1

Materials and Methods

FRAP microscopy: Images were acquired on a Leica TCS SP confocal microscope with a

Leica PL APO 100x/1.4 oil immersion lens. For GFP excitation we used the 488 nm line

of an Argon Krypton laser and fluorescence emission was collected between 500 to 560

nm. All experiments were done at 37°C. For the FRAP experiments a pre-bleach image

was acquired by averaging four consecutive images. Then a single spot on the cell was

bleached with a laser pulse, lasting between 0.1 and 0.5 s at 100% power without

scanning. The objective lens used produced a laser spot of approximately 0.2 µm on the

cell, but the bleached area was significantly larger, typically in the order of 1 to 2 µm.

Single section images were then collected at 5-s intervals. For this part of the imaging the

laser power was typically attenuated to 10% of the maximum.

Fluorescence Recovery After Photobleaching (FRAP): Plots were generated from

background subtracted images using the public domain software ImageJ

(http://rsb.info.nih.gov/ij) following the analysis described in (1). In brief the signal I was

measured in an area of interest (AOI) and normalised to the change in the total

fluorescence due to the bleach pulse and the imaging: I = (To/Io)* It/Tt; where To and Tt

is the total cellular fluorescence in the pre-bleach and post-bleach images, respectively,

and Io is the average intensity in the AOI in the pre-bleach image.).

Double staining microscopy: GFP and DAPI co-localization images were acquired using

a Deltavision system (Applied Precision, USA) and a cooled CCD camera (Photometrics

CH350L) on an Olympus IX70 inverted microscope, using a 100x/1.35 UPlanApo

objective lens.

2

Figure S1. The GFP tag does not interfere with HP1β chromatin binding in transgenic T

cells and does not affect binding to methylated histone H3 peptides in vitro. (A) T cells

were isolated from a transgenic mouse expressing GFP-HP1β or a non-transgenic control

mouse. Lysates were prepared from whole cells (WC) or cells sequentially extracted with

hypotonic buffer (CE), nuclear extraction buffer lacking (NE 0) or containing 400 mM

NaCl (NE400). The residual chromatin pellet (NE P) was also analyzed after addition of

SDS-PAGE sample buffer and sonication. Volumes of each fraction derived from the

same number of cells were analysed by Western blotting using a HP1β monoclonal

antibody that detects both HP1β and GFP-HP1β (lane M). Asterisk indicates a protein

that cross-reacts with the HP1β antibody and is found in both transgenic and control

mouse T cells. (B) Recombinant HP1β and GFP-HP1β were purified from E. coli as

HIS6-tagged fusion proteins and tested for their ability to bind to beads containing short

peptides derived from the N-terminal domain of histone H3. The peptides were

dimethylated (me2), trimethylated (me3) or acetylated (Ac) at either lysine-9 (K9) or

lysine-27 (K27). The amount of protein remaining bound to the H3 peptide beads after

extensive washes in binding buffer is shown alongside the equivalent of 100% of the

input protein (lanes labeled I). Lanes HM and LM contain protein size markers.

Figure S2. HP1α co-localizes with GFP-HP1β and DAPI dense heterochromatic foci in

transgenic T cells. Transgenic peripheral lymphocytes were fixed in paraformaldehye and

stained with anti-HP1α antibody (HP-1H5 mouse monoclonal IgG1 from P.

Chambon). (A) DAPI staining identifies heterochromatic foci. (B) GFP fluorescence

reveals co-localization of GFP-HP1β with these foci in T cells but not non-T cells also

present on the slide. (C) Immunofluorescent staining with HP1α antibodies reveal co-

localization with both DAPI dense and GFP foci.

3

Figure S3. Immunofluorescent staining of primary murine lymphocytes reveals

acetylated histone H3 is located in euchromatin and largely excluded from

heterochromatic foci. (A) DAPI staining identifies heterochromatic foci. (B) Acetylated

histone H3 is excluded from heterochromatic foci and present in the euchromatic regions

of the nuclei. (C) Merged images from (A) and (B). The anti-acetyl-histone H3 rabbit

polyclonal IgG was obtained from Upstate.

Figure S4. His6-GFP-HP1β forms both homo- and heterodimers with HP1α and HP1γ.

(A) Recombinant His6-GFP-HP1β purified from E. coli (lane 1, 100% of input protein)

was incubated with beads containing either GST alone (lane 2) or GST fusions of the

three mouse HP1 family members (lanes 3 to 5). After extensive washes, the amount of

His6-GFP-HP1β that remained bound to beads was detected by western blotting with

GFP antibodies. The same result was obtained by Nielson et al in the absence of GFP (2).

(B) His6-GFP-HP1β and His6-HP1β were individually purified from E. coli and their

native sizes analyzed by gel filtration through a Superdex 200 column in 50 mM Sodium

Phosphate pH 7.2, 150 mM NaCl, 0.01% Triton X-100, 1 mM DTT. The position of the

peaks of size markers co-fractionated with purified His6-GFP-HP1β and His6-HP1β is

shown. In both cases very little protein is present as a monomer.

Figure S5. GFP-HP1β is highly mobile and GFP fluorescence does not recover after

photobleaching. FRAP on paraformaldehyde fixed cells revealed that photobleaching is

irreversible within the experimental time frame (A and B), and reiterated bleaching of a

single point resulted in overall loss of fluorescence of the entire live nucleus (C). (A)

FRAP in a fixed cell. The pre-bleach image is followed by a time-lapse series of confocal

4

images taken following targeted bleaching of a fixed cell (incubated for 20 min in 1%

paraformaldehyde) the fluorescence intensity of the bleached spot does not recover. (B)

A graphical representation of the result obtained from 10 cells ± SEM. (C) A prebleach

image was obtained, this was following by targeted reiterated bleaching (0.4 s) of a

euchromatic spot every 5 s for 30 s. The cells were then imaged six times at 30 s

intervals. Each point represents the analysis of a minimum of 10 cells ± SEM. The graph

shows mean fluorescence intensity plotted against recovery time after the 30 s bleach

period. Live cells (filled squares): as a result of photobleaching the whole nucleus

becomes dimmer indicating rapid movement of GFP-HP1β through the bleach spot. After

150s there is no recovery in fluorescence intensity indicating that photobleaching of GFP-

HP1β was not reversible in this timeframe. Fixed cells (open triangles): significant

bleaching occurred only at the bleach spot [as in (A)]; there was no detectable recovery

of fluorescence with time (150 s) indicating the irreversible nature of the photobleaching.

References

1. R. D. Phair, T. Misteli, Nature 404, 604 (2000).2. A. L. Nielsen et al., Mol. Cell 7, 729 (2001)

A

NE0

NE400

NEP

WC

CE

NE0

NE400

NEP

M

GFP-HP1β

HIS6-HP1βHP1β

*

B

WC

1

WC

2

CE

3 4 5 6 7 8 9 10 11 12

10 11 121

HM

2

LM

3

I

4

H3K9me2

5

H3K9me3

6

H3K27me2

7

H3K27me3

8

H3K9ac

9

H3K27ac

I H3K9me2

H3K9me3

H3K27me2

H3K27me3

H3K9ac

H3K27ac

13 14 15 16

HIS6-GFP-HP1β

HIS6-HP1β

Size(kDa)

116-

68-

31-

46-

transgenic non-transgenic

Figure S1

A

B

C

Figure S2

A B C

Figure S3

GST

IN

5

His

6-G

FP

-HP

1ββββ −−−−

2325

2729

3133

35IN

His

6-G

FP

-HP

1ββββ −−−−

His

6-H

P1ββββ

−−−−

443

240

158

6845

Siz

eM

arke

rs(k

Da)

Fra

ctio

n

GST-HP1αααα

GST-HP1ββββ

GST-HP1γγγγ

A B

Fig

ure

S4

43

21

Figure S5