Current Biology, Volume 24

Supplemental Information

Inefficient Double-Strand Break Repair

in Murine Rod Photoreceptors with

Inverted Heterochromatin Organization Antonia Frohns, Florian Frohns, Steffen C. Naumann, Paul G. Layer, and Markus Löbrich  

light

rod

cone

amacrine cell

horizontal cell bipolar cell

ganglion cell

Müller glia cell

cone precursor

rod precursor

horizontal cell precursor

amacrine cell precursor

ganglion cell precursor

undetermined progenitors

iii

adult P 4

GC

L

INL

O

NL

GC

L

N

BL

i ii

Predicted light scattering

conventional

chromatin

Supplemental Figures

light

A

Figure S1 (related to Figure 1). (A) Schematic

diagrams of the organization and light

focusing effects of mouse retinae. (i) Diagram

of the adult mouse retina showing its three

nuclear layers (ONL, INL and GCL) and position

of its seven major cell types. (ii) Diagram of the

P4 retina showing the position of various

precursor cells within the NBL and GCL. (iii)

Predicted light scattering by rods with

conventionally organized (left) or inverted

chromatin pattern (right). Note the higher degree

of scattering and spreading of light into adjacent

columns for rods with conventionally organized

chromatin. The schematic diagram in (iii) was

modified from Solovei et al. [9].

inverted

chromatin

Lim1+2/53BP1/DAPI DAPI

Lim1+2 53BP1

G

CL IN

L O

NL

INL i

Lim1+2/53BP1/DAPI

Pax6/53BP1/DAPI Pax6/53BP1/DAPI DAPI

Pax6 53BP1

ii

GC

L IN

L O

NL

INL

B

GS/53BP1/DAPI GS/53BP1/DAPI DAPI

GS 53BP1

G

CL IN

L O

NL

INL iii

Chx10/53BP1/DAPI Chx10/53BP1/DAPI DAPI

Chx10 53BP1

iv

G

CL IN

L O

NL

INL

bipolar cell (Chx10)

horizontal cell (Pax6)

amacrine cell (Pax6)

Müller glia cell (DAPI)

DSB (53BP1)

INL

v

(B) 53BP1 foci in the INL of the adult retina at 15 min after in vivo irradiation with 1 Gy.

(i) Immunofluorescence images of horizontal cell-specific transcription factor Lim1 + 2 (green),

53BP1 (red) and DAPI (blue) co-staining. (ii) Immunofluorescence images of horizontal,

amacrine and ganglion cell-specific transcription factor Pax6 (green), 53BP1 (red) and DAPI

(blue) co-staining. (iii) Immunofluorescence images of Müller glia cell-specific protein

glutamine synthetase (green), 53BP1 (red) and DAPI (blue) co-staining. (iv)

Immunofluorescence images of bipolar cell-specific transcription factor Chx10 (green), 53BP1

(red) and DAPI (blue) co-staining. Scale bars: 20 µm. (v) Immunofluorescence image of a

serial staining procedure against horizontal and amacrine cell-specific transcription factor Pax6

(green), bipolar cell-specific transcription factor Chx10 (red), 53BP1 (white) and DAPI (blue).

Nuclei of Müller glia cells show only DAPI signals. Note the lack of 53BP1 foci in Chx10-

positive bipolar cells.

pATM 53BP1 pATM/53BP1/DAPI Zoom pATM/53BP1/DAPI

GC

L N

BL

pATM 53BP1 pATM/53BP1/DAPI Zoom pATM/53BP1/DAPI

GC

L IN

L O

NL

1 Gy, 15 min Ctrl

1 Gy, 15 min Ctrl

P 4

a

du

lt

C

i

ii

iii

IIN

L O

NL IN

L O

NL

pATM/DAPI pATM

1 Gy, 15 min - adult

A

T w

t

(C) 53BP1 and pATM foci in the adult and P4 retina. Immunofluorescence images of pATM

(green) and 53BP1 (red) co-staining in the adult (i) or P4 retina (ii) of irradiated or unirradiated

mice. Nuclei were counterstained with DAPI (blue). In the irradiated adult retina, 53BP1 and

pATM foci were restricted to cells within the INL (except for bipolar cells) and GCL. In the

irradiated P4 retina, both 53BP1 and pATM foci were present in all cells although a gradient with

diminished signals at the apical part was evident. Unirradiated samples exhibited almost no

spontaneous foci but a pan-nuclear 53BP1 signal in those cell types that formed 53BP1 foci

after irradiation. Zoomed images display the retinal sections indicated by boxes. Open boxes

are used when the zoom shows an area which extends beyond the displayed retinal section.

Scale bars: 20 µm. (iii) pATM antibody specificity. Immunofluorescence images of retinae from

wt and AT mice stained against pATM (green) at 15 min after 1 Gy. Nuclei were counterstained

with DAPI. Note the absence of IR-induced pATM foci in the retina of AT mice. Scale bar: 5 µm.

Rhodopsin/DAPI Rhodopsin/DAPI

Rhodopsin

DAPI

GC

L N

BL

P 4

Rhodopsin

H3

P4

- -

1 G

y

(D) Rod-specific marker in the P4 retina. (i) Immunofluorescence images of rod-specific

rhodopsin staining (red) in the P4 retina. Already determined but not yet fully differentiated rod

photoreceptors were identified by rhodopsin expression. Nuclei were counterstained with DAPI

(blue). Scale bar: 20 µm. (ii) Western blot for rhodopsin in retinal explants from P4 mice before

and 15 min after irradiation with 1 Gy. H3 was used as loading control.

i ii

D

wt

H2A

X-/

-

wt

53

BP

1-/

-

MEFs – 1 Gy MEFs – 1 Gy MEFs – 1 Gy

GAPDH

gH2AX

GAPDH

53BP1

DNA-PKcs

GAPDH

wt

MD

C1

-/-

MDC1

GAPDH

wt

DN

A-P

Kcs

-/-

- -

1 G

y

MDC1

a-Tubulin

MEFs – 1 Gy human glioma cells – 1 Gy P4 retina

i ii iii

iv v vi

Figure S2 (related to Figure 2). Antibody specificity. (i-v) Western blots for pATM (i), gH2AX (ii),

53BP1 (iii), MDC1 (iv), and DNA-PKcs (v) in wt and corresponding knockout cells at 15 min after 1 Gy.

Note the lack of signals in the corresponding knockout cells. GAPDH was used as loading control. (vi)

Western blots for MDC1 from retinal explants of P4 mice before and 15 min after irradiation with 1 Gy.

Since the MDC1 signal in Fig. 2A appeared to be decreased in the P4 retina after irradiation, we again

performed Western blotting for MDC1 with retinal explants of P4 mice. Note that MDC1 is similarly

expressed with and without irradiation. a-tubulin was used as loading control.

pATMSer1981

GAPDH

wt

AT

M-/

-

A

CAR/DAPI CAR/DAPI

CAR

DAPI

GC

L IN

L O

NL

15 min 8 h 24 h 15 min 8 h 24 h

retina brain

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

10 Gy 20 Gy 20 Gy 20 Gy

15 min 8 h 24 h

frac

tio

n o

f D

NA

-re

leas

ed

Retina

Brainbrain

retina

Ctr

l

10 G

y

20 G

y

20 G

y

20 G

y

Ctr

l

10 G

y

20 G

y

20 G

y

20 G

y

B a

du

lt

Figure S3 (related to Figure 3). (A) Cone-specific marker in the adult retina. Immunofluorescence

images of cone-specific cone arrestin (CAR) staining (green) in the adult retina. Nuclei were

counterstained with DAPI (blue). Scale bar: 20 µm. (B) Repair of IR-induced DSBs in the retina and

brain of adult mice analyzed by pulsed-field gel electrophoresis. Retina and brain tissues of adult

mice were transferred in culture, irradiated with 10 or 20 Gy, incubated for repair, and harvested at distinct

time points (15 min, 8 h and 24 h). After sample preparation, the DNA was separated on a gel. Note the

higher amount of broken DNA entering the gel for the 24 h-retina sample compared with the 24 h-brain

sample (upper panel). Quantification of the fraction of DNA released from the gel plug into the gel is a

measure for the level of DNA fragmentation (lower panel). Note that rod photoreceptors account for about

80% of all cells in the adult retina [12]. Data represent the mean +/- SEM from ≥2 experiments.

1 Gy , 15 min - gH2AX/DAPI

DMSO + ATMi + DNA-PKi + ATMi +DNA-PKi

ON

L-Z

oo

m

B

A

C

MRE11

pMRE11Ser264

pNBS1Ser343

NBS1

a-Tubulin

- -

1 G

y

adult

Figure S4 (related to Figure 4). (A) gH2AX and 53BP1

foci in AT and SCID mice. gH2AX or 53BP1 foci levels in

the kidney, in the INL or in rods of wt, AT or SCID mice at 5

and 15 min after in vivo irradiation with 1 Gy. SCID but not

AT mice showed robust foci formation. Data represent the

mean +/- SEM from ≥2 experiments. (B) gH2AX and

53BP1 foci in the absence of ATM, DNA-Pkcs or both.

Immunofluorescence images of gH2AX foci (green) in rod

photoreceptor cells at 15 min after in vitro irradiation with 1

Gy in the presence of DMSO, a chemical ATM inhibitor

(ATMi), a chemical DNA-PKcs inhibitor (DNA-PKi) or both

together. Nuclei were counterstained with DAPI (blue).

Note the absence of gH2AX foci after combined inhibitor

treatment. Scale bar: 5 µm. (C) MRN complex

components in the adult retina. Western blots showing

the expression and IR-induced phosphorylation of MRE11

and NBS1 in explants of adult retina before and 15 min

after irradiation with 1 Gy. a-tubulin was used as loading

control.

IN

L O

NL

pKAP1/DAPI pKAP1

1 Gy, 15 min - adult

w

t

KAP1 DAPI KAP1/DAPI

H3K4me3 DAPI H3K4me3/DAPI

O

NL

KAP1 DAPI KAP1/DAPI

H3K4me3 DAPI H3K4me3/DAPI

INL

C

H3K9me3 DAPI H3K9me3/DAPI H3K9me3 DAPI H3K9me3/DAPI

adult

gH2AX KAP1 gH2AX/KAP1/DAPI gH2AX pKAP1 gH2AX/pKAP1/DAPI

P 4 B

IN

L O

NL

A

T

A

pKAP1/DAPI pKAP1

le

ns co

rne

a cili

ary

e

pith

eliu

m

KAP1 DAPI KAP1/DAPI

adult D

KAP1 DAPI KAP1/DAPI

P 4

le

ns co

rne

a cili

ary

e

pith

eliu

m

Figure S5 (related to Figure 5). (A) pKAP1 antibody specificity. Immunofluorescence images of

retinae from wt and AT mice stained against pKAP1 (red) at 15 min after 1 Gy. Nuclei were

counterstained with DAPI. Note the absence of IR-induced pKAP1 staining in the retina of AT mice.

Scale bar: 5 µm. (B) Recruitment of KAP1 and pKAP1 to laser-induced DSBs. Hoechst pre-

sensitized retinal explants of P4 mice were fixed 5 min after micro-irradiation with a 405 nm diode

laser and subsequently stained against gH2AX and KAP1 or pKAP1. Enlarged images of single cells

of the explants showed that both KAP1 and pKAP1 (red) accumulated at laser-induced DNA damage

sites marked by gH2AX (green). Scale bar: 2 µm. (C) Localization of KAP1 in cells of the adult

retina. Immunofluorescence images of KAP1, H3K4me3, and H3K9me3 staining (red) in rod and

cone photoreceptors (left panels) and in cells of the INL (right panels) of the adult retina.

Counterstaining of nuclei with DAPI (blue) allowed the discrimination between rods and cones by

nuclear morphology (cones with several chromocenters are encircled in red). Note that all analyzed

cell types showed enrichment of KAP1 in DAPI-weak regions that stained positive for the euchromatin

marker H3K4me3 but negative for the heterochromatin marker H3K9me3. Rod photoreceptors

showed only marginal levels of KAP1 staining. Scale bar: 2 µm. (D) Localization of KAP1 in cells of

the tissue which surrounds the adult or P4 retina. Immunofluorescence images of KAP1 staining

(red) in cells of the tissue surrounding the retina of adult (left panels) or P4 (right panels) mice. Nuclei

were counterstained with DAPI (blue). KAP1 staining was most prominent in DAPI-weak regions of

adult mice and in DAPI-rich regions of P4 mice. Scale bar: 2 µm.

adult P4

SPOC1 DAPI SPOC1 DAPI

GC

L IN

L O

NL

Figure S6 (related to Figure 6). (A) pKAP1 antibody specificity. Western blot

for pKAP1 in wt and ATM-/- cells at 15 min after 1 Gy. Note the lack of signal in

ATM-/- cells. GAPDH was used as loading control. (B) SPOC1 in the adult and P4

retina. Immunofluorescence images of SPOC1 staining in unirradiated retinae.

Nuclei were counterstained with DAPI. The SPOC1 signal was strongest in the

ONL of adult retinae and absent in P4 retinae. Scale bar: 20 µm.

wt

AT

M-/

-

GAPDH

pKAP1Ser824

MEFs – 1 Gy

A

B

Supplemental Experimental Procedures

Cell culture

ATM-/-, H2AX-/-, 53BP1-/-, and MDC1-/- mouse embryonic fibroblasts (MEFs, kindly provided by P.

A. Jeggo, Brighton, UK) were cultured in Dulbecco’s modified Eagle medium (DMEM) with 10%

fetal calf serum (FCS), 1% L-glutamine, 1% NEAAs and 1% Pen/Strep. Human glioblastoma cells

(DNA-PKcs-deficient M059J and DNA-PKcs-proficient M059K, a gift from P. A. Jeggo) were

cultured in minimal essential medium (MEM) containing 10% FCS, 1% L-glutamine, 1% NEAAs

and 1% Pen/Strep.

Immunoblotting analysis

For Western blotting of retina samples, adult and P4 mice were sacrificed and their eyes were

removed. Isolation of the retina was performed in ice-cold F12 medium under the binocular.

Isolated retinae were placed in Dulbeccos Modified Eagle Medium, supplemented with 10%

(vol/vol) fetal calf serum, 1% (vol/vol) L-glutamine, 100 units/ml penicillin and 0.1 mg/ml

streptomycine. Retinae were cultivated for 30 min at 37 C, 90% humidity and 5% CO2. For

Western blotting of MEFs and human glioblastoma cells, wt and knockout cells were grown to

80% confluency. Cells and retinal explants were irradiated with 1 Gy and harvested after 15 min.

They were sonicated three times for 1 min each in cell lysis buffer (500 mM EDTA, 1 mM

β‐mercaptoethanol, 5% glycine, 0.5 mM PMSF, 1 μM Na3VO4, 1x protease inhibitor Complete®,

1 x PhosphoStop (Roche), pH 8,5). After centrifugation of the cell extracts for 30 min at 4 C with

15.7 g, the protein concentration was determined and the cell lysates were boiled with SDS

Laemmli loading buffer [4% (w/v) SDS, 200 mM DTT, 120 mM Tris/HCl, pH 6.8, 10 mM β-

mercaptoethanol, 20% (v/v) glycerin, 0.02% bromophenol blue] for 5 min at 95 C (target proteins

>200 kD at 80 C). Proteins were separated via SDS–PAGE and transferred to a nitrocellulose

membrane. The membrane was blocked for 1 h in 5% low fat milk or 5% BSA in TBS/0.1%

Tween-20 and immunoblotted with primary antibody in TBS/0.1% Tween-20/2.5% low fat milk

over night at 4 C, followed by HRP-conjugated secondary antibody incubation in PBS/0.1 %

Tween-20/5% low fat milk for 1 h. The immunoblots were developed using Lumi Light (Roche).

Signal detection was carried out with a chemi smart system (Vilber Lourmat).

Antibodies

Antibodies used for immunofluorescence were: gH2AX (Millipore 05-636) 1:400, 53BP1 (Bethyl

IHC-00001) 1:500, pATM (Rockland 200-301-400) 1:200, KAP1 (Abcam ab22553) 1:200, pKAP1

(Epitomics 3640-1) 1:200, H3K4me3 (Cell signaling 9727) 1:500, H3K9me3 (Abcam ab8898)

1:500, Rhodopsin (Cern901, kindly provided by Dr. W.J. deGrip, Nijmegen, the Netherlands)

1:500, Lim1+2 (DSHB 4F2) 1:100, Pax6 (DSHB) 1:50, Chx10 (Santa Cruz sc-365519) 1:100,

glutamine synthetase (BD Biosciences 610518) 1:100, CAR (Millipore ab15282) 1:100, Alexa

Fluor488 (Invitrogen A11001, A11008) 1:400, Alexa Fluor594 (Invitrogen A11005, A110012) 1:400,

Alexa Fluor647 (Dianova 715-605-150) 1:200. Antibodies for Western blotting were: gH2AX

(Millipore 05-636) 1:2000, 53BP1 (Bethyl IHC-00001) 1:1000, pATM[S1981] (Epitomics 2152-1)

1:1000, KAP1 (Abcam ab22553) 1:1500, pKAP1[S824] (Epitomics 3640-1) 1:10000, MDC1

(Novus Biologicals NB100-396) 1:1000, RNF8 (Abcam ab65739) 1:1000, Nrl (Santa Cruz

sc166087) 1:300, Ku80 (Santa Cruz sc81308) 1:1000, DNA-PKcs (Novus Biologicals NB100-658)

1:1000, LigIV (Santa Cruz sc28232) 1:2000, CtIP (Bethyl A300-488A) 1:1000, Rad51 (Abcam

ab63801) 1:2000, Artemis (GeneTex GTX100128) 1:1000, CHD3 (Cell Signaling Tech. 4241)

1:2000, CHD4 (Cell Signaling Tech. 4245) 1:2000, HDAC1 (Cell Signaling Tech. 5356) 1:1000,

HDAC2 (Cell Signaling Tech. 5113) 1:1000, RBAP46 (Cell Signaling Tech. 6882) 1:1000, MTA1

(Cell Signaling Tech. 5647) 1:1000, MBD3 (Cell Signaling Tech. 3896) 1:1000, SETDB1 (Bethyl

A300-121A) 1:1000, H3K9me3 (Abcam ab8898) 1:10000, H4K20me3 (Abcam ab9053) 1:3000,

SPOC1 (kindly provided by Dr. H. Will, Hamburg, Germany) 1:2000, H3 (Abcam ab10799)

1:3000, a-Tubulin (Santa Cruz sc8035) 1:2000, Erk2 (Santa Cruz sc153) 1:4000, GAPDH (Santa

Cruz sc25778), MRE11 (Abcam ab214) 1:1000, pMRE11[S264] (Abcam ab74148) 1:1000, NBS1

(Cell Signaling Tech. 3002) 1:1000, pNBS1[S343] (Cell Signaling Tech. 3001) 1:1000, Rhodopsin

(Cern901) 1:10000, HRP (Jackson ImmunoResearch 711-035-152) 1:80000, HRP (Santa Cruz

sc2031) 1:10000.

Pulsed-field gel electrophoresis

Pulsed-field gel electrophoresis was performed with explanted retinae and brains of adult mice.

Retinal explants were isolated and cultured as described for Western blotting. Brains were sliced

using razor blades before culturing. After 30 min in culture medium, samples were irradiated with

20 Gy at the same settings used for Western blotting and in vivo irradiation. At the end of the

repair time (15 min, 8 h and 24 h), samples were collected in ice-cold F12 medium. Retinae were

trypsinized for 5 min at 37 C (36 units/ml, Cell Systems). Digestion was stopped by adding FCS.

After several washes with ice-cold F12 medium, trituration was done within HBSS medium with a

fire-polished Pasteur pipette. Brain slices were cut into small pieces using micro-scissors and

incubated in an enzyme mix containing trypsin (90 units/ml, Cell Systems), hyaluronidase (600

units/ml, Roche) and collagenase (90 units/ml, Cell Systems) for 10 min. Trituration with fire-

polished Pasteur pipettes followed before cells were separated from residual tissue clumps with

cell strainers (40 and 20 µm, BD Bioscience). After centrifugation, cell pellets were embedded in

agarose plugs and lysed. Pulsed-field gel electrophoresis was carried out with a Power supply

system in 0.8% agarose gels. The gels were run at 14 C with linearly increasing pulse times from

50 to 5000 s for 66 h at field strength of 1.5 V/cm. Gels were stained with ethidium bromide and

photographed with a charge-coupled device camera under UV transillumination. Quantitative

analysis of released DNA fractions was performed with ImageJ.

Inhibitor treatment of retinal explants

For inhibitor studies, retinae were isolated and cultured as described for Western blotting. 10 µM

of the ATM inhibitor Ku60019 (Tocris) and 5 µM of the DNA-PK inhibitor Nu7441 (Tocris) were

added to the culture medium 1.5 h before IR. 15 min after irradiation, explants were placed in 5%

(vol/vol) neutral buffered formalin for 16 h. Formalin-fixed tissues were embedded in paraffin and

sectioned at a thickness of 4 μm.

Laser irradiation of retinal explants

Retinal explants were isolated and cultured as described for Western blotting. Explants were pre-

sensitized with 10 µg/ml Hoechst dye 33258 (Invitrogen) for 10 min at 37 C, 90% humidity and

5% CO2 followed by two washing steps with medium. Micro-irradiation was performed by using an

inverted confocal microscope (Leica TCS SP5 II) at 37 C and a 405 nm diode laser (laser power

100%) focused through a 63x Plan-Apochromat oil objective. Several fields of explants were

scanned in parallel lines. Retinal explants were fixed in 5% (vol/vol) neutral buffered formalin 5

min after irradiation and stained for immunfluorescence microscopy (see Immunofluorescence

analysis of tissues - without heating step in citrate buffer).

Serial immunostaining

For serial immunostaining of the retina (only applied for Fig. S1B, panel v), dewaxed sections

were first incubated with monoclonal mouse Chx10 antibody, followed by its detection with

secondary antibody. Then simultaneous staining with monoclonal mouse Pax6 and polyclonal

rabbit 53BP1 antibody was carried out, followed by their detection with secondary antibodies.

Nuclei were counterstained with DAPI.