H3.3 is deposited at centromeres in S phase as a
placeholder for newly assembled CENP-A in G phase.
Elaine M Dunleavy, Geneviève Almouzni, Gary H Karpen
To cite this version:
Elaine M Dunleavy, Geneviève Almouzni, Gary H Karpen. H3.3 is deposited at centromeres in
S phase as a placeholder for newly assembled CENP-A in G phase.. Nucleus, European Nuclear
Society, 2011, 2 (2), pp.146-57. <10.4161/nucl.2.2.15211>. <hal-00742956>
HAL Id: hal-00742956
https://hal.archives-ouvertes.fr/hal-00742956
Submitted on 17 Oct 2012
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A: Western analysis
tag
no tag
1X 2X 5X 1X 2X 5X
- CENP-A-SNAP
anti-CENP-A
- endogenous CENP-A
- Tubulin
anti-tubulin
- CENP-A-SNAP
anti-SNAP
B: TMR labelling and BTP blocking efficiency on whole cell nuclei
merge
TMR
anti-SNAP
DAPI
TMR
BTP
No chase
TMR
20 μM
C: TMR labelling and BTP blocking efficiency on chromatin fibers
TMR only
BTP block, No chase, TMR
merge
anti-CENP-A
TMR
Figure S1
H3.1-SNAP tagged cell line:
Immunofluorescence:
merge
anti-SNAP
DAPI
+ Triton-X
20 μM
Western analysis:
1X 2X 5X
- H3.1-SNAP
anti-H3
- endogenous H3
- H3.1-SNAP
anti-SNAP
short
exposure
long
exposure
H3.3-SNAP tagged cell line:
Immunofluorescence:
merge
anti-SNAP
DAPI
+ Triton-X
20 μM
no
Western analysis:
1X 2X 5X tag
- H3.3-SNAP
anti-H3
- endogenous H3
- H3.3-SNAP
anti-SNAP
short
exposure
long
exposure
Figure S2
A: BTP blocking efficiency on chromatin fibers at G1/S phase border
CENP-A
TMR
H3.1-SNAP
EdU
DAPI
CENP-A
TMR
H3.3-SNAP
EdU
DAPI
B: Replication-independent deposition of new H3.3 at centromeres
merge
TMR (new H3.3)
CENP-A
EdU
DAPI
H3.3 deposition in a
CENP-A domain
H3.3 deposition in an
interspersed domain
C: Replication and new H3 deposition in centromere flanking regions
new H3.1 deposition in flank
new H3.1 deposition in flank
merge
TMR (new H3.1)
CENP-A
EdU (5 hrs)
DAPI
new H3.3 deposition in flank
new H3.3 deposition in flank
merge
TMR (new H3.3)
CENP-A
EdU (5 hrs)
DAPI
Figure S3