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J. Biol. Chem., Vol. 276, Issue 26, 23974-23985, June 29, 2001

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A New Spectrin, IV, Has a Major Truncated Isoform That Associates with Promyelocytic Leukemia Protein Nuclear Bodies and the Nuclear Matrix^*

William T. Tse^§, Ju Tang, Ou Jin, Catherine Korsgren, Kathryn M. John, Andrew L. Kung, Babette Gwynn^¶, Luanne L. Peters^¶, and Samuel E. Lux^¶

From the  Division of Hematology/Oncology, Children's Hospital, and the Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115 and ^¶ The Jackson Laboratory, Bar Harbor, Maine 04609

Received for publication, October 12, 2000, and in revised form, March 6, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We isolated cDNAs that encode a 77-kDa peptide similar to repeats 10-16 of -spectrins. Its gene localizes to human chromosome 19q13.13-q13.2 and mouse chromosome 7, at 7.5 centimorgans. A 289-kDa isoform, similar to full-length -spectrins, was partially assembled from sequences in the human genomic DNA data base and completely cloned and sequenced. RNA transcripts are seen predominantly in the brain, and Western analysis shows a major peptide that migrates as a 72-kDa band. This new gene, spectrin IV, thus encodes a full-length minor isoform (SpIV1) and a truncated major isoform (SpIV5). Immunostaining of cells shows a micropunctate pattern in the cytoplasm and nucleus. In mesenchymal stem cells, the staining concentrates at nuclear dots that stain positively for the promyelocytic leukemia protein (PML). Expression of SpIV5 fused to green fluorescence protein in cells produces nuclear dots that include all PML bodies, which double in number in transfected cells. Deletion analysis shows that partial repeats 10 and 16 of SpIV5 are necessary for nuclear dot formation. Immunostaining of whole-mount nuclear matrices reveals diffuse positivity with accentuation at PML bodies. Spectrin IV is the first -spectrin associated with a subnuclear structure and may be part of a nuclear scaffold to which gene regulatory machinery binds.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Spectrin is an important component of the membrane skeleton attached to the inner leaf of the lipid bilayer of plasma membranes. First described in the erythrocyte (1), spectrins are found in all or almost all cells (2-4). In erythrocytes, an intact spectrin-based membrane skeleton is critical for the structural integrity of the plasma membrane. Defects in its components are associated with red cell fragility and premature destruction in the human diseases hereditary spherocytosis and elliptocytosis and their animal models (5). The function of a spectrin-based plasma membrane skeleton in non-erythroid tissues is less well defined, but it is hypothesized to be important in establishing and maintaining the asymmetric distribution of proteins in specialized plasma membrane domains, particularly in polarized cells (6).

Recently, components of a spectrin-based membrane skeleton have also been found in several intracellular organelles. Isoforms of spectrin and ankyrin exist in Golgi membranes (7-9), lysosomal membranes (10), and secretory vesicles (11-14). Spectrin also associates with actin-related protein 1 (centractin), a subunit of the dynactin complex, which associates with dynein and transports vesicles along microtubules in the secretory pathway (15). A spectrin-based membrane skeleton attached to intracellular organelles may provide a structural framework to anchor the vesicular transport machinery (16-19). The potential role of a spectrin-based membrane skeleton in the nucleus is unclear. There are interesting recent reports indicating that spectrin II is part of a nuclear protein complex involved in repair of DNA interstrand cross-links (20-22). Whether II-spectrin binds with a -spectrin partner in the nucleus to form a membrane skeleton is unknown. We have previously described spectrin III (see footnote 1 for nomenclature),¹ which associates with the Golgi and intracellular vesicles (9). We now identify another intracellular -spectrin, spectrin IV, which has a major truncated isoform (IV5) and a full-length isoform (IV1). Spectrin IV resides in the cytoplasm, where it may attach to vesicles, and in the nucleus, where it associates with PML² bodies and the nuclear matrix.

While this manuscript was in revision, Berghs et al. (23) independently described spectrin IV and four of its isoforms: IV1-IV4. Their IV1 isoform corresponds to our full-length spectrin IV, also named IV1. The 77-kDa isoform (IV5) described here is the major isoform of spectrin IV. It was not reported by Berghs et al. and could not have been detected with the antibodies they employed (23).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Molecular Cloning and DNA Analysis-- Search of the GenBank^TM data bases was performed using the NCBI BLAST similarity search programs. Nucleotide sequence analysis was done using the University of Wisconsin Genetics Computer Group sequence analysis programs. Screening and isolation of cDNA clones were done either by standard methods or by the GeneTrapper cDNA positive-selection method according to the manufacturer's instructions (Life Technologies, Rockville, MD). A hybridization oligonucleotide (5'-CCA ACG CCA CTG CCG CTT-3') and human brain plasmid cDNA library (Life Technologies) were used in the GeneTrapper method. Automated nucleotide sequencing was performed in the Children's Hospital Mental Retardation Research Center DNA Sequencing Core Facility using the dideoxynucleotide termination method. Polymerase chain reaction (PCR), and anchored PCR amplifications were done using the Advantage 2 polymerase kit (CLONTECH Laboratories, Inc., Palo Alto, CA). PCR templates used were Marathon-ready cDNAs prepared from retina or brain (CLONTECH). The cloning strategy and sequences of oligonucleotide primers used in the isolation of overlapping clones that constitute the full-length spectrin IV cDNA are available upon request.

Chromosome Localization-- Chromosome localization of the human spectrin IV gene utilized the Stanford G3 radiation hybrid panel (24) (Research Genetics, Huntsville, AL). Hybrid human-hamster clones were assayed for the presence of spectrin IV gene by PCR. Using the primers 5'-CGG CTG GCA GCT GTG AAC CAG ATG GTG-3' (forward) and 5'-AAC TGG CAC TGG GTC TCG GCT CAG GC-3' (reverse), a 268-bp product was generated in clones that carried the spectrin IV gene. The data were analyzed by the Stanford Human Genome Center RH Server on the Web. Chromosome localization of the mouse gene was done using the Jackson Laboratory BSS Interspecific ((C57BL/6JEi × SPRET/Ei)F1 × SPRET/Ei males) backcross panel (25). A 453-bp PstI fragment of human EST clone AA054636 detected a PstI restriction fragment length polymorphism in the mouse genome corresponding to a 3.7-kb hybridization band in C57BL/6JEi and 4.1-kb band in SPRET/Ei. The segregation pattern of the polymorphism in progeny of the cross was used to determine the map location of the gene. The typing data have been placed in the Mouse Genome Data base (accession number J:63322) and can be accessed on the Web.

Northern, PCR, and Whole-Mount in Situ Hybridization Analysis-- For Northern analysis, a 1.96-kb fragment of clone N164/N155 corresponding to bp 114-2071 was generated by PCR, subcloned, labeled, and used as a probe in hybridization with mRNA from various mouse tissues blotted on a charged nylon membrane (Multiple Tissue Northern blot, CLONTECH). High stringency hybridization and washes were performed according to the manufacturer's instructions (ExpressHyb, CLONTECH). The membranes were then exposed to Kodak X-OMAT AR film for 4 days to visualize the positive signals. For PCR analysis, complementary DNAs (1 ng) from various mouse tissues (Mouse Multiple Tissue cDNA Panel, CLONTECH; and Multiple Choice cDNAs, OriGene Technologies, Inc., Rockville, MD) were analyzed by PCR (94 °C, 40 s; 60 °C, 45 s; 72 °C, 3 min; 38 cycles) using the primers: 5'-CAA GCC CAG GTG CCC CTC-3' (forward) and 5'-GTT GTC ATT CCA TTG AGA AG-3' (reverse). The 107-bp product, representing the unique C-terminal sequence of spectrin IV5, was analyzed by agarose gel electrophoresis.

Whole mount in situ hybridization was done according to published protocols (26). Briefly, day 9.5 mouse embryos were isolated, fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) and bleached in methanol and hydrogen peroxide. They were then treated with proteinase K in PBS containing 0.1% Tween 20, refixed in 0.2% glutaraldehyde and 4% paraformaldehyde in PBS, and hybridized with digoxigenin-labeled RNA probes at 63 °C overnight. After high stringency washes, the embryos were incubated sequentially with alkaline phosphatase-conjugated anti-digoxigenin antibodies and 5-bromo-4-chloro-3-indoyl-phosphate chromogenic substrate for visualization of hybridization signals. The digoxigenin-labeled antisense RNA probes were generated by in vitro transcription with T7 polymerase using as the template a 1.96-kb fragment of clone N164/N155 (nucleotides 114-2071) generated by PCR and subcloned into a pBluescript SK vector. RNA transcripts in the sense direction were generated with T3 polymerase and used as negative controls.

Western Analysis-- An anti-peptide antiserum to IV-spectrin, termed SpB4-R15, was produced by immunizing rabbits with a keyhole limpet hemocyanin-conjugated synthetic peptide (Zymed Laboratories Inc., South San Francisco, CA). The peptide sequence was RLTTPPEPRPSASS, corresponding to codons 535-548 of clone N164/N155, a region that contains little homology to spectrins I, II, III, or V. The antibodies were affinity-purified by passing through an AminoLink Plus column (Pierce, Rockford, IL) containing a recombinant peptide of IV-spectrin repeat 15 fused to a GST protein, and eluted with 0.1 M glycine (pH 2.5). A cDNA encoding IV-spectrin repeat 15 was generated by PCR (forward primer 5'-GCG GGA TCC TCT CGG GAG CTT CAT AAG TTC-3'; reverse primer 5'-GCG GAA TTC GGA GCT GAC ATG CAG GCG GGC-3') and cloned into the BamHI and EcoRI sites of a pGEX-6P-1 vector (Amersham Pharmacia Biotech, Piscataway, NJ). The recombinant GST-IV-spectrin peptide was produced in the Escherichia coli BL21 strain and purified with glutathione-Sepharose beads according to the manufacturer's instructions (Amersham Pharmacia Biotech).

For Western analysis, mouse tissues were excised and homogenized on ice with a Polytron homogenizer (Brinkmann Instruments, Inc., Westbury, NY) in 0.32 M sucrose, 10 mM Tris (pH 8.0), 5 mM N-ethylmalemide, 2 mM EDTA, 5 µg/ml each of protease inhibitors leupeptin, pepstatin, aprotinin, and 0.4 mM diisopropyl fluorophosphate. Protein concentrations were determined by the method of Bradford with bovine serum albumin as the standard. Protein samples (35 µg) were run on a 3.5-17% non-linear gradient Laemmli SDS-polyacrylamide gel and electrophoretically transferred to nitrocellulose filter. The filter was incubated with 500 ng/ml affinity-purified SpB4-R15 antibody and goat anti-rabbit IgG (Bio-Rad Laboratories, Hercules, CA). Immunoreactive proteins were visualized with Lumi-Light Western blotting Substrate (Roche Molecular Biochemicals, Indianapolis, IN). Molecular sizes of the positive bands were determined by comparison with the molecular weight of red cell membrane proteins (27) run in parallel. Experiments using affinity-purified antisera from two rabbits immunized with the IV-spectrin peptide independently give similar results.

Immunofluorescence Microscopic Studies-- The affinity-purified rabbit polyclonal SpB4-R15 antibody was used in immunofluorescence microscopy studies. Canine kidney cells (MDCK), human neuroblastoma cells (SK-N-SH), and green monkey kidney cells (COS-7) were obtained from the American Type Culture Collection, Manassas, VA. Human embryonic kidney 293T and hepatoma Hep3B cells were kind gifts from Drs. Len Zon (Children's Hospital, Boston, MA) and David Livingston (Dana-Farber Cancer Institute, Boston, MA), respectively. Human mesenchymal stem cells were isolated, characterized, and cultured as described (28).

For immunofluorescence studies, cells were grown in slide chambers to subconfluency. They were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4) at room temperature (RT) for 10 min and permeabilized with 0.1% Triton X-100 in PBS at RT for 10 min. Alternatively, cells were fixed and permeabilized in 100% methanol at 20 ° for 10 min. The cells were then incubated with affinity-purified SpB4-R15 antibody at 1:10 to 1:100 dilutions at RT for 30 min, rinsed with PBS, and incubated with Cy3-conjugated goat anti-rabbit IgG for 30 min at RT. All second stage antibodies were from Jackson ImmunoResearch Laboratories, Inc., West Grove, PA. The cells were then rinsed and mounted in ProLong antifade reagent (Molecular Probes, Inc., Eugene, OR).

Double immunofluorescence studies were performed by adding a second mouse antibody in the first incubation step and an fluorescein isothiocyanate-conjugated goat anti-mouse IgG in the second step. The primary antibodies used in double label experiments with the SpB4-R15 antibody included anti-PML (mouse monoclonal PG-M3; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and anti-SUMO-1 (mouse monoclonal 21C7; Zymed Laboratories Inc.). Additional primary antibodies used to stain SpIV5-transfected cells included anti-nucleoporin p62 (mouse monoclonal 53; Transduction Laboratories, Lexington, KY), anti-CBP (rabbit polyclonal A-2; Santa Cruz Biotechnology), and anti-c-myc (mouse monoclonal 9E10, Santa Cruz Biotechnology). Secondary stage antibodies were Cy3-conjugated goat anti-mouse or anti-rabbit IgG. Antibody dilutions used were empirically determined and ranged between 1:100 and 1:1000.

Fluorescence microscopy was done using a Zeiss Axioskop microscope. Microscopic images were taken with Kodak Elite ASA 400 film, digitized with a Nikon CoolScan 2000 slide scanner, and processed with the Adobe Photoshop 5.5 program on a Power Macintosh G3 computer. Confocal microscopy was performed at the Brigham and Women's Hospital Confocal Microscopy Core Facility, using a Bio-Rad MRC-1024/2P confocal microscope interfaced with a Zeiss Axiovert microscope.

Expression Studies-- To make the SpIV5-green fluorescence protein (SpIV5-GFP) construct, a fragment of clone N164/N155 was generated by PCR and subcloned into a eukaryotic expression vector pcDNA4/HisMaxA (Invitrogen Corp., Carlsbad, CA) into which a cDNA for the enhanced GFP (CLONTECH) had first been inserted. The primers used in the PCR were DEL1 (forward: 5'-CTG ATG GCG CGG GAT AGC ACG CGG-3') and DEL3 (reverse: 5'-TTC CAT TGA GAA GGG GGC TGT-3'), and the PCR product corresponded to codons 2-678 of clone N164/N155. The resulting construct encoded a fusion protein consisting of six histidines and an Xpress epitope tag, followed by the SpIV5 peptide fused in-frame to GFP. The myc-tagged human CREB-binding protein construct was a kind gift of Dr. David Housman (Massachusetts Institute of Technology, Boston, MA). These constructs were transfected into 293T, COS-7, and Hep3B cell lines using LipofectAMINE (Life Technologies) or FuGENE 6 (Roche Molecular Biochemicals) reagents. Expression of the heterologous protein was analyzed after 24-48 h.

Deletion constructs of SpIV5-GFP were generated in a similar fashion, using PCR primer pairs as followed: SpIV5NR-GFP (DEL1 (forward) and DEL4 (reverse: 5'-CCT GGG CTT GTC GGC TGC CCC-3')), SpIV5R16-GFP (DEL1 (forward) and DEL5 (reverse: 5'-GGA GCT GAC ATG CAG GCG GGC-3')), SpIV5R10-GFP (DEL2 (forward: 5'-AGG CCA GCA AAG CAG ACC AGC TG-3') and DEL3 (reverse)) and SpIV5R15-GFP (DEL1 (forward), DEL6 (forward: 5'-CGG GCC CAG CTG CTG GCC GCC ACA GCC GAC GCC CTG CGC TTC-3'), DEL7 (reverse: 5'-GAA GCG CAG GGC GTC GGC TGT GGC GGC CAG CAG CTG GGC CCG-3') and DEL3 (reverse)). The deleted domains are: SpIV5NR-GFP, codons 649-678; SpIV5R16-GFP, codons 617-678; SpIV5R10-GFP, codons 2-84; SpIV5R15-GFP, codons 509-616. The deletion constructs were transfected into mammalian cell lines, and expression of the truncated peptides was analyzed as for the SpIV5-GFP construct.

Nuclear Matrix Analysis-- Preparation of nuclear matrix was performed as published previously (29). Briefly, cells grown on chamber slides were treated sequentially with cytoskeleton buffer (10 mM Pipes (pH 6.8), 100 mM NaCl, 300 mM sucrose, 3 mM MgCl₂, 1 mM EGTA, 0.5% Triton X-100, 4 mM vanadyl ribonucleoside complex, 1 mM Pefabloc) at 4 °C for 3 min, 25 units/ml DNase I in nuclease buffer (cytoskeleton buffer with 50 mM NaCl) at RT for 30 min, 0.25 M ammonium sulfate in nuclease buffer at RT for 10 min three times, high salt buffer (nuclease buffer with 2 M NaCl) at RT for 5 min three times, and then 100 µg/ml RNase A and 40 units/ml RNase T₁ in nuclease buffer at RT for 60 min. The extracted cells were fixed in methanol at 20 °C for 10 min and immunofluorescence microscopy was performed. Pefabloc, DNase I, RNase A, and RNase T1 were purchased from Roche Molecular Biochemicals; vanadyl ribonucleoside complex was from Life Technologies; all other reagents were from Sigma-Aldrich, St. Louis, MO.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

A Spectrin-like Gene Is Localized to Human Chromosome 19q13.2 and Mouse Chromosome 7-- Nucleotide sequences of I-, II-, I-, II-, and III-spectrins were used as query sequences to search the GenBank^TM EST data base for clones that were similar but not identical to known spectrin chains. One EST clone from a human retinal library was identified that had sequence similarity to repeats 10 and 11 of -spectrins (GenBank^TM accession number AA054636). Screening of bacteriophage cDNA libraries by conventional methods using a 453-bp PstI fragment of this clone as a probe failed to yield positive clones. Subsequently, the GeneTrapper positive selection method was used (Life Technologies). Using sequence information obtained from the clone, an oligonucleotide probe was synthesized, biotinylated, and hybridized with single-stranded DNA from a human brain cDNA library. Six unique clones that contained sequences complementary to the oligonucleotide probe were recovered using streptavidin-coated paramagnetic beads. All of the clones were polyadenylated, but four appeared to be incomplete or partially spliced and are not described here. The complete coding sequence reported here is a composite of the two unique clones, N164 and N155 (Fig. 1A). Clone N164 extends from nucleotide 1-2172 of the composite sequence and is followed by a poly(A) tail. Clone N155 extends from nucleotide 10-2418 and is also polyadenylated. However, neither clone contains a consensus polyadenylation signal close to the polyadenylation site, and both are too short to account for the transcripts observed on Northern blots (described below). We suspect they are minor transcripts and that a significant portion of the 3'-untranslated repeat has not been cloned.

View larger version (37K): [in this window] [in a new window]   Fig. 1.   Identification of a new -spectrin. A, clones N164 and N155 together represent a 2418-bp cDNA that encodes a 77-kDa peptide containing five full spectrin repetitive motifs and a partial repeat at each end. Coils A, B, and C of the triple helical coil units of the spectrin-repetitive motifs are indicated. The repetitive motifs are most similar to repeat 10 (R10) to repeat 16 (R16) of the other -spectrins. The C-terminal 30 residues of the 77-kDa peptide do not conform to a repeat structure (NR). The synthetic peptide used to generate the rabbit polyclonal antiserum SpB4-R15 is located in the 15th repeat (R15). B, predicted amino acid sequence of the spectrin-like peptide is shown. The phasing of the spectrin-repetitive units shown was established by crystallographic data (30). The consensus sequence is from the same reference. Amino acid residues identical to the consensus sequence in the spectrin-like peptide are shaded. C, predicted secondary structure of the 77-kDa spectrin-like peptide, folded into sequential triple helical coiled coil structures but with two helical coils (coils B and C) near the N terminus of the peptide and a single coil (coil A) near the C terminus. D, potential tandem multimerization of the 77-kDa spectrin-like peptide with multiple peptides joining in a head-to-tail fashion through interaction of the helical coils at the ends. E, schematic representation of the cDNA of the two isoforms of spectrin IV: a full-length -spectrin (SpIV1) and a truncated isoform represented by clones N164 and N155 (SpIV5). Exon boundaries are indicated by vertical lines, and the larger exons are numbered. Note that the truncated SpIV5 isoform begins with an alternatively spliced 5'-untranslated repeat, numbered exon 18a, and terminates with a unique sequence of 90 bp that results from extension into intron 27 (filled box).

Sequence analysis showed that clones N164 and N155 contained an identical 2034-bp open reading frame that potentially encodes a peptide 678 residues in length, with a calculated molecular weight of 77,197 Da (Fig. 1B). The first methionine in this open reading frame (bp 128-130) was taken as the start codon. The amino acid sequence of this peptide is very similar to repeats 10-16 of other known -spectrins, with ~45-65% identity over the repeat domain. Analysis of the secondary structure of this spectrin-like peptide predicts the formation of multiple -helical coils that fold into triple helical coiled-coil units, a characteristic feature found in all spectrin peptides (30). The spectrin-like peptide represented by clone N164/N155 is predicted to have five full repetitive motifs (repeats 11-15), flanked on each side by two partial repeats (repeats 10 and 16). Partial repeat 10 consists of two coils of a triple helical coiled-coil unit (helices B and C) and partial repeat 16, a single coil (helix A) (Fig. 1C). These coils may potentially allow head-to-tail interaction of multiple peptides to form concatemers (Fig. 1D), analogous to the way - and -spectrins interact to form heterotetramers. However, in the latter case -spectrin ends with two helical coils (helices A and B) and -spectrin starts with a single coil (helix C).

The repeat domain of the spectrin-like peptide is followed by a C-terminal, non-repeat domain (NR) of 30 residues, which is rich in proline residues and dissimilar to the C termini of other -spectrins. No similar sequence is reported in the protein data bases.

To show that clones N164 and N155 are transcripts of a new spectrin-like gene and not an alternative transcript of other known -spectrins, we determined the chromosomal location of the new spectrin-like gene. PCR primers derived from the sequence of clone N164/N155 were used to screen the Stanford G3 human-hamster radiation hybrid genomic DNA panel for the presence or absence of its gene in different cell lines (24). Tight linkage (lod score 11.25) of the gene was found to DNA marker SHGC-33106, a part of the biliverdin reductase B gene (also called NADPH-flavin reductase), which maps to chromosome 19q13.13-q13.2 (31). This chromosomal location differs from other human -spectrin genes, which are located on chromosomes 2, 11, 14, and 15, indicating that clone N164/N155 is a transcript of a new -spectrin-like gene. Of note, the chromosome location of this new gene is close to that of -actinin 4 (32). It has been previously shown that the spectrin I and III genes both localize close to an actinin gene (9, 33), suggesting that sequential duplications of a chromosome region containing a primordial -spectrin gene and -actinin gene may have given rise to the neighboring locations of these genes.

We next mapped the location of this spectrin-like gene in the mouse. Analysis of the segregation pattern of a PstI restriction fragment polymorphism in 94 progeny of The Jackson Laboratory BSS interspecific backcross panel (25) localized the gene to mouse chromosome 7 near the centromere (7.5 centimorgans), a region of the mouse genome homologous to human chromosome 19q13.1. The location of the gene is also different from other known -spectrin genes, which map to chromosomes 11, 12, and 19. Mouse spectrin V has not yet been mapped.

This gene location approximates the position of the spontaneous mouse mutation reduced pigmentation (rp) (34), in which pigment abnormalities and lysosomal dysfunction suggest an underlying defect in intracellular vesicle biogenesis (35). To determine if a mutation in the spectrin-like gene may be the cause of the rp defect, an informative subset of homozygous F2 rp animals from an intercross between C57Bl/6J-rp/rp and M. musculus castaneus (Cast/Ei) were analyzed for recombination between the rp locus and part of the spectrin-like gene detected with a clone N155-derived probe. Of the 70 meioses analyzed, four recombination events were identified, indicating that the rp locus and the spectrin-like gene are distinct genes separated by ~5.7 centimorgans on chromosome 7.

The Spectrin-like Gene Is a Bona Fide -Spectrin, Designated Spectrin IV-- We then searched the GenBank^TM high throughput genomic sequence (htgs) data base for genomic DNA clones that contain this spectrin-like gene. Two genomic clones (accession numbers AC021625 and AC020929), derived from chromosome 19, contained sequences that matched clone N164/N155. Analysis of these clones revealed additional sequences similar to -spectrins that were not represented in clones N164/N155, indicating that a longer isoform of the spectrin-like gene exists.

Using the putative exon sequences deduced from the genomic DNA clones, PCR and anchored PCR primers were designed and used to amplify human retinal and brain cDNA templates to obtain overlapping fragments of this long isoform, which were completely sequenced. Clones were obtained that together constitute an 8069-bp cDNA, with an open reading frame that encodes a 289.0-kDa peptide similar in structure to other -spectrins. The predicted peptide contains a highly conserved actin/protein 4.1-binding domain, followed sequentially by 17 spectrin-repetitive motifs, a unique domain of 307 amino acids, a pleckstrin homology domain, and a conserved C terminus (Fig. 2).

View larger version (107K): [in this window] [in a new window]   Fig. 2.   Amino acid sequence of the full-length spectrin IV isoform. The sequence of full-length spectrin IV (SpIV1) is aligned with the sequences of spectrins I, II, and III (SpI, SpII, and SpIII, respectively). Residues that are identical in more than two of the four spectrins are boxed in black. The first residues of the actin/4.1-binding domain, each of the 17 spectrin repeats, and the pleckstrin homology domain are indicated above the sequences with arrowheads. The beginning of the 77-kDa isoform (N164/N155) is also indicated in repeat 10. The amino acids in repeat 15 that are marked by dots were used to generate the SpB4-R15 peptide antibody. The three tandem repeats in the spectrin IV non-homologous domain near the C terminus are indicated beneath the sequence by three arrows. The muscle isoform of spectrin I (SpI2) is used in the alignment, because it is the full-length isoform. Spectrin V (91) is not included, because its structure is different from the other  spectrins in several aspects.

Compared with the corresponding repeats of other -spectrins, repeat 4 of this peptide has two small insertions, 15 and 8 amino acids in length. Repeat 5 has a 3-residue insertion, repeat 7 has a 6-residue deletion, repeat 8 has two 2-residue insertions, and repeat 15 has a 2-residue insertion. All of these insertions and deletions are positioned near the predicted turns of the triple helical coiled-coil unit of spectrin repeats, thus preserving their overall conformational structure.

The unique domain after repeat 17 is longer than the corresponding domain of other -spectrins and contains, near its beginning, three tandem repeats, which are underlined by arrows in Fig. 2. The consensus sequence of these repeats, which includes amino acids present in at least two of the three repeats, is RRRPERQESADXXEEXXR and is unlike any other known motifs found in data bases. Some of these insertions, particular those in repeat 4, and the unique domain are potential interaction sites for binding partners of the spectrin-like peptides. Of note, repeat 4 lies within the region (repeats 2-7) previously defined as a membrane binding site in SpII (36).

The predicted amino acid sequence of clone N164/N155 is identical to codons 1325-1972 of the long isoform. The non-repeat (NR) segment in the C terminus of the N164/N155 peptide corresponds to a translated portion of intron 27, similar to the way the truncated C terminus of the erythrocyte isoform (SpI1) of spectrin 1 originates (37). The 5'-terminal portion of the N164/N155 cDNA is not present in the DNA sequence of the long isoform and represents an alternatively spliced 5' exon (exon 18a) located between exons 18 and 19 of the gene. Comparison between the cDNA and genomic sequence of the spectrin-like gene reveals an exon-intron structure identical to that of spectrin I gene (38), with 36 exons that span >145 kb, except that exon 31, which contains most of the unique three-repeat domain, is longer in size.

These results indicate that the new spectrin-like gene is a bona fide -spectrin gene, which we designate as spectrin IV (human gene SPTBN3 and mouse gene Spnb4) and which has both a full-length isoform (SpIV1; GenBank^TM accession number AF311855) and a truncated isoform represented by clone N164/N155 (SpIV5; GenBank^TM accession number AF311856). Data on the exon-intron structure of the two spectrin IV isoforms are available in the annotations of the GenBank^TM entries, and are shown schematically in Fig. 1E. A search of the GenBank^TM data bases for other clones derived from SpIV revealed only three additional clones: BE107551, which matches sequences in the 5'-end of SpIV1 (bp 440-900), D81941, which corresponds to sequences near the 3'-end of SpIV1 (bp 7778-8023), and AL133093, which matches bp 838-2174 of SpIV5. After this work was completed, a partial, uncharacterized cDNA clone, KIAA1642, was deposited in the GenBank^TM data base (accession number AB046862) (39). Clone KIAA1642 is identical to SpIV1 from bases 1258 to 7249, except for three single nucleotide substitutions at positions 2238, 4093, and 5523. The substitution at base 4093 results in a Ser  Gly change at codon 1331, whereas the other two substitutions do not affect the amino acid sequence.

Spectrin IV Is Expressed Predominantly as the Truncated IV5 Isoform-- To analyze the expression pattern of spectrin IV, a 1.96-kb fragment of clone N164/N155 that encodes spectrin repeats 10-16 (nucleotides 114-2071) was generated by PCR and subcloned into a pBluescript vector. The fragment was used as a probe in Northern analysis of a blot of poly-(A⁺) RNA isolated from different mouse tissues (Fig. 3A). A major 5-kb hybridizing band and minor 9- and 3-kb bands are found in brain. Expression of spectrin IV in other tissues was at a level too low to be detected by the analysis.

View larger version (78K): [in this window] [in a new window]   Fig. 3.   Spectrin IV is primarily expressed as a truncated form. A, Northern analysis of mouse RNA using a human spectrin IV probe shows a major 5-kb hybridizing band and minor 9-kb and 3-kb bands, predominantly in the brain. B, whole-mount in situ hybridization of day 9.5 mouse embryos with an antisense spectrin IV probe shows positive signals in the forebrain (FB), midbrain (MB), hindbrain (HB), and optic vesicle (OV) (right embryo). Hybridization of an embryo at the same age with a sense spectrin IV probe shows no detectable signals (left embryo). C, Western analysis of protein extracts (35 µg) from mouse tissues shows a prominent 72-kDa band in all tissues tested, a major 30-kDa band in spleen, and a minor 68-kDa band in the heart. No band was observed at 289-kDa under these conditions, but, as shown in D, faint bands were detectable at 272, 240, 166, and 146 kDa when the Brain lane was exposed for a long time. These may correspond to SpIV1 (289 kDa) and some of the other SpIV isoforms detected by Berghs et al. (23).

Whole-mount in situ hybridization was also performed using mouse embryos at post-coital day 9.5. Spectrin IV-specific riboprobes in both the sense and antisense orientations were generated using as a template the 1.96-kb fragment of N164/N155 cloned in the pBluescript vector. Hybridization using riboprobes in the antisense orientation revealed positive signals mainly in the forebrain and hindbrain and in the developing eye (optic vesicle) (Fig. 3B). No other tissues were positive at this stage of development at the level of detection of the method. Control hybridization using riboprobes in the sense orientation showed no signals, indicating that the signals seen with the antisense riboprobes were specific.

PCR analysis of cDNAs prepared from multiple mouse tissues using primers designed to amplify the 90-bp segment encoding the unique 30 amino acids at the C terminus of the truncated isoform (SpIV5) showed prominent expression in post-coital day 7 mouse embryos (data not shown).

These results indicate that spectrin IV transcripts are found predominantly in the brain, especially in the developing embryo, although there must be a low level of spectrin IV expression in other tissues undetectable by the techniques used, because, as shown below, the protein is detectable in multiple tissues.

A polyclonal antiserum against spectrin IV (SpB4-R15) was generated by immunizing rabbits with a 14-residue synthetic peptide derived from a segment of repeat 15 that has limited homology to other -spectrins. The human sequence matches the corresponding mouse sequence in 13 out of 14 residues (data not shown), indicating that the polyclonal antiserum against the human spectrin IV peptide should also recognize the mouse peptide. The antiserum was affinity-purified with a recombinant protein containing repeat 15 of spectrin IV fused to glutathione S-transferase (GST) and used in Western analysis of proteins from mouse tissues. A major 72-kDa band was detected in all tissues (Fig. 3C). Spleen also contained a major band at 30-kDa, and there was a minor 68-kDa band in the heart. The major 72-kDa band is slightly smaller than the expected size of the truncated isoform SpIV5, probably due to aberrant migration of the isoform in SDS-polyacrylamide gels. It is notable that other spectrins also migrate below their actual molecular weight on SDS gels. At the sensitivity where the 72-kDa band was easily detected, no band was seen at 289 kDa, the size of the full-length isoform SpIV1 (Fig. 3C). However, with prolonged exposure faint bands were detected at 272, 240, 166, and 146 kDa (Fig. 3D). These could correspond to IV1 (289 kDa) and the IV3 (149 kDa) and IV4 (242 kDa) isoforms described by Berghs and his colleagues (23), along with an unknown isoform (166 kDa). The IV2 isoform would not be detected with the SpB4-R15 antisera. The results indicate that the major isoform of spectrin IV is the truncated isoform SpIV5.

Spectrin IV Is Present in the Nucleus and Associates with the PML Nuclear Bodies-- The affinity-purified spectrin IV antibody SpB4-R15 was used in indirect immunofluorescence microscopy of mammalian cells. Staining of human neuroblastoma SK-N-SH cells and canine kidney MDCK cells showed diffuse, micropunctate signals in the cytoplasm and the nucleus with no involvement of the plasma membrane (Fig. 4, upper panel). The prominent nuclear staining suggested that spectrin IV might actually be present inside the nucleus. To better delineate the nuclear distribution of spectrin IV, we examined its distribution in human mesenchymal stem cells (MSCs), multipotential stromal cells derived from bone marrow that appear as large and flat fibroblast-like cells in culture (28). Staining of the MSCs with the SpB4-R15 antibodies showed a similar micropunctate pattern in the cytoplasm and the nucleus. In addition, 10-30 prominent foci of spectrin IV staining were clearly seen in the nuclei of these cells above the background of diffuse nucleoplasmic staining (Fig. 4, middle panel, left).

View larger version (55K): [in this window] [in a new window]   Fig. 4.   Spectrin IV is located in the cytoplasm and nucleus, and associates with PML nuclear bodies. Upper panel, immunofluorescence staining pattern of (A) SK-N-SH and (B) MDCK cells shows micropunctate staining of both the cytoplasm and the nucleus. Middle panel, staining of human mesenchymal stem cells (MSCs) with the spectrin IV antiserum (left) and with a mouse monoclonal antibody against PML (middle). Both show discrete nuclear bodies that coincide perfectly when the images are merged (right). Lower panel, MSCs stained with the spectrin IV antiserum (left) and a SUMO-1 antibody (middle) also show colocalization of signals in nuclear dots (right). In addition, there appears to be coincidence of spectrin IV and SUMO-1 signals in the cytoplasm.

The pattern of nuclear foci seen in the MSCs is reminiscent of that seen with antibodies against PML, a protein that is involved in the pathogenesis of acute promyelocytic leukemia and that associates with a specific nuclear structure, the PML bodies (40-43). We therefore performed double immunofluorescent staining of MSCs using antibodies against both PML and spectrin IV. Staining of MSCs with an antibody against PML protein also showed 10-30 distinct nuclear dots in each cell, which coincided perfectly with the nuclear dots revealed by the spectrin IV antibody (Fig. 4, middle panel). These results indicate that spectrin IV is present in the nucleus and colocalizes with PML nuclear bodies.

PML protein has been shown to translocate into the nucleus to form nuclear bodies only after post-translational modification by a small ubiquitin-like modifier, SUMO-1 (also called PIC1 or GMP1) (44, 45). We co-stained MSCs with antibodies against SUMO and spectrin IV. The SUMO antibody revealed a faint, diffuse staining of both the cytoplasm and nucleus, with marked accentuation of signals in 10-30 nuclear dots (Fig. 4, lower panel). The diffuse staining pattern and the nuclear dots revealed with the SUMO antibody coincides very well with the staining pattern of the spectrin IV antibody, indicating that spectrin IV colocalizes in cells with SUMO-modified proteins, such as PML and Sp100 proteins, or that spectrin IV is itself modified by SUMO-1.

Expression of a Spectrin IV-GFP Fusion Protein Forms Nuclear Dots-- To demonstrate a specific association of spectrin IV with nuclear bodies, a plasmid construct was made that encoded the SpIV5 isoform fused to the green fluorescence protein (SpIV5-GFP). When the construct was transfected into COS-7 monkey kidney cells, 40-60 prominent, spherical, GFP-positive dots were evident inside the cell nuclei (Fig. 5). The nuclei were outlined in this experiment with an antibody against p62 nucleoporin, a component of the nuclear pore complex on the nuclear membrane (46). The possibility that these dots lie in the cytoplasm on top of the nuclei was excluded by confocal microscopy. In a small number of cells, however, a few GFP-positive dots were also found in the cytoplasm, mainly in the perinuclear region (Fig. 5, lower panel, arrows). The SpIV5-GFP nuclear dots were relatively uniform in size within each transfected cell but varied in size in relation to the level of expression of the fusion protein. Cells with bright fluorescence signals had fewer and larger nuclear dots, whereas cells with dim fluorescence had smaller and more numerous dots.

View larger version (46K): [in this window] [in a new window]   Fig. 5.   The 77-kDa isoform of spectrin IV (SpIV5) is located primarily in the nucleus. Left, exogenous expression of SpIV5 fused to green fluorescence protein (Spectrin IV-GFP) in COS-7 cells results in GFP-positive dots in the nucleus, as shown by confocal microscopy. Middle, the outline of the nucleus is marked in red with an antibody against nucleoporin, a component of the nuclear pore complex. Right, this indicates that the GFP-positive dots are almost all located within the nucleus. Lower panel, occasionally a few GFP-positive dots are also found in the cytoplasm, especially in the perinuclear region (arrows).

We next investigated whether the SpIV5-GFP fusion protein colocalized with PML nuclear bodies. Because the PML antibody used in our experiments does not stain nuclear dots in COS-7 cells, we performed the expression experiments using human embryonic kidney 293T cells. When the SpIV5-GFP construct was transfected into 293T cells, about 30-90 GFP-positive dots were seen in the nucleus of each transfected cell (Fig. 6, A and D). Staining of the 293T cells with a PML antibody revealed about 10-30 PML dots in each untransfected cell and about 20-50 PML dots in each transfected cell (Fig. 6, B and E). All the PML dots in transfected cells were positive for GFP but only about half of the GFP-positive dots in these cells were positive for PML (Fig. 6, C and F), indicating that the nuclear dots expressing SpIV5-GFP include all the PML-containing dots.

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Spectrin IV5-GFP nuclear dots and PML bodies are related. Upper panel, 293T cells transfected with the SpIV5-GFP construct (Spectrin IV-GFP) contain multiple GFP-positive nuclear dots. Co-staining of the transfected cells with PML antibody shows that all PML dots are positive for SpIV5-GFP (arrows) while some of the GFP-positive dots are not PML positive (arrowheads). Lower panel, a scatterplot showing the relationship between the spectrin IV5-GFP dots and PML dots in transfected (filled circles) and untransfected cells (open circles). The number of PML dots is increased in transfected cells and is roughly proportional to the number of GFP-positive dots in these cells (dashed line).

To demonstrate a quantitative relationship between expression of SpIV5-GFP and PML, the number of PML- and GFP-positive nuclear dots were counted in 40 transfected cells and compared with the number of PML bodies in 40 neighboring untransfected cells. Transfected cells have 31.9 (±8.7 (S.D.)) PML nuclear bodies per cell while untransfected cells average 17.8 (±7.4 (S.D.)). The number of GFP-positive nuclear bodies is 61.5 (±31.6 (S.D.)) per cell, confirming that ~50% of the GFP-positive nuclear bodies are positive for PML proteins. The number of PML nuclear bodies in transfected cells is roughly proportional to the number of GFP-positive bodies in these cells (Fig. 6, lower panel; correlation coefficient = 0.66), but there is a wide variation among cells. This analysis indicates that overexpression of the SpIV5-GFP fusion protein leads to a corresponding increase in the number of PML bodies in cells, from either enhanced formation or dispersal of these structures, suggesting that spectrin IV may be involved in the genesis of PML bodies.

CREB-binding Protein Concentrates in Spectrin IV-containing Nuclear Dots-- Because both COS-7 and 293T cells contain the SV40 T antigen, which increases the expression of the transfected plasmid but may interfere with the function of the PML bodies (47), we transfected the same SpIV5-GFP construct into human hepatoma Hep3B cells, which do not contain T antigen. Co-staining of the transfected Hep3B cells with PML and spectrin IV antibodies shows the same nuclear dot relationship (Fig. 7, upper panel), indicating that colocalization of the spectrin IV and PML nuclear dots is not an artifact or a unique feature of the T antigen-expressing cell lines.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Spectrin IV5-GFP colocalizes with PML and CREB binding protein (CBP). Upper panel, expression of the SpIV5-GFP fusion protein (Spectrin IV-GFP) in Hep3B cells shows GFP-positive nuclear dots (left) that include all PML nuclear bodies (middle). There is slight misalignment of the GFP- and PML-positive signals in some nuclear dots that are either above or below the plane of focus. Lower panel, cotransfection in Hep3B cells of constructs encoding the SpIV5-GFP fusion peptide (left) and myc-tagged CBP (middle) results in nuclear dots that express both peptides (right).

CREB-binding protein (CBP), a transcription coactivator important in regulation of expression of many genes (48), also concentrates in PML nuclear bodies (49). We investigated whether expression of spectrin IV would recruit CBP into the same nuclear dots. We expressed the SpIV5-GFP construct in Hep3B cells and stained the cells with A22, an antibody against an N-terminal epitope of CBP that reveals nuclear dots in cells (49). CBP staining in these cells was mostly diffuse and nucleoplasmic, but in occasional cells distinct dots were seen above the background. These CBP-positive nuclear dots coincided with the GFP-positive dots containing the spectrin IV5-GFP fusion protein, indicating that CBP and spectrin IV5 resided in the same nuclear bodies (data not shown). To more clearly demonstrate this association, an expression vector containing a human CBP cDNA fused to an myc tag was transfected into Hep3B cells together with the SpIV5-GFP construct. Staining of the transfected cells with an antibody against the myc tag revealed multiple nuclear dots that coincided with GFP-positive signals derived from the SpIV5-GFP peptide (Fig. 7, lower panel), showing that CBP is recruited into the same nuclear bodies that contain SpIV5.

The N- and C-terminal Helical Coils of SpIV5 Are Needed to Form Nuclear Dots-- The C- and N-terminal helical coils of the SpIV5 peptide can potentially interact to allow these peptides to form multimers (Fig. 1D). To determine whether these structures are essential for nuclear body formation, we made DNA constructs that encode different truncated forms of SpIV5 fused to a GFP tag (Fig. 8). A full-length SpIV5-GFP construct expressed in 293T or COS-7 cells generated prominent nuclear bodies (Fig. 8, images A). In the case of 293T cells these coincided with PML bodies, as described before (Fig. 6). A construct with the C-terminal non-repeat (NR) domain deleted gave the same expression pattern (images B). In contrast, a construct with both coil A of repeat 16 and the NR domain deleted formed a diffuse pattern in both the cytoplasm and nucleus (images C). Similarly, deletion of coils B and C of the N-terminal repeat 10 resulted in faint intranuclear blobs that did not associate with PML bodies (images D). An internal deletion of repeat 15 produced nuclear bodies similar to those produced by the full-length construct, indicating that repeat 15, which in spectrin I binds ankyrin (50), is not essential for nuclear bodies to form (images E).

View larger version (58K): [in this window] [in a new window]   Fig. 8.   Localization of SpIV5-GFP in nuclear dots requires the partial spectrin repeats at each end. A, expression of the complete 77-kDa spectrin IV5 construct results in nuclear dots. B, deletion of the C-terminal non-repeat domain of SpIV5 does not interfere with nuclear dot formation but slightly increases background staining in the nucleus. C, in contrast, a deletion that also includes repeat 16 completely abolishes nuclear dots. D, deletion of the N-terminal partial repeat 10 similarly eradicates nuclear dots. E, an internal deletion that removes repeat 15, in contrast, does not alter formation of nuclear dots. Upper micrograph panel, transfected 293T cells. Lower panel, transfected COS-7 cells.

These results show that the N- and C-terminal helical coils of the SpIV5 isoform are both necessary for nuclear dot formation. Because these sites are potential sites for tandem multimerization of SpIV5 peptides, such multimerization may be required for formation of nuclear bodies or association with PML-containing structures.

Spectrin IV and Its Nuclear Dots Associate with the Nuclear Matrix-- PML bodies are retained in preparations of the nuclear matrix (51, 52). We investigated whether spectrin IV also resides in the nuclear matrix. MSCs grown on microscopic slides were treated sequentially with non-ionic detergent, DNase I, high salt buffers, and RNase, which extracts most soluble and chromatin-associated proteins from the cells, leaving only proteins that are tightly bound to the nuclear matrix (29). The extracted cells were fixed with methanol at 20 °C and stained with antibodies for indirect immunofluorescent analysis.

Staining of the nuclear matrices with anti-spectrin IV antibody (Fig. 9A) revealed a well-demarcated, finely reticular pattern throughout the nuclear matrix, with accentuation of signals at 10-30 distinct nuclear dots, similar to the nuclear staining pattern seen in non-extracted MSCs (Fig. 4), but with the cytoplasmic staining almost completely eliminated. Co-staining of the extracted MSCs with the PML antibody (Fig. 9B) showed 10-30 bright nuclear dots that align perfectly with the dots revealed with the spectrin IV antibody (Fig. 9C). Co-staining with the SUMO-1 antibody showed the same colocalization of nuclear dots positive for spectrin IV and SUMO-1 (data not shown). In contrast, a control antibody against p62 nucleoporin showed no signals, because nucleoporin does not associate with the nuclear matrix (53). The resistance of spectrin IV to well established extraction procedures used for preparation of nuclear matrix indicates that it is a nuclear matrix protein.

View larger version (66K): [in this window] [in a new window]   Fig. 9.   Spectrin IV is retained in the nuclear matrix. Upper panel, nuclear matrix preparation of mesenchymal stem cells (MSC) co-stained with antibodies against (A) spectrin IV and (B) PML shows, in C, that spectrin IV nuclear dots and PML bodies associate with the nuclear matrix. Lower panel: D and F, nuclear matrix preparations of 293T cells transfected with the SpIV5-GFP construct (Spectrin IV-GFP) show that the GFP-positive nuclear dots resist extraction. E and G, phase contrast micrographs showing the extracted nuclei.

To confirm that spectrin IV-positive nuclear bodies associate with the nuclear matrix, we also prepared whole-mount nuclear matrices from 293T cells transfected with the SpIV5-GFP constructs. The post-extraction nuclear matrices of the transfected cells retained the prominent GFP-positive nuclear dots (Fig. 9, lower panel), indicating the nuclear bodies formed from expression of SpIV5-GFP also tightly associate with the nuclear matrix. These results show that both endogenous spectrin IV and exogenously expressed spectrin IV5 retain their association with the nuclear matrix and PML bodies.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We have identified a new -spectrin, which has a major isoform that is truncated at the N-and C-terminal ends and has the potential of forming end-to-end multimers. Spectrin IV associates with the nuclear matrix and PML nuclear bodies and localizes to cytoplasmic vesicles.

The finding of spectrin IV in the nucleus of cells was unexpected. There have been reports of reactivity in mammalian cell nuclei to antibodies against - or -spectrin (54, 55), but the findings have not been fully explored. There is intriguing evidence that suggests spectrin II is present in the nucleus and is deficient in cells derived from Fanconi anemia patients (20-22), but whether it binds with a -spectrin partner in the nucleus has not been investigated. The finding of a new -spectrin in the nucleus, as reported here, suggests that a spectrin-based skeleton may be an important component of the nuclear structure.

Immunostaining of spectrin IV in mesenchymal stem cells (MSCs) reveals nuclear dots that coincide perfectly with PML nuclear bodies (Fig. 4). PML nuclear bodies, alternatively known as Kr-bodies, nuclear domains 10, or PML oncogenic domains (41, 42), are 0.1- to 1-µm spherical bodies in the nucleus that associate with the Sp100 protein, an autoantigen in patients with primary biliary cirrhosis (56), and the PML protein, a RING finger protein disrupted in acute promyelocytic leukemia (APML) that carries the t (15;17) translocation (57-59). The PML protein has been shown to negatively regulate growth and suppress tumor formation (60), mediate apoptosis through caspase-dependent and independent pathways (61), repress or activate gene transcription (49, 62), and mediate ras-induced premature senescence (63, 64). Whether these functions of PML require that it be located in nuclear bodies has not been resolved.

PML nuclear bodies are highly dynamic structures that vary in size and number under different conditions. Their distribution is cell-cycle-dependent (65) and is affected by viral proteins (66) and interferon treatment (67). The aberrant PML-RAR chimeric peptide in APML with t (15;17) disrupts PML bodies, which are restored when the cells are treated with retinoic acid or arsenic, two agents useful in APML therapy (57-59). Conjugation of the PML protein by the small ubiquitin-like peptide SUMO-1 is necessary for its translocation into the nucleus to form nuclear bodies (44, 45). Beyond this, the structural basis for the formation and dynamic distribution of PML bodies in the nucleus is still unclear.

A large number of proteins associate with PML nuclear bodies (40, 41, 43). A sampling includes: CREB-binding protein (49), Rb (62), p53 (63, 64), HP1 (68), p17^Kip1 (69), Daxx (70), and the BLM (Bloom syndrome) DNA helicase (71). These proteins are involved in nuclear processes such