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First published online December 20, 2006
doi: 10.1242/10.1242/jcs.03327

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C-terminal domains deliver the DNA replication factor Ciz1 to the nuclear matrix

¹ CRISTAL, School of Medicine, Leeds University, LS2 9JT, UK
² Biology Department, University of York, Heslington, York, YO10 5YW, UK

View larger version (42K): [in this window] [in a new window]   Fig. 1. (A) Schematic representation of the mouse Ciz1 protein, which is approximately 70% homologous to human Ciz1. Several of the amino acid sequence features detected by Prosite (http://us.expasy.org/prosite/) are indicated in the key. Analysis of expressed sequence tags that map to the Ciz1 Unigene Cluster Hs.212395 (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=unigene) indicates that at least four exons are alternatively spliced in transcripts from a wide range of tissues. Three of these (indicated in red) are absent from a Ciz1 variant cloned from a mouse embryo-derived cDNA library, designated ECiz1 (Coverley et al., 2005). From left to right these are part exons 2, 6 and 8. This analysis focuses on full-length Ciz1 (845 amino acids) and the ECiz1 variant (708 amino acids). Green ovals indicate the presence of a GFP tag attached to the N terminus of both variants. (B) Ectopic GFP-Ciz1 and GFP-ECiz1 form foci in the nucleus of NIH3T3 cells. Representative confocal images of GFP fluorescence taken 20 and 44 hours after transfection are shown. Bar, 10 µm.

View larger version (31K): [in this window] [in a new window]   Fig. 2. Formation of sub-nuclear speckles requires both N- and C-terminal domains. (A) Removal of 273 amino acids from the C-terminal end of Ciz1 to generate GFP-Ciz1 N572, or (B) from ECiz1 to generate GFP-ECiz1 N572, alters the sub-nuclear distribution of both fragments, leading to formation of large intra-nuclear bodies. The Ciz1-derived fragment accumulates in these bodies within the first 20 hours of transfection, whereas the ECiz1-derived fragment takes up to 2 days. (C) The C-terminal 275 amino acids (GFP-C275) is initially excluded from the nucleus, but forms a non-focal pattern in the nucleus of most cells within 2 days of transfection, and remains associated with chromosomes during mitosis. (D) GFP-708-830, which spans the MH3 homology domain, behaves in a similar way to C275, and also partitions with condensed chromosomes during mitosis. Constructs were transfected into NIH3T3 cells and representative confocal fluorescent images taken at the indicated times. Bar, 10 µm.

View larger version (52K): [in this window] [in a new window]   Fig. 3. Endogenous Ciz1 resists salt extraction. (A) Western blot of total protein (T) from cycling NIH3T3 cells after separation using 8% SDS-PAGE. The Triton X-100 soluble fraction (S) and insoluble fraction (P) are also shown. Endogenous Ciz1 isoforms (p100, p125a and p125b) are detected with Ciz1 polyclonal antibody 1793. Mcm2 is shown as a control. (B) Sequential extraction of Triton X-100-resistant nuclei with increasing NaCl in CSK buffer. The protein present in the supernatant fraction (SN) is shown. PCNA was used as the control. (C) Ciz1 in extracted nuclei detected by immunofluorescence (red). Nuclei are counterstained with Hoechst 33258 (blue). (D) Equivalent numbers of Triton X-100-resistant nuclei from a cycling population, a population treated with thymidine for 24 hours prior to harvesting, and a quiescent population of NIH3T3 cells were sequentially extracted with 0.5 M and 2 M NaCl. Intervening wash steps, with 0.5 M NaCl are marked with W. A significant proportion of Ciz1 p100 isoform from all three populations is resistant to 2 M NaCl, remaining in the pellet fraction (P) after extraction. SN, supernatant fractions.

View larger version (51K): [in this window] [in a new window]   Fig. 4. Ciz1 resists nuclease extraction. (A) Western blot showing partitioning of Ciz1 in Triton X-100-resistant nuclei after digestion with DNase 1. PCNA is released into the soluble fraction (S). Digestion does not release Ciz1 into the soluble fraction; all Ciz1 isoforms are retained in the pellet (P). (B) Nuclei were visualized by bright-field microscopy at 600x magnification (top panels) or by fluorescence microscopy after staining with Hoechst 33258 (lower panels, blue). Bar, 10 µm. (C) Immunofluorescence image of Ciz1 (red) in DNase 1 or mock-treated nuclei after extraction with 0.5 M or 2 M NaCl as indicated. (D) Ciz1 in nuclei treated with the protein-protein cross-linker DTSP before DNase 1 treatment and extraction with 2 M NaCl. (E) Colocalization of matrix-associated newly synthesized DNA (green) with matrix-associated Ciz1 foci (red) in an early S phase nucleus. Newly synthesized DNA was detected by incorporation of biotinylated-dUTP during in vitro synthesis reactions. Merged image shows very significant overlap (yellow) in this nucleus. Images of Hoechst fluorescence (blue) were taken under equivalent conditions so that they reflect the level of nucleic acid remaining in mock treated and DNase 1 digested nuclei.

View larger version (22K): [in this window] [in a new window]   Fig. 5. Ectopic Ciz1 is immobilized through C-terminal interactions. (A) Ectopically expressed GFP-ECiz1 (green) in unextracted nuclei, and after extraction with Triton X-100, or Triton X-100 followed by DNase 1 and 0.5 M NaCl. (B) Ectopically expressed GFP-C275 after the same treatments and (C) GFP-ECiz1 N471 in untreated nuclei and after extraction with Triton X-100. DNA is stained with Hoechst 33258 (blue) and imaged to reflect the DNA content after each treatment.

View larger version (27K): [in this window] [in a new window]   Fig. 6. GFP-ECiz1 N471 resists detergent extraction after cross-linking with DTSP, but remains susceptible to salt extraction whereas GFP-C275 is resistant to both treatments. Twenty-four hours after transfection with the indicated C- or N-terminal Ciz1 fragments, cells were cross-linked with DTSP then extracted with Triton X-100 (left panels) or Triton X-100 with 0.5 M NaCl (right panels). Total nuclei were visualised after staining with Hoechst 33258 (blue) and those expressing GFP are indicated with arrows. Bar, 10 µm.

View larger version (28K): [in this window] [in a new window]   Fig. 7. Cell-cycle dependent immobilization of GFP-Ciz1. (A) Histogram shows the proportion of transfected cells that retain GFP-fluorescence in the nucleus after extraction with Triton X-100. Twenty-four hours after transfection, full-length ECiz1 is retained in all transfected cells, GFP-ECiz1 N471 in virtually none, whereas GFP-C275 and GFP-708-830 was retained in 27% and 29%, respectively. (B) The proportion of transfected cells with immobilized GFP-C275 increases to almost 80% by 48 hours after transfection, but this is significantly reduced when cells are prevented from cycling during the second 24 hours by exposure to thymidine. (C) The proportion of cells with immobilized GFP-C275 increases during late G1 or early S phase. Cells were arrested with thymidine for 24 hours, released for 10 hours then given a second thymidine block for a further 14 hours. Arrested cells were transfected with GFP-C275 then released for the indicated times. The cell cycle profile of each population is shown. (D) The proportion of cells with immobilized GFP-C275 increases during late G1 or early S phase, but remains constant in populations arrested for a further 10 or 22 hours in S phase. Transfection frequencies were the same at the beginning and end of the time course (not shown). Histograms show the average of three data sets with standard errors.

View larger version (24K): [in this window] [in a new window]   Fig. 8. (A) Schematic representation of Ciz1 attached to an insoluble nuclear structure via a C-terminal `anchor' (yellow circle). Red triangles indicate alternatively spliced sequences that are conditionally absent from the N-terminal (replication active) half of Ciz1. (B) Speculative model showing possible role for Ciz1 as a mediator between replication factories (grey circles) and chromatin-associated replication complexes (grey barrels).