D., Mitterweger, A., and Becker, P.B. 2008. The

chromosomal high-affinity binding sites for the Drosophila dosage compensation

complex. PLoS Genet 4(12): e1000302.

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