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Genes to Cells (2009) 14, 975-990. doi:10.1111/j.1365-2443.2009.01324.x
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Proteome analysis of human nuclear insoluble fractions

 Hideaki Takata1{dagger}, Hitoshi Nishijima1,2,*,{dagger}, Shun-ichiro Ogura3, Takehisa Sakaguchi1, Paula A. Bubulya4, Tohru Mochizuki3 and Kei-ichi Shibahara1,2,*

1 Department of Integrated Genetics, National Institute of Genetics, Research Organization of Information and Systems, Mishima 411-8540, Japan
2 Department of Genetics, School of Life Science, The Graduate University for Advanced Studies, Mishima 411-8540, Japan
3 Shizuoka Cancer Center Research Institute, Shizuoka 411-877, Japan
4 Department of Biological Sciences, Wright State University, Dayton, OH 45435, USA


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
The interphase nucleus is a highly ordered and compartmentalized organelle. Little is known regarding what elaborate mechanisms might exist to explain these properties of the nucleus. Also unresolved is whether some architectural components might facilitate the formation of functional intranuclear compartments or higher order chromatin structure. As the first step to address these questions, we performed an in-depth proteome analysis of nuclear insoluble fractions of human HeLa-S3 cells prepared by two different approaches: a high-salt/detergent/nuclease-resistant fraction and a lithium 3,5-diiodosalicylate/nuclease-resistant fraction. Proteins of the fractions were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry, identifying 333 and 330 proteins from each fraction respectively. Among the insoluble nuclear proteins, we identified 37 hitherto unknown or functionally uncharacterized proteins. The RNA recognition motif, WD40 repeats, HEAT repeats and the SAP domain were often found in these identified proteins. The subcellular distribution of selected proteins, including DEK protein and SON protein, demonstrated their novel associations with nuclear insoluble materials, corroborating our MS-based analysis. This study establishes a comprehensive catalog of the nuclear insoluble proteins in human cells. Further functional analysis of the proteins identified in our study will significantly improve our understanding of the dynamic organization of the interphase nucleus.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
The interphase nucleus in mammalian cells is a highly ordered and compartmentalized structure with dynamic flexibility (Spector 2003; Lanctot et al. 2007; Misteli 2007). Indeed, a view of chromosome territories is emerging, in which individual chromosomes occupy discrete and non-overlapping three-dimensional domains in the nucleus. Moreover, particular regions of chromosomes can move with respect to nuclear structures and to other chromosomal regions upon their transcriptional activation (Lanctot et al. 2007). In addition, a number of nuclear bodies exist for distinct functions (Lamond & Spector 2003; Handwerger & Gall 2006), and a growing number of functional sites containing specific machineries are produced rapidly in the nucleus when required (Spector 2003). To understand the mechanisms that control the dynamic organization of nuclear domains and chromosomes is a great challenge for modern cell biology. To date, two different conflicting, although not mutually exclusive, models have been proposed: a deterministic (scaffold) model and a self-organization model (Cook 2002; Misteli 2007).

In the deterministic model, stable structural elements pre-exist to support the formation of nuclear/chromosome organization (Nickerson 2001; Berezney 2002). The ‘nuclear matrix’, originally defined as residual material remaining after extraction of nuclease-treated nuclei with high ionic strength buffers and detergents (Berezney & Coffey 1974; Mirkovitch et al. 1984), was described as a framework that maintains many of the architectural features of the nucleus (Nickerson 2001; Berezney 2002). Indeed, functional nuclear domains, including RNA transcription sites, DNA replication sites and chromosomal territories, retain their spatial positions even after the removal of the soluble nuclear proteins, strongly supporting this model (Berezney 2002). In addition, a number of observations suggested that the ‘nuclear matrix/scaffold’ functions as a structural constraint to anchor chromatin loops (Saitoh & Laemmli 1993). However, the concept of the ‘nuclear matrix’ is controversial, because principal structural components of the ‘nuclear matrix’ have not yet been identified, and many nuclear components including mRNAs move simply by diffusion (Pederson 2000).

On the other hand, in the self-organization model, the morphological appearance of nuclear compartments is a reflection of ongoing functions (Cook 2002; Misteli 2007). Once new functional sites are generated within the nuclear space, structural elements can form de novo even without pre-existing stable structures, and the resulting structural features support ongoing activities in a self-reinforcing manner. Recent photobleaching experiments have revealed that most nuclear proteins, including structural components of heterochromatin and ‘residential’ proteins of nuclear bodies, diffuse relatively freely and rapidly throughout the nucleoplasm (Misteli 2007). In addition, most nuclear structures can form de novo. For example, immobilization of both structural and functional components of Cajar body (CB) is sufficient for the formation of functional CB (Kaiser et al. 2008). The self-organization model is especially suited for the explanation of the dynamic and flexible properties of the interphase nucleus and its chromosomes.

Recent advances in mass spectrometry (MS) techniques combined with the complete sequencing of the human genome have facilitated the proteomic analyses of purified subnuclear fractions (Andersen & Mann 2006), including nucleoli (Andersen et al. 2002), the nuclear envelope (Schirmer et al. 2003) and nuclear speckles (Saitoh et al. 2004). These studies have given rise to new concepts about these compartments and implications for their roles. Furthermore, recent studies revealed that polymeric forms of actin are indeed present in the nucleus (McDonald et al. 2006). The actin/myosin I transport machineries are implicated in long-range chromosome movements induced by transcriptional activation (Chuang et al. 2006). These observations have inferred potential roles of proteins that are traditionally defined as architectural components of cells in facilitating the dynamic organization of the interphase nucleus, at least within nuclear microenvironments.

Much attention has been focused on the possible existence of nuclear architectural components, and an intensive proteomic analysis of nuclear insoluble proteins would probably give new clues about its composition. Further characterization of nuclear architectural components is essential to comprehensively evaluate what nuclear components potentially constitute any form of ‘nuclear architecture’. Toward this end, we analyzed two separate nuclear insoluble protein preparations by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) and identified 502 different proteins, including 37 proteins that are previously uncharacterized with regard to their functions. This report presents a rigorous proteomic analysis of nuclear insoluble fractions that identifies many new potential candidates for architectural components that are required for the formation of nuclear compartments or higher order chromatin structures.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Historically, several approaches have been employed to strip the nucleus of histones and other chromatin components to prepare nuclear insoluble fraction. However, there is still controversy on which reagents are suitable for its preparation, either for the sufficient depletion of chromatin components and soluble nuclear proteins while preserving in situ structure and function of nuclear architecture (Berezney & Coffey 1974; Capco et al. 1982; Mirkovitch et al. 1984; Nickerson 2001). To avoid obtaining results that could be biased by using a single type of procedure, we applied two different methods to prepare nuclear insoluble fractions for subsequent proteomic analysis.

Preparation and characterization of the high-salt-resistant nuclear insoluble fraction

The first nuclear insoluble fraction that is resistant to high-salt, non-ionic detergent and nuclease treatment was prepared as schematized in Fig. 1a by adapting the procedures developed mainly for the electron microscopic analysis of the ‘nuclear matrix’, wherein fibrogranular nuclear structures were observed (Capco et al. 1982; He et al. 1990). First, human HeLa-S3 cells were permeabilized with a buffer containing 0.5% Triton X-100 in a nearly physiological ionic strength to separate nuclei from cytoplasmic components. The isolated nuclei were then digested with DNase I and eluted sequentially with 0.25 M ammonium sulfate and 2 M NaCl. This sequential elution of digested nuclei allows stripping of chromatin efficiently while the residual structures observed under electron microscopy are preserved (He et al. 1990). Indeed, the amount of the four major histones decreased significantly (Fig. 1b), and stained DNA disappeared (Fig. 1d). The pellets were then treated with RNase A to obtain an RNA-depleted final fraction of high-salt-resistant nuclear insoluble proteins. Matrin-3 is a nuclear factor originally identified from similar fractions of rat liver nuclei (Nakayasu & Berezney 1991). To confirm the efficacy of our fractionation procedure, we tested the enrichment of Matrin-3 using Western blot analysis. As shown in Fig. 1c, it was about 50-fold enriched in the high-salt-resistant nuclear insoluble fraction compared with that in a whole cell extract. However, proliferating cell nuclear antigen (PCNA), a DNA polymerase clamp and one of the major nuclear soluble factors that we used here as a negative control, was abundant in whole nuclei but completely absent in the high-salt-resistant nuclear insoluble fraction (Fig. 1c). In addition, immunofluorescence analysis of nuclear proteins was performed. Heterogeneous nuclear ribonuclear proteins (hnRNP)-U and lamin-B1 remained clearly detectable even after RNase treatments (Fig. 1d and Fig. S1f, Supporting Information). However, nucleolin and fibrillarin – general markers of nucleoli – were reduced significantly after RNase treatments, although remnant nucleoli remained as observed by phase-contrast imaging (Fig. S1a,b). SC-35, a marker component of nuclear speckles, and CENP-A, a centromere-specific histone H3 variant, disappeared almost completely after DNase I treatment and subsequent elution with 0.25 M ammonium sulfate (Fig. S1c,e). Coilin, a marker component of Cajal body, was also removed by DNase I treatment, although faint fluorescent signals of coilin remained in the insoluble fraction (Fig. S1d). Taken together, these observations indicated that many of the soluble nuclear components were extracted efficiently during preparation of the high-salt-resistant nuclear insoluble fractions.


Figure 1
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  		Figure 1  Preparation and analysis of the high-salt-resistant nuclear insoluble fractions. (a) Flowchart of the preparation procedure. (b) An SDS-PAGE separation of the proteins extracted from total cells and from subcellular fractions are indicated at the top of the gel. Ten micrograms of proteins from each fraction, prepared as described in (a), were separated on a 4–20% gradient polyacrylamide gels and stained with CBB. Arrows indicate the positions of Matrin-3, lamin-A/C, lamin-B1 and histones. Sizes of the molecular weight markers are indicated in kDa. (c) Western blot analysis to show the enrichment of Matrin-3. The same amounts of proteins from each fraction indicated in (b) were probed using anti-Matrin-3 antibody. Anti-PCNA antibody was used as a control for nuclear soluble factors. Diluted proteins from salt-insoluble fraction were detected with anti-Matrin-3 to estimate the degree of enrichment of Matrin-3 in insoluble fraction. (d) Immunofluorescence labeling to validate the efficacy of preparation procedures. HeLa-S3 cells were centrifuged onto APS-coated glass slides using a cytospin. The mounted cells were treated as described in (a), followed by fixation and staining with DAPI or with specific antibodies against hnRNP-U. Scale bar: 5 µm. Cell, whole cell; Nucleus, isolated nucleus; Salt-insoluble, high-salt-resistant nuclear insoluble fraction.

 
Preparation and characterization of the LIS-extracted nuclear insoluble fractions

In the second approach, we employed the chaotropic ion lithium 3,5-diiodosalicylate (LIS) to elute histones and other chromatin proteins. This was carried out primarily with the intention to preserve the nature of the ‘chromosome scaffolds’ that are predicted nuclear structures associated with chromosomes at scaffold attachment regions in DNA (SAR, also referred as a matrix attachment regions; see Fig. 2a) (Mirkovitch et al. 1984). First, human HeLa-S3 cells grown in suspension were homogenized with a buffer containing 0.1% digitonin, and the isolated nuclei were extracted in the presence of LIS. The treatment of the nuclei with LIS induced the formation of a ‘nuclear halo’, in which freed DNA looped out into the surrounding space (Fig. 2d). The resulting nuclear halo was treated with DNase I and restriction enzymes. After these treatments, the amounts of the four major histones decreased significantly in the Coomassie brilliant blue R-250 (CBB) stained gel (Fig. 2b), and DNA staining disappeared (Fig. 2d). Resulting pellets were treated with RNase A and centrifuged to obtain final pellets of LIS-extracted nuclear insoluble fractions. Topoisomerase II{alpha} and hnRNP-U (also termed scaffold attachment factor A) are major components of similarly prepared LIS-resistant nuclear insoluble fractions and were found previously to associate with SAR (Earnshaw et al. 1985). Therefore, we assessed the efficacy of our fractionation procedure by monitoring the amounts of topoisomerase II{alpha} and hnRNP-U. As shown in Fig. 2c of Western blot analysis, topoisomerase II{alpha} and hnRNP-U were highly enriched in the LIS-extracted nuclear insoluble fractions (around 20-fold) when compared with whole cell extracts. By contrast, the signal for the regulator of chromosome condensation 1 (RCC1), one of the major chromatin binding proteins and used as a negative control, was extensively depleted by LIS treatment. Immunofluorescence analysis was also performed. Signals corresponding to hnRNP-U remained observable throughout the course of the fractionation (Fig. 2d), whereas the signals of nucleolin, fibrillarin, SC-35, coilin and CENP-A disappeared to a non-detectable level (Fig. S2a–e). Thus, we concluded that major nuclear factors were excluded efficiently, whereas conventional ‘chromosome scaffold’ factors identified in the previous study (Earnshaw et al. 1985) were enriched in our LIS-extracted nuclear insoluble fraction.


Figure 2
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  		Figure 2  Preparation and analysis of the lithium 3,5-diiodosalicyalate (LIS)-extracted nuclear insoluble fraction. (a) Flowchart of the preparation procedure. (b) An SDS-PAGE separation of the proteins extracted from total cells and subcellular fractions are indicated at the top of the gel. Ten micrograms of proteins from each fraction prepared as described in (a) were separated on a 4–20% gradient polyacrylamide gel and stained with CBB. Positions of topoisomerase II{alpha} (topoII{alpha}), hnRNP-U and histones are indicated. Bands of increased intensity that co-migrate with histones in the LIS-extracted nuclear insoluble fraction were RNase A. (c) Western blot analysis to show the enrichment of topoII{alpha} and hnRNP-U. The same amounts of proteins from each fraction indicated in (b) were probed using anti-topoII{alpha} or hnRNP-U antibodies. Diluted proteins from LIS-extracted nuclear insoluble fraction were detected with anti-topoII{alpha} and hnRNP-U antibodies to estimate the degree of enrichment in insoluble fraction. Anti-RCC-1 antibody was used as a control for chromatin-associated factors. (d) Immunofluorescence labeling to validate the efficacy of preparation procedures. Nuclei isolated from HeLa-S3 cells were plated on poly-L-lysine-coated cover slides by centrifuging and then treated as described in (a), followed by fixation and staining with Hoechst 33342 or specific antibodies against hnRNP-U. Scale bar: 5 µm. Cell, whole cell; Cytosol, supernatant after digitonin treatment; Nucleus, isolated nucleus and LIS-insoluble, LIS-extracted nuclear insoluble fraction.

 
Proteomic analysis of the two nuclear insoluble fractions

For proteomic analysis, the two nuclear insoluble fractions described above were resolved on gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels, and the separated proteins were stained with CBB (Figs 3 and 4). These gels were sequentially cut at approximately 2 mm intervals, although some slices were wider because of the absence of any prominent band at those positions. Gel slices were in-gel digested with trypsin and protein mixtures were extracted from the gel. The resulting peptide mixtures were analyzed separately using LC-ESI-MS/MS. In our analysis, false-positive identifications of the proteins were considerably low (see the Experimental procedures section for details).


Figure 3
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  		Figure 3  Proteins identified in the high-salt-resistant nuclear insoluble fraction. The prepared protein mixtures were separated on a 4–20% gradient SDS-PAGE gel and stained with CBB. The gel was then divided into 56 gel slices to be subjected to proteome analysis (LC-ESI-MS/MS). Gel slices excised are indicated with the numbers corresponding to those in Table S1. The gene names for the identified proteins are shown in each slice from left to right with a higher Mascot score. Gene names with Mascot scores of less than 100 are shown by green letters: for clarity they are indicated only once even if they were detected in more than one gel slice. A total of 333 different proteins were identified from the 56 gel slices. Marker protein sizes are indicated.

 

Figure 4
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  		Figure 4  Proteins identified in the LIS-extracted nuclear insoluble fraction. The prepared protein mixtures were separated on 4–20% gradient SDS-PAGE gels and stained with CBB. The gel was then divided into 50 slices to be subjected to proteome analysis (LC-ESI-MS/MS). A total of 50 gel slices excised for protein identification are indicated, with the numbers corresponding to those in Table S2. The gene names for the identified proteins are shown in each slice from left to right with a higher Mascot score. Gene names with Mascot scores of less than 100 are shown by green letters: for clarity they are indicated only once even if they were detected in more than one gel slice. A total of 330 different proteins were identified from the 50 gel slices. Marker protein sizes are indicated.

 
After these analyses, 333 different proteins in the high-salt-resistant nuclear insoluble fraction and 330 different proteins in the LIS-extracted nuclear insoluble fraction were identified as shown in Figs 3 and 4 respectively. Upon comparison of nuclear insoluble proteins prepared by our two separate methods, 161 proteins were found in both fractions. Therefore, the total number of proteins identified in our analysis was 502. Matrin-3, topoisomerase II{alpha} and hnRNP-U, used to monitor the enrichment of insoluble nuclear factors (Figs 1c and 2c ), were detected readily in both fractions. Other members of hnRNPs and lamins (A/C and B1), known as major components in nuclear insoluble fractions (Luderus et al. 1992), were also identified commonly in our two fractions.

As expected, factors identified in these two insoluble fractions were not entirely overlapping (172 proteins and 169 proteins were non-overlapping in high-salt-extracted versus LIS-extracted fractions respectively), reflecting the difference of extraction procedures (Fig. 5a). In the high-salt-resistant nuclear insoluble fraction, chromatin and chromatin-related proteins were efficiently removed, whereas non-chromatin binding proteins, such as factors related to RNA metabolism, were enriched (Fig. 5b). By contrast, the LIS-extracted nuclear insoluble fraction was prepared with an intention to preserve a putative ‘chromosome scaffold’, and therefore chromatin-related or chromosome scaffold-associated proteins reported previously (Gassmann et al. 2005) were more included (Fig. 5c). The information obtained in this study is summarized in Tables S1 and S2. In addition, detailed information on the peptides identified in this study is summarized in Tables S3 and S4.


Figure 5
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  		Figure 5  Profiling of proteins identified in the high-salt-resistant and LIS-extracted nuclear insoluble fractions. (a) A Venn diagram indicates the separately identified proteins in each fraction and the overlap between the two. Numbers indicate proteins identified in each group, and the other numbers in parentheses are those of the proteins classified as ‘function unknown’. Salt-insoluble, high-salt-resistant nuclear insoluble fraction; LIS-insoluble, LIS-extracted nuclear insoluble fraction. (b and c) The proteins identified in the high-salt-resistant nuclear insoluble fraction in (b) and in the LIS-extracted nuclear insoluble fraction in (c) were classified based on their known and hypothetical functions, including those classed as ‘function unknown’. The number of the proteins identified in each category was indicated in parentheses.

 
Functional classification of the identified proteins

We carried out extensive bibliographic and bioinformatic analyses for each of the 502 proteins, but many of them are still not well defined with regard to their biological functions. Therefore, we searched for homologs in other species with help from YOGY, a web-based, integrated database used to retrieve protein homologs (http://www.sanger.ac.uk/PostGenomics/S_pombe/YOGY). We obtained many proteins that displayed strong similarity with some proteins in budding yeast (Tables S1 and S2). Some of the homologs have been analyzed well, with assigned functions based on biological analysis. These in silico approaches allowed us to assign known and/or hypothetical functions to many of the identified proteins. Based on these analyses, functional classifications were deduced, revealing a wide range of implicated functions for various nuclear insoluble proteins (Fig. 5b,c). To our surprise, ~7% of the total proteins we identified (37 proteins) displayed no known functions and have been categorized as ‘function unknown’.

Functional domains frequently found in the identified proteins

Motif analysis is a useful tool for predicting the function(s) of proteins. We searched for motifs using the Pfam database (http://pfam.sanger.ac.uk/search) and summarized the results in Table 1. The motifs most commonly found in our analysis included the RNA recognition motif (RRM), WD40 repeats, HEAT repeats and the SAP domain. The RRM is a protein motif mainly required for association with RNA, implicated in a variety of functions related to RNA metabolism (Maris et al. 2005). Thirty-five RRM-containing proteins were isolated in our fractions, despite extensive RNase A treatment during fractionation. Two RRM-containing proteins were classified as ‘function unknown’, including MKI6IP in the high-salt-resistant nuclear insoluble fraction and MKI6IP and RBM25 in the LIS-extracted nuclear insoluble fraction. The WD repeat (WDR) proteins generally contain four or more repeating units of approximately 40 amino acids that usually have a tryptophan (W)–aspartic acid (D) dipeptide at their carboxyl terminus. The WDR fold into a propeller-like structure with blades, each consisting of four-stranded β sheets (Smith et al. 1999). The WDR proteins are found in all eukaryotes and are implicated in a wide range of functions. Among 30 different WDR proteins obtained in this study, three proteins were classified as ‘function unknown’, including WDR75 in the high-salt-resistant nuclear insoluble fraction and SMU1, THOC6 and WDR75 in the LIS-extracted nuclear insoluble fraction.


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  		Table 1  The numbers of identified proteins that contain unique functional domains
 
Another interesting protein motif often found in the identified factors was the HEAT repeat. This corresponds to tandemly arrayed units forming a rod-like helical structure and appears to function in protein–protein interaction (see the Discussion section; Andrade et al. 2001). A careful alignment revealed that the HEAT repeat is also similar to the structural maintenance of chromosome (SMC) repeat (Neuwald & Hirano 2000). A total of 20 different HEAT/SMC repeat-containing proteins were obtained here, including one functional unknown protein, TEX10, identified both from the high-salt-resistant and LIS-extracted nuclear insoluble fractions. The SAP motif, originally identified within sequences of chromatin-associated proteins such as scaffold attachment factors A (also known as hnRNP-U) and B, is a putative DNA binding motif functionally associated with chromosome structure (Aravind & Koonin 2000). The identified proteins containing SAP motifs were hnRNP-U in the high-salt-resistant nuclear insoluble fraction and ACIN1, SAFB1, hnRNP-U, Ku70 and DEK (DEK oncogene protein) in the LIS-extracted nuclear insoluble fraction.

Subnuclear distribution of SON and DEK

To substantiate the MS-based results, we analyzed the subcellular distributions of two identified proteins, SON and DEK. SON protein (SON) is a large 2564 amino acid protein identified in the proteomic analysis of nuclear speckles (Saitoh et al. 2004). SON was identified only in the high-salt-resistant nuclear insoluble fraction and its detailed function has not yet been determined. Immunofluorescence localization of SON revealed that SON localizes in the nuclear speckles in intact cells and in isolated nuclei, as expected (Fig. 6a). Noticeably, the dot-shaped signals of SON remained in the high-salt-resistant nuclear insoluble materials (Fig. 6a), although the signals of other nuclear speckle factors such as SC-35 were diminished after the same treatments (see Fig. S1c).


Figure 6
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  		Figure 6  Subcellular localizations of SON and DEK. (a) HeLa-S3 cells were centrifuged onto APS-coated cover slides using a cytospin. The cells on glass slides were treated as described in Fig. 1a and fixed with 4% paraformaldehyde. The fixed samples were visualized or stained with specific antibodies against SON or DAPI for DNA. (b) Nuclei were isolated from HeLa-S3 cells stably expressing GFP-fused DEK. Isolated nuclei were attached to the poly-L-lysine-coated glass slides and treated as described in Fig. 2a, followed by fixation with 4% paraformaldehyde. The fixed samples were stained with Hoechst 33342 (DNA) or immunolabeled with anti-topoisomerase II{alpha} antibody (TopoII{alpha}), as indicated. Difference in localization of DEK (green) and topoisomerase II{alpha} (magenta) in LIS-extracted nuclear insoluble fractions was shown by overlapping image using a laser scanning confocal microscope (bottom). (c) HeLa-S3 cells stably expressing GFP-fused DEK were centrifuged onto APS-coated glass slides using a cytospin. DNA was counterstained with DAPI. Deconvolved images of cells at different phases of mitosis were corrected by chromatic aberration of microscope that was measured using fluorescent beads with a diameter of 0.2 µm. Colocalization of DEK (green) and DNA (magenta) were shown in white areas. Enlarged images of the white rectangle regions were also shown at the bottom. (d) Metaphase chromosome spreads were prepared as detailed in the Experimental procedures section and deconvolved images were obtained as in (d). The panel on the right is an enlarged image of the white rectangle region. Scale bars: 5 µm (left) and 1 µm (right). Cell, untreated cells; Nucleus, isolated nuclei, Salt-insoluble, high-salt-resistant nuclear insoluble material; LIS-insoluble, LIS-extracted nuclear insoluble material. Scale bar: 5 µm.

 
DEK is a conserved protein that contains a SAP motif and was originally isolated as a part of a fusion protein expressed in cells derived from patients with acute myeloid leukemia-harboring (t6;9) chromosomal translocations (von Lindern et al. 1992). DEK was identified only in the LIS-extracted nuclear insoluble fraction, and its function remains largely unknown. To visualize DEK, enhanced green fluorescent protein (GFP)-tagged DEK (GFP–DEK) was expressed stably in HeLa-S3. GFP–DEK was located exclusively in the nucleus at interphase and also colocalized with DNA, consistently with a previous report (Fig. 6b) (Kappes et al. 2001). In addition, GFP–DEK remained associated with the LIS-extracted nuclear insoluble material, and their localizations were different from those of topoisomerase II{alpha} (Fig. 6b), a major component of insoluble nuclear materials in interphase. Interestingly, GFP–DEK signals were prominent in regions of sparse chromatin and were not detected at the nuclear periphery (Fig. 6c). In mitosis, the GFP–DEK protein remained associated with mitotic chromosomes from prometaphase to telophase (Fig. 6c). Of note, the dense signals of GFP–DEK did not overlap with those of 4',6-diamidino-2-phenylindole (DAPI)-stained DNA of the chromosomes. Rather, GFP–DEK was enriched in regions where chromatin is sparse and around the periphery of chromosomes. This is evident from deconvolved images of interphase and mitotic cells (Fig. 6c) and of spread mitotic chromosomes (Fig. 6d), as well as in 3D images reconstructed from confocal laser scanning microscopy (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
This study represents the first intensive proteomic analysis of nuclear insoluble fractions by LC-ESI-MS/MS reported to date. As the sensitivity of protein detection by LC-ESI-MS/MS used in our study is high, we might have identified factors that are present in the nuclear insoluble materials in minor amounts at some specific site of the nucleus or time of the cell cycle, or inevitable contaminants. Nevertheless, the results of this study are useful, as it provides detailed catalogs of the nuclear insoluble fractions to seek for the potential candidate proteins responsible for various aspects of nuclear architectures.

As expected, numerous proteins implicated in a variety of nuclear functions were identified in our study, and extensive bioinformatic and bibliographic analyses of those proteins gave some hints for understanding nuclear organization. For instance, many identified proteins have binding or functional affinity to RNAs or RNPs, such as hnRNPs, and RRM- and DEAD box RNA helicase-containing proteins. The mechanisms of RNA transport/retention and the physiological implications of retained RNA in the nucleus remain largely unknown, but RNA molecules and/or RNPs have long been suggested to play some structural and regulatory roles in nuclear architectures (Nickerson 2001). Indeed, RNase A treatment of unfixed nuclei abolished chromosome territories in human cells, suggesting some important roles of RNAs and/or RNPs in their stability (Ma et al. 1999). Additionally, pericentric staining detected with both anti-HP-1{alpha} and anti-methylated histone H3-K9 antibodies disappeared after the RNase A treatment of permeabilized unfixed mammalian cells (Maison et al. 2002).

Most of the proteins identified previously as a component of the mitotic chromosome scaffold (Gassmann et al. 2005) were included in our lists (63 of 78 proteins; 53 of which were in the LIS-extracted nuclear insoluble fraction). Given that architectural factors exist in the interphase nucleus to support the organization of some nuclear compartments, their attachments with mitotic chromosomes at mitosis would be beneficial because they could facilitate the reassembly of the original compartments of the nucleus promptly at the exit of mitosis. How individual chromosomes are kept together in a distinct mass in the interphase nucleus is an important question to be resolved. We expect that some architectural components might be present to support organization of chromosome. If this is the case, such components should remain associated with the chromosomes during the course of mitosis.

Among the functional domains often found in this study, the HEAT/SMC repeats are highly suggestive of their potential implications in nuclear/chromosome organization. A total of 20 proteins contained HEAT/SMC repeat. The HEAT/SMC repeats are predicted to function as a flexible scaffold on which other proteins can assemble, and good examples are condensin and cohesin complexes (Neuwald & Hirano 2000). Whether or not stable scaffold structures exist for interphase nuclear organization, some HEAT/SMC repeat-containing proteins – once recruited – might serve as platforms for the subsequent assembly of other functionally related components to form nuclear compartments.

Detailed analysis of the cellular distribution of GFP–DEK gave novel insights into the cellular distributions and potential functions of DEK. GFP–DEK signals were clearly enriched in regions where chromatin is decondensed or sparse in the interphase nuclei. GFP–DEK associated with mitotic chromosomes, as shown previously (Kappes et al. 2001), but the condensed signals of GFP–DEK were concentrated in regions of sparse chromatin or in chromosome peripheries. The DEK protein binds preferentially to superhelical and cruciform DNA (Waldmann et al. 2004). Therefore, we suggest that DEK might function as an architectural protein to support the formation of some type of chromosomal domains. Further analysis of DEK protein could reveal novel mechanisms for nuclear and chromosome organization.

Microscopic analysis of the localization of SON and DEK showed their associations with the nuclear insoluble materials, corroborating our MS analysis. Importantly, we found a novel role of SON as a component required for the proper localization of other pre-mRNA-processing factors, partly by observing morphological changing in nuclear speckles in SON-depleted cells (details are discussed in the other paper, A. Sharma, H. Takata, K.-I. Shibahara, A. Bubulya and P. A. Bubulya, unpublished data). As SON remained associated with nuclear speckle-like residual structures even after intensive treatment of the nuclei with high-salt, detergent and nuclease, we speculate that SON is a key ‘nuclear speckle scaffold’ factor to support nuclear speckle assembly.

An exciting but long-controversial question is whether architectural structures exist to support the dynamic organization of the interphase nucleus (Pederson 2000; Nickerson 2001; Berezney 2002). In other words, to what extent can the self-organizing properties of the nucleus promote and maintain its own architectural stability and dynamic behavior? So far, there is no clear evidence of networks or long filamentous structures in the nucleus in situ (Pederson 2000). Indeed, even through the intensive searching, any novel factors potentially capable of constituting filamentous structures could not be identified besides lamins, actins and other major cytoskeletal components. As it is difficult to exclude cytoskeletal contamination completely during preparation of nuclear insoluble fractions, we were initially concerned about having cytoplasmic contamination in our nuclear insoluble fractions. However, recent studies demonstrated that actins and myosins do exist in the nucleus, and they are implicated in a wide range of functions (Hofmann et al. 2006; Jockusch et al. 2006). An excellent study predicted unique polymeric forms of actins to be present in the nucleus (McDonald et al. 2006). In this context, immunofluorescence analysis using the monoclonal antibody (2G2) that also recognizes actins present in the nucleus indicated that indeed there was 2G2-labeled actin in the LIS-extracted nuclear insoluble materials (H. Nishjima, H. Takata, K.-I., Shibahara, unpublished data), suggesting a role for nuclear actin in the organization of the interphase nucleus. In addition, nuclear myosin I is an alternative splicing product of the cytoplasmic myosin I (Pestic-Dragovich et al. 2000). Many components of cytoskeletal systems have multiple alternatively spliced forms, but not all of the dispersed exons are assembled or annotated to the draft human genome sequence. Therefore, comparison of peptides from our tandem MS data with the human genome sequence database is important for finding novel nuclear isoforms of cytoskeletal components.

In conclusion, this proteomic study of human nuclear insoluble fractions identified 502 different proteins with 37 previously uncharacterized proteins, two of which (SON and DEK) we showed to be associated with nuclear insoluble materials in vivo. Notably, we found that SON plays a key role for the integrity of nuclear speckles as a ‘nuclear speckle scaffold’ (A. Sharma, H. Takata, K.-I. Shibahara, A. Bubulya and P. A. Bubulya, unpublished data). Although much work remains to be carried out to determine the functions of individual nuclear insoluble proteins that we have identified here, the data presented in this study will be invaluable to identify other nuclear architectural components required for the dynamic organization of the interphase nucleus.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Cell culture

Human HeLa-S3 cell lines were maintained in Joklik’s modification of minimal essential medium (Sigma-Aldrich, St Louis, MO, USA) supplemented with 5% (v/v) fetal bovine serum (Nissui Seiyaku, Tokyo, Japan), 100 U/mL penicillin, 100 µg/mL streptomycin and 2 mM L-glutamine (Sigma-Aldrich) at 37°C under 5% CO2 in air in a humid incubator.

Reagents and antibodies

See Appendix S1 in Supporting Information for details.

Preparation of the high-salt-resistant nuclear insoluble fraction or material

The high-salt-resistant nuclear insoluble fraction was prepared from human HeLa-S3 cells as described with some modifications (He et al. 1990). Briefly, HeLa-S3 cells harvested at 5.0 x 105 cells per mL were incubated at 4°C for 3 min in cytoskeleton (CSK) buffer [10 mM PIPES–NaOH pH 6.8, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, 0.5% Triton X-100, 1 µg/mL leupeptin, 1 µg/mL aprotinin, 1 µg/mL pepstatin and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. After nuclei were carefully checked by phase-contrast microscopy to avoid cytoplasmic contamination, soluble proteins were separated from nuclei by centrifugation at 600 g for 3 min. To remove DNA from the nuclei, the pellets were treated with DNase I at 200 U/mL (Takara Bio Inc., Ohtsu, Japan) at 25°C for 30 min in digestion buffer (10 mM PIPES–NaOH pH 6.8, 50 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, 0.5% Triton X-100 and 1.2 mM PMSF). Ammonium sulfate was then added to the digested mixtures from a 1 M stock solution to a final concentration of 0.25 M, and the mixtures were incubated at 25°C for 5 min, followed by centrifugation at 600 g for 3 min. For further extraction, the pellets were incubated in the digestion buffer containing 2 M NaCl at 4°C for 20 min, followed by centrifugation at 600 g for 3 min. To remove RNA-related components, the pellet was incubated with 0.2 mg/mL of RNase A in the digestion buffer at 25°C for 30 min, and the mixtures were pelleted by centrifugation at 600 g for 3 min to obtain a final fraction; hereafter, this is referred to as ‘high-salt-resistant nuclear insoluble fraction or material’. The fractions were directly dissolved in SDS sample buffer [50 mM Tris–HCl pH 6.8, 2% sodium dodecyl sulfate (SDS), 10% glycerol and 1%β-mercaptoethanol] for 1D SDS-PAGE or stored at –80°C.

Preparation of the LIS-extracted nuclear insoluble fraction or material

The LIS-extracted nuclear insoluble fraction was prepared from human HeLa-S3 cells as described with some modifications (Neri et al. 1999). In brief, HeLa-S3 cells, harvested at 5.0 x 105 cells per mL, were washed once in phosphate-buffered saline (PBS) (pH 7.4, free of Mg2+ and Ca2+). To isolate nuclei, the washed cells were resuspended in isolation buffer (7.5 mM Tris–HCl pH 7.4, 40 mM KCl, 1 mM EDTA–KOH pH 7.4, 0.1% water-soluble digitonin from Wako Pure Chemical, Osaka, Japan, 1% thiodiglycol, 0.1 mM spermine, 0.25 mM spermidine, 0.5 mM PMSF and 1 µg/mL each of aprotinin, pepstatin and leupeptin) and lyzed in a dounce homogenizer (Wheaton, Millville, NJ, USA) using a B pestle until cytoplasmic contamination was not detectable by phase-contrast microscopy. The nuclear pellet was washed twice by nuclear washing (NW) buffer (3.75 mM Tris–HCl pH 7.4, 20 mM KCl, 0.5 mM EDTA–KOH, pH 7.4, 0.1% digitonin, 0.05 mM spermine, 0.125 mM spermidine, 1% thiodiglycol, 0.5 mM PMSF and 1 µg/mL each aprotinin, pepstatin and leupeptin) by centrifugation at 200 g. The isolated nuclei were then resuspended in NW buffer (but without EDTA–KOH) at a genomic DNA concentration of 200 µg/mL and incubated on ice in the presence of 0.5 mM CuSO4 for 10 min. The resulting nuclei were extracted with LIS dissolved immediately before use (Wako Pure Chemical) in extraction buffer (20 mM HEPES–NaOH pH 7.4, 100 mM lithium acetate, 1 mM EDTA–NaOH pH 7.4, 0.1% digitonin and 0.5 mM PMSF). Ten milliliters of LIS solution was used for 200 µg of DNA. Extraction was carried out for 10 min at room temperature, and the mixtures were sedimented at 1500 g for 10 min. The loosely compacted pellet was washed six times to remove LIS using Tris-digestion buffer (20 mM Tris–HCl pH 7.4, 20 mM KCl, 70 mM NaCl, 10 mM MgCl2, 0.1% digitonin, 0.05 mM spermine, 0.125 mM spermidine, 0.5 mM PMSF and 1 µg/mL each of aprotinin, pepstatin and leupeptin). After washing, the LIS-extracted nuclei was resuspended in 10 mL of Tris-digestion buffer, and DNA was digested for 2 h at 37°C, under constant agitation, using DNase I at 100 U/mL and EcoRI, HindIII and XhoI at 50 U/A260 unit of LIS-extracted nuclei. The DNA-digested nuclei were further treated with RNase A (0.2 mg/mL) and MNase (3 U/mL; Sigma-Aldrich) for 1.5 h at room temperature. The mixtures were sedimented at 1200 g for 10 min and washed four times with the Tris-digestion buffer to obtain a final fraction; hereafter, this is referred to as ‘LIS-extracted nuclear insoluble fraction or material’. The residues were directly dissolved in SDS–urea sample buffer (50 mM Tris–HCl pH 6.8, 2% SDS, 4 M urea, 10% glycerol and 100 mM dithiothreitol) for 1D SDS-PAGE or stored at –80°C.

Mass spectrometry and data analysis

After electrophoresis, the gel was stained with CBB. After imaging, the area from the top to the bottom of each lane of the CBB-stained gel was cut, as indicated in Figs 3 and 4, and each gel slice was further cut into small pieces (~0.5 mm cubes). The protein was in-gel digested using trypsin (Trypsin Gold, Mass Spectrometry Grade trypsin; Promega, Madison, WI, USA), as described (Shevchenko et al. 1996), or using XL-TrypKit (APRO Life Science Institute Inc., Naruto, Japan). The recovered tryptic peptides were concentrated and desalted by passing through a µ-C18 ZipTip (Millipore, Billerica, MA). The peptides were eluted in 50% acetonitrile (ACN)/0.1% trifluoroacetic acid. After drying the solution in a SpeedVac, the peptides were resuspended in 5 µL of 0.1% formic acid. Protein identification was performed using LC-ESI-MS/MS (NanoFrontier L; Hitachi High-Technologies Co, Tokyo, Japan). Using the autosampler, 3-µL aliquots of tryptic digest samples were loaded onto a preconcentration column (200 µm inner diameter, 5 mm in length; GL Science, Tokyo, Japan) at a flow rate of 20 µL/min, and the peptides were separated and introduced into the mass spectrometer through a C18-packed PicoFritTM column (75 µm inner diameter, 10-cm column equipped with a silica tip, 15 µm inner diameter; New Objective, Woburn, MA, USA). The column was previously equilibrated with solvent A (2% ACN and 0.1% formic acid). Peptides were eluted with a linear gradient from 100% solvent A to 60% solvent B (98% ACN and 0.1% formic acid) at a flow rate of 200 nL/min. Spray voltage was set at 1.8 kV, and capillary temperature was set at 140°C. The mass spectrometer was operated in positive ion mode. The MS scan range was 200–2000 m/z, and multiply charged ions with MS intensity higher than 10 counts were automatically chosen for MS/MS. In the case where there were many ions with intensity higher than 10 counts, the number of ions chosen for MS/MS simultaneously was set to 2. The acquisition time for both MS and MS/MS scan cycles was 1 s. Exclusion time was set to 2 min. Following data acquisition, the raw data files acquired for each LC-MS/MS run were converted to Mascot generic files using Data Processing software (NanoFrontier L Data Processing, P/N 3807051-03). The resulting Mascot generic files were searched against National Center for Biotechnology Information nonredundant (NCBInr) databases (NCBInr 20080322 including 6342781 proteins entries and 2165052802 residues) using the MASCOT search engine (version 2.2.01; Matrix Science Ltd., London, UK). Searches were performed with the following criteria: species restrictions to Homo sapiens; the expectation value (P) <0.05; tryptic specificity allowing one missed cleavage; MS tolerance was set to ±75 parts per million (p.p.m.) and MS/MS as ±0.2 Da; a variable modification was oxidation (Met) and carbamidomethylation (Cys) and a fixed modification was not allowed. Identification was considered positive if the protein was obtained with at least two independent peptides with an expectation value of P < 0.05. The false discovery rate for detected peptides was 4.2% when the expectation value was set at 0.05 (Fig. S3). As proteins with at least two high-expectation value peptides (P < 0.05) per protein were selected, false-positive identifications of the proteins were considerably minimized. If peptides were matched to multiple members of a protein family, or if a protein appeared under different names and accession numbers, one entry with the highest score was selected.

Expression of the enhanced green fluorescent protein-fusion protein

See Supporting Information for details.

Immunofluorescence localization of proteins

For the high-salt-resistant nuclear insoluble fraction, harvested HeLa-S3 cells were resuspended in PBS and transferred to amino propyl silane (APS)-coated glass slides (Matsunami, Osaka, Japan) using a cytospin centrifuge (Shandon, Pittsburgh, PA, USA). The cells on the glass slides were treated accordingly as described above, followed by fixation with 4% paraformaldehyde for 10 min at 37°C and permeabilized with 0.2% Triton X-100 in PBS. After incubation with 1% Bovine Serum Albumin (BSA) in PBS for 15 min, samples were incubated with primary antibodies for 1 h at room temperature, followed by incubation with secondary antibodies for 1 h. After washing, the cells were stained with 1 µg/mL DAPI. For the LIS-extracted nuclear insoluble fraction, nuclei were isolated from HeLa-S3 cells grown in suspension as described above, resuspended in NW buffer without EDTA and incubated on ice in the presence of 0.5 mM CuSO4 for 10 min. The treated nuclei were attached onto poly-L-lysine-coated Lab-Tek II Chamber Slide (Nunc, Naperville, IL, USA) by centrifugation at 100 g. The nuclei on the slides were treated as described above. The resulting samples were fixed with 4% paraformaldehyde for 10 min at room temperature and permeabilized with 0.2% Triton X-100 in PBS. After 1 h of incubation with blocking buffer (PBS supplemented 2% BSA, 0.4% gelatin and 0.2% Tween-20), samples were incubated with primary antibodies for 1 h at room temperature, followed by incubation with secondary antibodies for 1 h. DNA staining was performed with 5 µg/mL Hoechst 33342 in PBS supplemented 0.2% Tween20. In both cases, samples were mounted using VECTASHIELD (Vector, Burlingame, CA, USA) antifade mounting medium. Images were acquired using a Zeiss Axiovert 200 M fluorescence microscope or a Zeiss LSM510 META laser scanning confocal microscope (Carl Zeiss Inc., Thornwood, NY, USA). For deconvolved images, the Z-stack images were obtained at a 0.2-µm interval by Zeiss Axiovert 200 M using a 100x objective and were processed using AxioVision 4.5 (Carl Zeiss Inc.).

For preparing metaphase chromosome spread samples, HeLa-S3 cells stably GFP-fused DEK were synchronized at metaphase with 0.1 µg/mL colcemid for 3 h. The cells were collected and swelled using hypotonic treatment (75 mM KCl) for 15 min. The cells were centrifuged onto APS-coated cover slides using a cytospin. After fixation with 4% paraformaldehyde, DNA was stained with 1 µg/mL DAPI.


   Acknowledgements
 
We thank Ms Kagen Kunita and Ms Akane Morohoshi for technical assistance. The anti-RCC1 antibody was kindly donated by Dr Takeharu Nishimoto of Kyusyu University. This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas and City Area Program from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) and grants from the Transdisciplinary Research Integration Center, Research Organization of Information and Systems, the Seed Of Excellence Foundation in Shizuoka Prefecture, the Takeda Science Foundation and the Naito Foundation, Japan. HT is a postdoctoral fellow in the National Institute of Genetics.


   Footnotes
 
Communicated by: Fuyuki Ishikawa Back

{dagger}Both authors contributed equally to this work. Back

* hnishiji{at}lab.nig.ac.jp or kshibaha{at}lab.nig.ac.jp


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 Introduction
 Results
 Discussion
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Received: 13 April 2009
Accepted: 13 May 2009




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