QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 4 March 2003
doi: 10.1242/jcs.00324

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ladomery, M. Articles by Hastie, N. Search for Related Content PubMed PubMed Citation Articles by Ladomery, M. Articles by Hastie, N.

Expression in Xenopus oocytes shows that WT1 binds transcripts in vivo, with a central role for zinc finger one

Michael Ladomery^1,*, John Sommerville², Sarah Woolner^1,, Joan Slight¹ and Nick Hastie^1,

¹ MRC Human Genetics Unit, Western General Hospital, Crewe Rd, Edinburgh EH4 2XU, UK
² School of Biology, Bute Medical Buildings, University of St Andrews, St Andrews, Fife KY16 9TS, UK
^* Present address: Centre for Research in Biomedicine, Faculty of Applied Sciences, University of the West of England, Coldharbour Lane, Bristol BS16 1QY, UK
Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK

View larger version (30K): [in a new window]   Fig. 1. Zinc finger two is required for nuclear targeting. (A) Epitope tagged mouse WT1 constructs. Act, activation domain; Dim, dimerisation domain; Rep, repression domain; RRM, putative RNA recognition motif; T7, tag; ZF, zinc finger. Insertions due to alternative splicing: `17 aa' and `KTS'. (B) Constructs were transiently transfected into Cos7 cells. DAPI stain (left) and corresponding immunofluorescence of tagged protein (right) are shown.

View larger version (75K): [in a new window]   Fig. 2. Zinc finger one is required for co-sedimentation with RNP in mammalian cells. Extracts obtained from transfected Cos7 cells were applied to 20-60% Nycodenz gradients. Western blots: (a) WT1 (+KTS); (b) WT1 (–KTS); (c) RF34; (d) RF234; (e) RF1234; (f) CTF1 (+KTS); (g) native EGR1; (h) U5 snRNP associated splice factor p116; (i) WT1 pre-mRNA (RT-PCR).

View larger version (80K): [in a new window]   Fig. 3. Localisation of tagged mouse WT1 in nuclear structures of Xenopus oocytes. (A-D) Lampbrush chromosome bivalent showing the location of WT1 (+KTS) as FITC image (A), location of p116 as TRITC image (B), corresponding DAPI (C) and phase-contrast (D) images. In addition to locating to the lateral loops of the chromosomes, both WT1 (+KTS) and endogenous p116 locate to Cajal bodies (arrows in A, B and D) and to smaller nuclear particles. (E,F) In images obtained at higher power, WT1 (+KTS) is seen to be specific to the B-snurposomes (arrows) located on the surfaces of the Cajal bodies (E, FITC and F, phase contrast). (G-I) Expression of WT1 (–KTS) shows that it is restricted to the lateral loops of the chromosomes (G) and is not found in Cajal bodies (arrows in I) or in other nuclear particles. The chromosomal DNA axes are stained with DAPI (H).

View larger version (42K): [in a new window]   Fig. 4. Nuclear import and localisation of T7-tagged WT1; CT (C-terminus +KTS, amino acids 233-449); CTF1 (+KTS); and EGR1, 18 hours after injecting expression plasmids into the GVs of stage IV oocytes. (A) Western blot. Sets of 20 nuclei and cytoplasms were isolated under oil, and protein extracts equivalent to four nuclei (N) and one cytoplasm (C) were loaded. (B-M) Nuclear spreads were immunostained with anti-T7 tag and counterstained with DAPI. Although CT (B-D) immunostained lampbrush chromosome loops similarly to full-length WT1 (Fig. 3), expression of CTF1 appeared to cause chromosome compaction, loop retraction and more intense labelling of Cajal bodies (arrows in E and G). Expression of EGR1 also resulted in chromosome compaction with immunostaining (H) close to the DNA axes (I). As seen in phase contrast (J), lateral loops were less obvious and Cajal bodies (arrows in J) were not immunostained (H). Inclusion of WT1-derived zinc finger one into EGR1 (EGR+F1), however, mimicked full-length WT1 (–KTS) (K-M). Bar, 10 µm.

View larger version (74K): [in a new window]   Fig. 5. Nuclear import and localization of co-expressed isoforms: T7-tagged WT1 (+KTS) and Myc-tagged WT1 (–KTS) at 30 hours after plasmid injection. Western blot (A) showing the expression of both full-length constructs, and (B) of full-length Myc-tagged WT1 (–KTS) and T7-tagged CT (C-terminus +KTS). The slower migration of WT1(–KTS) is due to the larger size of the Myc versus the T7 tag. Co-expression of full-length isoforms results in the absence of signal in snurposomes: FITC image detecting the T7 tag (C), corresponding DAPI (D) and phase-contrast (E) images, highlighting Cajal bodies (arrows in E). In contrast, co-expression of only the C-terminus of the +KTS isoform, together with full-length –KTS, results in immunostaining of snurposomes: FITC image detecting the T7-tag (F), corresponding DAPI (G) and phase-contrast (H) images, highlighting B-snurposomes and Cajal bodies (arrows in F and H). Bar, 10 µm.

View larger version (56K): [in a new window]   Fig. 6. Effect of the expression of T7-tagged WT1 (–KTS) and EGR1 on transcription from lampbrush chromosomes. For immunostaining, expression plasmids were co-injected with BrUTP (0.1 µg) or [3H]-labelled uridine (0.1 µCi) into the GVs of stage IV oocytes. (A-C) Immunostaining of a chromosome bivalent from oocytes expressing WT1 with anti-BrU showing extensive labelling of lateral loops (A); the DAPI-stained DNA axes (B) and the phase-contrast image (C) are shown. (D-F) Immunostaining of a chromosome bivalent from oocytes expressing EGR1, with anti-BrU showing compacted chromosomes and limited labelling of lateral loops (D). The DAPI-stained DNA axes (E) and the phase-contrast image (F) are also shown. Bar, 10 µm. (G) Incorporation of [3H]-labelled uridine into noninjected oocytes (open circles), noninjected oocytes incubated in the presence of 5 µg/ml actinomycin D (black circles), and oocytes expressing WT1 (open squares) and EGR1 (black squares). Each time-point represents incorporation per oocyte averaged from five oocytes.

View larger version (102K): [in a new window]   Fig. 7. Localisation of ectopically expressed CT (WT1 C-terminus +KTS), CTF1, EGR1 and EGR1+F1 in the extrachromosomal nucleoli of stage IV oocytes. On expressing the T7-tagged proteins for 30 hours, immunostaining was seen to extend to the nucleoli. Although CT (A-C) and WT1 (not shown) located throughout the nucleolus, CTF1 was more localised to the DNA-containing fibrillar centres as seen in DAPI staining (E). Overexpressed EGR1 gave discrete localisation (G) at the DAPI-stained fibrillar centres (H). By contrast, EGR+F1 resembled CT (J-L). Bar, 10 µm.

View larger version (48K): [in a new window]   Fig. 8. Stability of interaction of WT1, CT and CTF1 with the nascent RNA transcripts of lampbrush chromosomes. Isolated nuclei were spread and washed extensively in solutions containing 200 mM, 400 mM and 600 mM NaCl. Although the chromosomes became more condensed in the higher salt conditions, most of the anti-T7 signal on the loop matrix (A,D,G) was retained, albeit in a more compact form. The corresponding DAPI (B,E,H) and phase-contrast (C,F,I) images are shown. Bar, 10 µm. (J,K,L) Salt stability of RNP immunoprecipitated from oocytes expressing WT1, CT and CTF1 (see text for details).

View larger version (72K): [in a new window]   Fig. 9. Nuclear extract from AC29 mouse mesothelioma cells was applied to anion exchange and a salt wash gradient applied. (A) Protein stain, showing flow through (FT) and increasing salt elutions. (B) Western blot showing the distribution of native WT1, the splice factors p116 and U2AF65, and EGR1. (C) Presence of RNA in fractions FT, 7, 9 and 11. Small nuclear RNA species, end-labelled by T4 RNA ligase, are shown above. The arrow points to U1 snRNA. WT1 pre-mRNA is shown below (RT-PCR).

View larger version (64K): [in a new window]   Fig. 10. Comparison of zinc fingers in mouse WT1 and other transcription factors with closely related Krüppel class zinc fingers. Note that WT1 has four zinc fingers, whereas the others have three. WT1 zinc fingers 1-3 align with corresponding zinc fingers in SP1, SP3, MGIF, GKLF and KLF2. EGR1 zinc fingers align with WT1 zinc fingers 2-4. WT1 zinc finger one has distinct amino acids (shown in bold).