spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 3 March 2009
doi: 10.1242/jcs.034793

Journal of Cell Science 122, 937-946 (2009)
Published by The Company of Biologists 2009
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplementary Material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Briers, S.
Right arrow Articles by Sutherland, H. G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Briers, S.
Right arrow Articles by Sutherland, H. G.
Right arrowPubmed/NCBI databases
*Domain*Gene
*GEO Profiles*HomoloGene
*Protein*UniGene
*Substance via MeSH
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

KRAB zinc-finger proteins localise to novel KAP1-containing foci that are adjacent to PML nuclear bodies

Stephanie Briers*, Catherine Crawford*, Wendy A. Bickmore and Heidi G. Sutherland{ddagger}

MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Crewe Road, Edinburgh EH4 2XU, UK


Figure 1
View larger version (43K):
[in this window]
[in a new window]

  		Fig. 1. Gene-trap of Zfp647 and subcellular localisation of Zfp647 fusion proteins with β-gal and GFP. (A) Schematic diagram of gene-trap of Zfp647 locus in the ES492 cell line. Arrows indicate position of gene-trap integration into the Zfp647 locus to generate a fusion protein with β-geo (β-gal and neomycin). (B) Deconvolved images from single optical sections of immunofluorescence on ES492 cells before (–RA) and after (+RA) 6 days of differentiation with retinoic acid. Gene-trapped Zfp647 was detected with antibody that detects β-gal (red in merge). Co-staining was with an antibody that recognises KAP1 (green in merge). DNA was counterstained with DAPI (blue in merge). Double arrowheads indicate colocalisation of Zfp647-β-gal fusion protein with KAP1 at pericentric heterochromatin. Scale bars: 5 µm. (C) NIH3T3 cells transiently transfected with: GFP-tagged Zfp647 construct lacking the zinc fingers (GFP-Zfp{Delta}ZF); a construct lacking the KRAB domain (GFP-Zfp647{Delta}KRAB); or full-length Zfp647 (GFP-Zfp647). GFP signal is on the left, DAPI staining is on the right. Double arrowheads show concentrations of GFP-Zfp647 at constitutive heterochromatin (DAPI-bright foci). Arrow indicates a concentration of GFP-Zfp647 at a site that does not correspond with constitutive heterochromatin. Scale bars: 5 µm. (D) Quantification of the various subcellular localisation patterns of β-gal- and GFP-tagged Zfp647.

 

Figure 2
View larger version (54K):
[in this window]
[in a new window]

  		Fig. 2. Detection of endogenous Zfp647 in cell extracts. (A) Western blot with antibody raised in sheep against Zfp647 on extracts from embryos wild-type (+/+) or homozygous mutant (–/–) for the gene-trap into Zfp647. (B) Western blot with antibody raised in rabbit against Zfp647 on extracts from embryos wild-type (+/+) or homozygous mutant (–/–) for the gene-trap into Zfp647, and in whole cell (T), cytoplasmic (C) and nuclear (N) extracts of mouse NIH3T3 cells. (C) GelCode Blue-stained gel of identical amounts of protein samples as loaded in Fig. 2A,B. The position of the histone proteins is indicated.

 

Figure 3
View larger version (55K):
[in this window]
[in a new window]

  		Fig. 3. Detection of endogenous Zfp647 in cells. (A) Immunofluorescence on undifferentiated (–RA) and day 6 differentiated (+RA) OS25 cells with antibodies that recognise Zfp647 (red in merge) and KAP1 (green in merge). DNA was counterstained with DAPI (blue in merge). Double arrowheads indicate KAP1 concentrated at domains of constitutive heterochromatin. Arrows indicate nucleoplasmic foci of Zfp647 and KAP1 that overlap. Scale bars: 5 µm. (B) Percentage of cells with KAP1 localised at the constitutive pericentric heterochromatin with days of differentiation of OS25 ES cells. (C) Deconvolved images from single optical sections of immunofluorescence on undifferentiated (–RA) and day 6 differentiated (+RA) OS25 cells using antibodies that recognise Zfp647 (red) and KAP1 (green). DNA was counterstained with DAPI (blue). Associated nucleoplasmic foci of Zfp647 and KAP1 are shown enlarged in insets. Scale bars: 5 µm. (D) Percentage of cells that show foci of Zfp647 (white bars) and Zfp647 foci associated with KAP1 foci (black bars) with days of differentiation of OS25 ES cells.

 

Figure 4
View larger version (61K):
[in this window]
[in a new window]

  		Fig. 4. Colocalisation of NT2 and KAP1 in nucleoplasmic foci. (A) Immunofluorescence on 12.5 dpc primary mouse embryonic fibroblasts (MEFs) with antibodies that recognise Zfp647 (green in merge) and KAP1 (red in merge). DNA was counterstained with DAPI (blue in merge). Double arrowheads indicate KAP1 concentrated at domains of constitutive heterochromatin. Arrows indicate nucleoplasmic foci of Zfp647 and KAP1 that overlap, and others are shown enlarged in inset. Scale bar: 5 µm. (B) Immunofluorescence on undifferentiated (–RA) and day 8 differentiated (+RA) OS25 cells with antibodies that recognise the KRAB-Zfp NT2 (green in merge) and KAP1 (red in merge). DNA was counterstained with DAPI (blue in merge). Double arrowheads indicates KAP1 concentrated at domains of constitutive heterochromatin. Arrows indicate nucleoplasmic foci of KAP1 and NT2, and are shown enlarged in the inset. Scale bars: 5 µm.

 

Figure 5
View larger version (77K):
[in this window]
[in a new window]

  		Fig. 5. HP1s concentrate at KAKA foci. (A) Deconvolved images from single optical sections of immunofluorescence on day 6 differentiated OS25 cells with antibodies that recognise Zfp647 (red in merge) and either HP1{alpha} or β (green in merge). DNA was counterstained with DAPI (blue in merge). Double arrowheads indicate HP1 concentrated at domains of constitutive heterochromatin. Arrows indicate nucleoplasmic foci of ZFP647 with HP1, and are shown enlarged in insets. (B) Immunofluorescence as in A but in NIH3T3 cells and examined by confocal microscopy. Double arrowhead indicates HP1 concentrated at a domain of constitutive heterochromatin. Arrows indicate nucleoplasmic foci of Zfp647 with HP1, and are shown enlarged in insets. (C) Deconvolved image from single optical section of immunofluorescence on differentiated OS25 cells with antibodies that that recognise SETDB1 (red in merge) and KAP1 (green in merge). DNA was counterstained with DAPI (blue in merge). Nucleoplasmic foci of Zfp647 and SETDB1 that coincide are shown enlarged in insets. Scale bars: 5 µm.

 

Figure 6
View larger version (63K):
[in this window]
[in a new window]

  		Fig. 6. Association of KAKA foci with PML-NBs. Deconvolved images from two optical sections of day 6 differentiated OS25 cells, separated by 0.5 µm in the z-axis, after immunofluorescence with antibody that recognises Zfp647 (red) and, in green, either CREST anti-serum (A), antibody that recognises PML (B), SUMO1 (C) or SUMO2/3 (D). DNA was counterstained with DAPI (blue). Scale bars: 5 µm.

 

Figure 7
View larger version (56K):
[in this window]
[in a new window]

  		Fig. 7. KRAB-ZFP, HP1 and KAP1 localisation in Suvar39h knockout cells. (A) Immunofluorescence on day 8 differentiated ES cells null for both Suv39h1 and h2 (Suv39h dn), and on the wild-type parental controls for these cells (wt41) with antibodies that detect Zfp647 (green in merge) and HP1β (red in merge). DNA was counterstained with DAPI (blue in merge). Arrows indicate nucleoplasmic foci of Zfp647 and HP1β, and some are shown enlarged in inset. Double arrowheads indicate HP1β concentrated at domains of constitutive heterochromatin in wild-type cells, but delocalised in Suv39h dn cells. Scale bars: 5 µm. (B) As in A but with antibodies that recognise Zfp647 (green in merge) and KAP1 (red in merge). Arrows indicate nucleoplasmic foci of Zfp647 and KAP1. Double arrowheads indicate KAP1 concentrated at domains of constitutive heterochromatin in wild-type cells, but delocalised in Suv39h dn cells. Scale bars: 5 µm.

 

Figure 8
View larger version (46K):
[in this window]
[in a new window]

  		Fig. 8. KAP1 and sumoylation. (A) Immunofluorescence on day 8 differentiated ES cells null for both Suv39h1 and h2 (Suv39h), and on the wild-type parental controls for these cells (wt41) with antibodies that detect KAP1 (green in merge) and SUMO1 (red in merge). Double arrowhead indicates concentration of SUMO1 with domains of constitutive heterochromatin in wild-type cells, but delocalised in Suv39h dn cells. Arrows indicate SUMO1 in PML-NBs that are juxtaposed to KAKA foci. Scale bars: 5 µm. (B) Western blot with antibody that detects KAP1 in extracts prepared from undifferentiated (–RA) and day 10 differentiated (+RA) ES cells null for both Suv39h1 and h2 (Sv39dn), and on the wild-type parental controls for these cells (wt41). Arrow indicates the unmodified form of KAP1 and double arrow indicates sumoylated KAP1. The panel below shows the GelCode Blue-stained histones as a loading control.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2009