Human Molecular Genetics, 2001, Vol. 10, No. 18 1995-2011
© 2001 Oxford University Press
Large-scale identification of mammalian proteins localized to nuclear sub-compartments
MRC Human Genetics Unit, Crewe Road, Edinburgh EH4 2XU, UK
Received May 15, 2001; Revised and Accepted June 26, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Many nuclear components participating in related pathways appear concentrated in specific areas of the mammalian nucleus. The importance of this organization is attested to by the dysfunction that correlates with mis-localization of nuclear proteins in human disease and cancer. Determining the sub-nuclear localization of proteins is therefore important for understanding genome regulation and function, and it also provides clues to function for novel proteins. However, the complexity of proteins in the mammalian nucleus is too large to tackle this on a protein by protein basis. Large-scale approaches to determining protein function and sub-cellular localization are required. We have used a visual gene trap screen to identify more than 100 proteins, many of which are normal, located within compartments of the mouse nucleus. The most common discrete localizations detected are at the nucleolus and the splicing speckles and on chromosomes. Proteins at the nuclear periphery, or in other nuclear foci, have also been identified. Several of the proteins have been implicated in human disease or cancer, e.g. ATRX, HMGI-C, NBS1 and EWS, and the gene-trapped proteins provide a route into further understanding their function. We find that sequence motifs are often shared amongst proteins co-localized within the same sub-nuclear compartment. Conversely, some generally abundant motifs are lacking from the proteins concentrated in specific areas of the nucleus. This suggests that we may be able to predict sub-nuclear localization for proteins in databases based on their sequence.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The mammalian nucleus is not homogeneous; rather it is organized into domains associated with different facets of nuclear function, and so proteins in common pathways often colocalize into specific areas of the nucleus (1–3). The importance of this organization is revealed by the mis-localization of nuclear proteins in human genetic disease (4,5), in cancers (6) and in virally infected cells (7).
The full complexity of the mammalian nucleus is yet to be revealed. Many abundant nuclear proteins have been detected and identified biochemically. Developments in protein microcharacterization and mass spectrometry have allowed the identification of components of large nuclear complexes/structures such as the spliceosome (8) or interchromatin granules (ICGs), which are components of the nuclear splicing speckles (9). Mutant genetic screens have identified proteins necessary for genome and nuclear functions but rely on recognition of appropriate mutant phenotypes and are best suited to the most genetically tractable organisms. Visual screens that identify mammalian proteins based only on the criterion of their sub-cellular location provide an alternative approach, and the large size of the mammalian nucleus makes it particularly amenable to a visual dissection. Such screens take one of two general forms. In the first, cDNA or genomic libraries under the control of an exogenous promoter are fused to green fluorescent protein or ß-galactosidase (ßgal) reporters and transfected into cells (10–14). However, these fusion proteins are expressed at non-physiological levels, which may alter or mask normal protein localization, and in some cases may be cell lethal. The relevant genes must also be represented in the cDNA libraries used to make the fusion constructs and so these screens may be biased toward abundantly expressed genes. In the second approach, reporter constructs are integrated into endogenous genes so that the resulting fusion proteins are expressed from the normal gene promoter. The tagging of the endogenous gene with the reporter is also mutagenic, thus enabling mutant phenotypes to be investigated (15).
The sequencing of the human and mouse genomes now makes genome-based approaches to understanding nuclear proteins more feasible. In turn, determining sub-nuclear localization can give important clues to function for novel proteins present in databases and currently of unknown function. Previously we have shown that a gene trap screen is capable of identifying chromosomal and nuclear proteins in mouse cells. A ß-galactosidase-neomycin phosphotransferase (ßgeo) reporter gene (lacking its own promoter and ATG) is integrated into an intron of an expressed gene so that it can be spliced in frame into the gene transcript (15). The resulting fusion proteins can recapitulate the sub-nuclear localization of wild-type proteins. However, we are not able to trap genes that contain no introns or proteins in which the nuclear localization signal (NLS) is at the N-terminus. The sequence of the trapped gene is identified by 5' rapid amplification of cDNA ends (5' RACE) from the fusion transcript (Fig. 1) and database searching.
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Our pilot screen established the efficacy of the approach (15). Refinements to 5' RACE sequencing and the enormous increase in human and mouse gene sequences present in databases suggested that the screen could be scaled up to identify large numbers of nuclear proteins. Here we report the identification and characterization of more than 100 different mouse genes whose protein products locate to nuclear compartments, including the nucleolus, splicing speckles, chromosomes (including heterochromatin), other nuclear foci and the nuclear periphery. More than half of the genes isolated in this screen encode novel proteins, or proteins whose sub-cellular localization was previously unknown. The analysis of conserved domains and motifs reveals common signatures amongst proteins concentrated in the same nuclear compartment, whilst the unexpected occurrence of some functional protein motifs points to new biochemical activities in some compartments.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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At least one in 10 proteins expressed in embryonic cells target to the nucleus
We conducted large-scale gene trap screens in both F9 cells and E14 mouse embryonic stem (ES) cells and used 5-bromo-4-chloro-3-indoyl ß-D-galactoside (X-Gal) staining to identify those with nuclear fusion proteins. Approximately two-thirds of the 703 neomycin resistant (neoR) F9 clones and 646 ES cell clones had visible X-Gal staining. Colonies with no visible staining are due to the greater sensitivity of G418 selection compared with X-Gal staining and to inactivation of ßgal activity in some fusion proteins (15). Approximately 40% of clones showed staining restricted to the cytoplasm and so were discarded. In addition
10% of clones had an intense spot of staining close to, but outside of, the periphery of the nucleus. These are most likely clones in which the fusion protein has misfolded and mislocalized (15). Ten percent of neoR clones (17% of clones with visible staining) were found to have either a nuclear distribution of X-Gal stain, or had staining in association with chromosomes or the chromosome segregation machinery in mitotic cells and these were selected for further study. Finally 4% of clones had staining in the cytoplasm as well as the nucleus. Only those with the most prominent nuclear staining were studied.
The identity of trapped genes and establishing a database of nuclear proteins
Sequences of trapped genes were obtained by 5' RACE and compared with those in databases using the BLAST algorithm. These results together with sub-nuclear distribution determined from immunostaining using a ßgal antibody (see below) are set out in Table 1. Approximately 35% of the genes encode previously identified nuclear proteins of known cellular location. One quarter of the genes encode proteins of known sequence but previously unknown sub-cellular localization and 37% of sequences identified only mouse or human expressed sequence tags (ESTs) or cDNA sequences. The remaining clones match genomic sequences only.
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Predicted protein products were analysed for the presence of NLSs using Psort II (http://psort.nibb.ac.jp/form2.html). The incidence of other conserved protein domains and motifs was assessed using InterProScan (http://www.ebi.ac.uk/interpro/scan.html) and SMART (http://smart.embl-heidelberg.de). The site of gene trap integration relative to the full-length sequence of wild-type proteins is indicated to illustrate how many amino acids/motifs are missing from the trapped protein (Table 1). For ES cell lines this may indicate whether the gene trap is likely to represent a knockout of gene function. To gather together the information from our screens we have created a nuclear protein relational database (NPD) that will be made publicly available.
Fusion proteins locate to different sub-nuclear compartments
To determine the distribution of fusion proteins within the nucleus at high resolution, cells were immunostained using an antibody recognizing ßgal. In about half of the clones the immunofluorescence signal was diffusely spread across the whole nucleus, but excluded from the nucleolus (nucleoplasmic). Figure 2 shows clone ES510, in which a gene encoding a zinc finger protein has been trapped, as an example of this staining pattern.
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In other clones the fusion proteins concentrated in known nuclear compartments. Amongst the most common of these (10% of clones) was the nucleolus—revealed by staining with an antibody that recognizes fibrillarin. F9/21D6 in Figure 2 is a novel protein that concentrates in the nucleolus around the dense fibrillar centres where fibrillarin is found.
Concentration of trapped proteins in multiple nuclear speckles was also common. The coincidence of some of these (10% of clones) with sites of splicing factor accumulation (splicing speckles) was confirmed by co-immunofluorescence with Y12 antibodies recognizing snRNPs (e.g. clone F9/5A7, in which a putative splicing factor hcc1 has been trapped in Fig. 2; see Fig. 3 for further examples).
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For another 10% of clones, the sites of ßgal immunostaining were coincident with 4,6-diamidino-2-phenylindole (DAPI) stain. Depending on its intensity, this pattern of staining can be indicative of a fusion protein that is associated with either interphase chromosomes, or more specifically with the heterochromatin that is found in large pericentric blocks in mouse cells and appears as bright DAPI foci (15,16). An example of the latter staining pattern is shown for ES113, a novel Krüppel-associated box (KRAB)-containing protein in Figure 2. KRAB boxes are involved in transcriptional repression through their interaction with KAP-1, and some KRAB-containing proteins have been shown to co-localize with heterochromatin (17).
Four percent of clones showed a concentration of staining at the nuclear periphery, which was confirmed by co-staining with an antibody that recognizes B-type lamins (e.g. F9/17A4, a trap of the nuclear pore complex protein NUP153 in Figure 2).
Trapped proteins located in other nuclear bodies, foci or fine speckles were also identified. Some of these (e.g. the novel proteins trapped in ES32 and F9/19B6) concentrate in patterns different to those described for other known nuclear bodies, e.g. Cajal bodies, and so may represent novel sub-nuclear domains. The foci containing traps of Bard1 (F9/16A3 in Figure 2) and Nbs1 (F9/29A6), both of which contain BRCT domains (Table 1 section IV), might correspond to BRCA1 foci (18).
Some proteins were found not in the nucleus per se but associated with the chromosome or cell division apparatus, e.g. centromeres in F9/20D4 cells and the mitotic spindle in F9/11D4 cells (traps of CenP-E and clathrin heavy chain, respectively) (Fig. 4).
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Eight of the fusion proteins, for which the subcellular localization of the endogenous protein was known, have completely mislocalized. These show a nuclear diffuse staining pattern and are listed in section VIII at the end of Table 1. Some are known components of nuclear sub-compartments, e.g. fibrillarin trapped in ES403 should show nucleolar staining, and can therefore provide information on motifs required for correct targeting to nuclear compartments. The mislocalized proteins are usually missing a large proportion of the endogenous protein after the gene trap and probably enter the nucleus by unmasking a cryptic NLS in the remainder of the protein. These are very likely to be knockouts of gene function, and some of these genes are known to be mutated in human syndromes, e.g. the thiamine transporter in thiamine-responsive megaloblastic anaemia (TRMA) with diabetes and deafness syndrome and the 70 kDa peroxisomal membrane protein in Zellweger syndrome (ES100 and F9/4D12 in section VIII of Table 1). Undoubtedly, some of the novel proteins identified in this screen will also be mislocalized relative to the wild-type protein but based on the proportion of the known proteins mislocalized this is likely to be a minority.
Dynamic changes in protein localization when transcription is inhibited
In vivo, many proteins move rapidly about the nucleus, and their apparent concentration into nuclear compartments reflects their steady-state association/disassociation with other nuclear proteins/complexes (19). Perturbing this equilibrium changes the apparent localization of such proteins. Hence, many proteins appear to alter their sub-cellular distribution in response to changes in transcription, during the cell cycle or accompanying differentiation. Analysing fusion protein dynamics can point toward the biological pathways in which the endogenous proteins function.
Many proteins involved in pre-mRNA processing concentrate in 20 to 40 nuclear foci or ‘speckles’ that correspond with ICGs and perichromatin fibrils detected by electron microscopy (3). Splicing is interrupted when transcription is blocked, and some splicing proteins [such as the serine/arginine-rich (SR) protein SC35 and ‘Sm’ snRNPs] then appear to concentrate into fewer, but larger, foci within the nucleus (9,20). We found that the trapped proteins from lines F9/2B4, WF9/5A7, F9/19C3 and F9/29D5 show the same behaviour as ‘Sm’ snRNPs in actinomycin D (ActD) treated cells (Fig. 3 and section II of Table 1). The trapped protein in F9/2B4 is the 100 kDa U5 snRNP (21) and the U5 snRNA has a similar behaviour (20). WF9/5A7 (Fig. 2) is the homologue of hCC1, a protein known to co-localize with human splicing factors (22). F9/29D5, a trap of plenty-of-prolines-101, was also identified amongst human proteins purified from ICGs (9).
Examination of gene trap proteins that are coincident with splicing components reveals additional complexity and heterogeneity in the morphology of the splicing compartment. The protein trapped in F9/21B2 cells is the homologue of human TRAP150 (Table 1 section II) and often appeared in long streaks in nuclei (Fig. 3, arrows), but moved into Sm snRNP-containing foci after treatment with ActD. Foci in F9/12C2 cells, although coincident with Sm antigens, are more rounded in shape and punctate. These foci enlarge and become fewer in number upon ActD treatment but they only partially overlap, and/or are adjacent to, Sm foci detected by Y12 staining (Fig. 3, arrows). The protein trapped in F9/12C2 is novel and contains an RNA recognition motif (RRM) (Table 1, section II). Similarly, the foci in F9/19A3, a trap of Rheb—a RAS-like GTPase, only partially overlap Sm foci in ActD treated cells (Fig. 3, arrows). The protein of unknown function trapped in ESKN60 cells also changed from a speckled pattern to engorged foci in response to ActD (Table 1, section IV).
Some nuclear proteins, including hnRNP A1 and a subset of SR proteins, continually shuttle in and out of the nucleus because they accompany the nuclear export of processed mRNAs. When transcription ceases these proteins accumulate in the cytoplasm (23,24). Other proteins such as U2AF, the U1 snRNP and p80 coilin form ring-like structures around the nucleolus when transcription is inhibited (20,25). We detected nucleo-cytoplasmic shuttling of Zap, a protein of unknown function trapped in F9/11C4 (Table 1, section IV), but a fraction of ZAP fusion protein also remains around the nucleolar remnants of ActD treated cells (Fig. 3). Fusion protein movements in response to ActD were also detected in other lines. In F9/21D6, a novel nucleolar protein (Table 1, section I) shows perinucleolar staining in ActD treated cells, the speckles seen in F9/19B6 cells disperse in treated cells, and the foci in ES32 cells form ring-like structures in the presence of ActD (Table 1, section IV).
Changes in fusion protein localization related to the cell-cycle or to differentiation
Many nuclear complexes disperse in mitotic cells, but some stay associated with segregating chromosomes. In ES38, the nucleolar protein Ubf had been trapped toward the C-terminus (Table 1, section I). Like the wild-type protein (26), the trapped protein remained associated with chromosomes throughout mitosis, concentrated in paired spots just below the centromeres of a few mouse chromosomes (most likely those carrying active arrays of rRNA genes). Other nucleolar proteins involved in pre-rRNA processing, including fibrillarin, are partially dispersed into the mitotic cytoplasm but with some protein coating the segregating chromosomes. This behaviour was seen for the novel nucleolar proteins that have been trapped in ES74 (Fig. 4), ES448 and F9/21D6 lines (Table 1, section I).
In ES446 cells we have trapped into the mouse gene encoding the high-mobility group protein Hmgi-c. The presence of AT-hook motifs in the protein sequence suggests that this may be a DNA binding protein (Table 1, section III). In interphase nuclei, the protein co-localizes with the DAPI stain suggesting that the protein distributes over both euchromatin and heterochromatin. However, during mitosis we have found that the protein concentrates at the pericentric heterochromatin of metaphase and anaphase chromosomes (Fig. 4).
Some proteins that are not nuclear associate with chromosomes or the chromosome segregation apparatus during mitosis and we also examined these (Table 1, section IV). In F9/20D4 cells the trapped protein CenP-E retains almost its entire C-terminal kinetochore binding domain (27) and recapitulates the dynamics of the normal protein (Fig. 4). We trapped the gene encoding clathrin heavy chain twice and the resulting fusion proteins are interrupted within the distal leg, or after the proximal leg, of the protein in F9/11D4 and F9/25A4 cells, respectively. Neither of these fusion proteins should be able to participate in clathrin trimerization to form triskeleton hubs and the F9/11D4 protein should not bind to clathrin light chains (28). Nevertheless in both cases, the fusion proteins were perinuclear (probably in Golgi-derived coated pits around the centrosome) and at the plasma membrane in interphase cells. We found that both the trapped clathrin fusion proteins, and wild-type clathrin (data not shown), co-localize with tubulin along the length of the mitotic spindle (Fig. 4). Tubulin is a component of coated pits and clathrin co-purifies with microtubules (29). It remains to be determined whether clathrin is present on the mitotic spindle through a passive interaction with tubulin, or whether it plays an active role in spindle function.
We identified proteins whose expression changes accompanying differentiation. In F9/12C4 we trapped the gene encoding Mis5 (a homologue of the human MCM6 replication licensing factor). Nuclear localization of this member of the minichromosome maintenance (MCM) family is dependent on complex formation with other MCMs that have NLSs and the trapped protein retains the zinc finger and ATPase motifs of the wild-type protein (Table 1, section IV). The variable levels of Mis5 protein in nuclear foci reflect cell cycle changes in the level of MCM proteins (30). Treating F9/12C4 cells with retinoic acid (RA) decreased the proportion of cells with abundant staining, consistent with an increase in cells that have exited the cell cycle in response to the induction of differentiation (data not shown). Similarly, the withdrawal of leukaemia inhibitory factor (LIF) and the addition of RA down-regulates the expression of the novel nuclear zinc finger protein trapped in ES205 cells (Table 1, section VII). mRNA levels from this gene are known to be reduced in F9 cells in response to RA-induced differentiation (31). In ES32 cells the novel trapped protein is present in both the cytoplasm and nucleus of undifferentiated cells but becomes concentrated into the nucleus in differentiated cells (Table 1, section IV).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Novel mammalian proteins in the nucleolus and splicing speckles
If the most frequent patterns of discrete sub-nuclear localization in our screen are from compartments that contain the greatest number of different protein components then, apart from the chromosomes themselves, the nuclear compartments involved in RNA processing (the nucleolus and the splicing speckles) are also complex (Table 1).
The nucleolus is much more than just a site of ribosome biogenesis. It is involved in the biogenesis of small RNAs transcribed by RNA polymerase III, and it may be where some factors involved in cell cycle control and checkpoints locate (32). Nucleolar proteins already known to be mutated in human disease include TCOF in Treacher Collins (4) and ataxin 7 in a form of spinocerebellar ataxia (33). Several proteins located in the nucleolus are involved in cancers (32,34). Here we have identified eight novel nucleolar components (Table 1 section I) and the motifs that they contain suggest roles in RNA processing (see below).
We identified six proteins that are novel to the nuclear compartments occupied by concentrations of splicing factors (Table 1, section II). Components of splicing complexes, assembled in vitro from nuclear extracts of human cells, have been characterized by nanospray mass spectrometry and MALDI (8). In this way 21 known and 19 novel spliceosome-associated proteins were identified. However, only one of these (the U5 snRNP 100 kDa protein) corresponds with a protein co-localizing with splicing factors in our screen. The analysis performed by Neubauer et al. (8) was designed to isolate core-spliceosomal proteins that interact stringently with a pre-mRNA substrate. Therefore it may not have enriched for proteins with a more structural or regulatory role in splicing, or those that are loosely associated with the spliceosome. This may be the type of protein we detect here. Similarly, human proteins present in purified ICGs have been determined by HPLC and ion-trap electrospray mass spectrometry (9). Only one of those reported proteins corresponds with one that we have identified here concentrated in splicing speckles (a novel proline-rich protein in line F9/29D5). Only five of the proteins reported by Mintz et al. (9) are in common with those identified by Neubauer et al. (8). Although the sequences of more than 80 mammalian proteins that concentrate in the nucleus with splicing factors have now been reported in the literature, it is clear that we are far from understanding the full complexity of gene products that locate in this nuclear compartment. The multitude of proteins that appear to participate in, or regulate, splicing may reflect the importance of alternative splicing in generating protein complexity from mammalian genomes with surprisingly few genes.
Proteins associated with chromosomes in interphase and mitotic cells
We identified 10 mammalian proteins that visually co-localize with chromosomes in the nucleus and seven of these remain associated with mitotic chromosomes (Table 1, section III). Many of these proteins appear to be concentrated at the sites of constitutive heterochromatin. At least two of these proteins have a proven link with human genetic disease.
The gene trapped in F9/18D6 cells is the mouse homologue of the gene mutated in ATRX syndrome. Motifs present in ATRX suggest that the protein may be a chromatin-remodelling factor, and so finding the protein concentrated in heterochromatin was surprising. However, the wild-type protein has the same heterochromatic localization in human and mouse cells (16), and ATRX individuals have altered methylation patterns at the sequences that underlie heterochromatin (35). The site of gene trap integration within the ATRX gene, and the co-localization of trapped and wild-type proteins (16), suggests that either the plant homeodomain (PHD) or the coiled-coil region is targeting the protein to heterochromatin.
Another chromatin remodelling factor, ACF, has been trapped in CT146 cells and, like ATRX, is also concentrated at heterochromatin (36). The site of gene trap integration suggests that the WAC domain (36) is responsible for heterochromatin targeting. The WAC domain is named for its presence in William’s syndrome transcription factor (WSTF), ATP-utilizing chromatin assembly and remodelling factor (ACF) and chromatin binding protein 146 (cbp146). A gene within the common deletion region for Williams–Beuren syndrome (36) encodes a very closely related human protein with a WAC motif at a similar position and we predict that this may also be protein concentrated at heterochromatin.
In ES446 cells we trapped the gene encoding Hmgi-c. This protein has been implicated in adipocyte homeostasis since a deficiency of fat tissue is found in mice with targeted disruption of Hmgi-c and this is reponsible for the pygmy phenotype (37). In humans, translocation-induced fusions of HMGI-C are associated with lipomas (38). Fusions to HMGI-C are also found in a variety of other mesenchymal tumours and in chronic lymphocytic leukemia, and HMGI-C is one of the most commonly rearranged genes in human neoplasms (38). The role of HMGI-C in tumorigenesis is unclear. Hmgi-c expression correlates throughout fetal development with high proliferative activity (39). Here we show that the protein localizes to the centric heterochromatin in mitosis (Fig. 4), suggesting that it might have a role in chromosome segregation. Interestingly, the site of gene trap integration in ES446 is into intron 3 of Hmgi-c, after the three AT hooks but before the C-terminal acidic domain (40), equivalent to the translocation breakpoints in human cancers (38).
In CT45 cells we have trapped the mouse homologue of a recently described human gene CECR2, located in the segment of proximal HSA22q that is present in multiple copies in Cat eye syndrome (41). Our trap suggests that this bromodomain-containing protein locates with chromatin in both interphase and metaphase cells.
Motifs shared between proteins in sub-nuclear domains
Although many of the proteins we found are novel and of unknown function, they contain domains or motifs that point to potential biochemical activities or functions (Table 1). By analysing the distribution of motifs/domains amongst the proteins identified here, and amongst proteins of known sub-nuclear location in databases, it became clear that mammalian proteins located in the same nuclear compartment often contain common signatures. Conversely, some generally common protein motifs are rare or absent from the proteins concentrated at some sub-nuclear sites.
Two of the novel nucleolar proteins we trapped contain motifs characteristic of the DEAD/H box family of proteins (Table 1, section I). Many DEAD box proteins are RNA helicases but some may function as RNPases (42). We analysed more than 60 known mammalian nucleolar proteins from the literature and found that the DEAD box helicase was also the most common motif recurring amongst them (Table 2), even though it is only the 47th most common motif amongst human proteins (http://www.ensembl.org/IPtop500.html) and it is rare in the proteins that we trapped in other nuclear compartments (Table 1).
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Five of the proteins that we found concentrated in splicing speckles contain an arginine serine-rich (RS) domain (Table 1). This domain is common amongst proteins known to play a role in the regulation of pre-mRNA splicing (43) and is also abundant in the proteins purified in ICGs (9). However, few of the human proteins reassembled in vitro into spliceosomal complexes (8) appear to contain this domain. The other abundant motif present in all screens to identify splicing factors is the RRM (Table 2), which is also relatively abundant in the human proteome (8,9,43,44).
Three of the trapped proteins that visually co-localize with interphase or mitotic chromosomes contain a PHD finger (Table 1, section III). This C4HC3 zinc finger-like motif has been implicated in protein–protein interaction, and it may be specialized for a role in the chromatin-mediated control of gene expression (45). PHD fingers are not abundant in the human proteome (44) (http://www.ensembl.org/Iptop500.html). Apart from the chromodomain, the PHD finger was the most common recurring motif amongst chromosome-associated proteins in the literature (Table 2), but the only chromodomain protein that we identified was the diffusely located SRG3 component of a SWI/SNF chromatin-remodelling complex (Table 1, section VII). Two of the PHD-containing chromosomal proteins that we found also have more C-terminal bromodomains and the chromosomal protein in CT45 cells also contains a bromodomain (Table 1 Section III). Some bromodomains have been shown to bind to acetylated lysine residues in the N-terminal tails of histone H3 and H4 (46) and the bromodomain is not abundant in the human proteome (Table 2) (http://www.ensembl.org/Iptop500.html). The combination of a PHD finger with a bromodomain appears to be a good indicator of likely chromosomal association for novel proteins in human or mouse cells.
The most common motif we identified amongst the proteins diffusely distributed in the nucleus (Table 1, section VII) was the C2H2 Krüppel-type zinc finger (Table 2). We found only one protein each in the nucleolus, on chromosomes and in unidentified foci that contain this motif. Since this class of zinc finger is the second most abundant motif amongst human proteins (44), the fact that it is not commonly found within proteins located at other discrete sites within the nucleus is highly significant. Given the putative DNA binding ability of this class of zinc fingers it is especially surprising that it is not common amongst proteins with a chromosomal localization. It may be a signature of proteins that can bind DNA, but that do not stably associate with chromatin. One-third of proteins with a C2H2 zinc finger are thought to have an N-terminal KRAB. This vertebrate-specific motif is thought to be involved in mediating transcriptional repression (17). Four of the nuclear-diffuse proteins that we trapped (ES205, F9/30A6, ES510 and ES9) have KRAB boxes and all contain more C-terminal C2H2 zinc fingers (Table 1, section VII). However, two of the trapped proteins that co-localize with interphase chromosomes and heterochromatin, but that do not remain associated with chromosomes at mitosis (ES113, ES261), also have KRAB boxes (Table 1, section III). Complete cDNA sequences are not available in databases for these two genes, so it remains to be determined if they contain C2H2 zinc fingers beyond the KRAB boxes and the sites of gene trap integration. For both ES113 and ES261 (Table 1, section III) fusion protein associated with heterochromatin is seen only in a subset of cells, suggesting that interaction of fusion protein with components of heterochromatin is cell cycle or differentiation dependent. Our data suggest that sequences in addition to the KRAB box may be necessary for targeting to heterochromatin, and that localization to heterochromatin of mouse embryonic cells is only seen for some KRAB box-containing proteins, and only some of the time.
The groups of proteins that we identified in either the chromosomal, nucleolar, splicing speckle or diffuse sub-domains of the nucleus also differ in their physical parameters. The proteins associated with chromosomes were the largest (average molecular weight = 119 kDa), and surprisingly were not especially basic (average pI = 7.6). In contrast, proteins from the two compartments involved in RNA metabolism (nucleolus and slicing speckles), were smaller (73 and 92 kDa on average, respectively) and more basic (pI = 8.3 and 9.3, respectively). Proteins diffusely located across the nucleoplasm had a similar average pI to chromosomal proteins (7.3), but were considerably smaller (81 kDa). We found the same trends amongst proteins described in the literature as belonging to these compartments (data not shown). Different pI values for proteins located at different sub-cellular locations have been reported for the Drosophila proteome, and suggested to reflect different functional requirements of cytoplasmic, nuclear and membrane-associated proteins (47). Our analysis suggests that similar constraints also operate at different sites within the nucleus of mammalian cells.
Proteins targeted to the same sub-nuclear compartment often seem to share common characteristics and signatures. These combinations of motifs are more rare in other compartments. Therefore, it may become possible to predict new constituents of nuclear compartments from databases based solely on the combination of protein motifs that they contain and their physical parameters (48).
Sub-nuclear localization as a guide to protein function in vivo
For some known proteins, the sub-nuclear localization that we determined was not expected, and demands a rethink about the putative functions of such proteins. For example, human homologues of two of our proteins co-localizing with splicing factors have been reported to interact with thyroid hormone receptor or its associated complexes and so were considered to have roles in transcriptional regulation. CT37 (TRIP12) was isolated in a yeast two-hybrid screen with thyroid hormone receptor (49), and the protein trapped in F9/21B2 cells has been purified biochemically as a component (TRAP150) of a thyroid hormone receptor associated complex (50). However, our localization (Fig. 3) (15) suggests that these proteins may interact with splicing factors in the nucleus, and this is supported by the presence of an RS domain, commonly found in splicing regulators, in TRAP150 (Table 1, section II).
Sites of gene trap integration
We have independently trapped a number of genes more than once, suggesting that certain regions of the genome are more susceptible to vector integration. The most common location trapped in both ES cells and F9 EC cells was in the same intron of a putative erythroid differentiation regulator gene on the X/Y pseudoautosomal region (F9/7A1 etc in Table 1, section VII). There may be a chromatin structure in this region of the sex chromosomes in embryonic cells that makes them susceptible to integration/recombination. Other loci trapped multiple times include a KRAB zinc finger protein (ES9 etc), karyopherin ß2 (WF/2C9 and ES140), a novel protein that localizes to splicing speckles (F9/19D5 and ESKN319), p52 transcriptional coactivator (F9/3G5 and ES149), clathrin heavy chain (F9/11D4 and F9/25A4) and a novel protein with similarity to the Drosophila muscleblind protein (F9/9D3 and ES644) (Table 1). Whilst in the latter three examples the vector integrated into different regions of the gene, in the others integration was into the same intron and irrespective of the cell line used. Three gene trap events occurred in equivalent gene regions to human cancer-associated translocation breakpoints, e.g. intron 7 or 8 of the Ewing’s sarcoma gene (ES361 in Table 1, section VII) (51), intron 5 of p52 transcriptional co-activator (F9/3G5 in Table 1, section III) (52) and intron 3 of Hmgi-c (ES446 in Table 1, section III) (38). The properties of these regions that make them susceptible to chromosomal breakage may be conserved between mouse and humans.
Functional genomics is trying to bridge the gap between the number of gene sequences in databases and the number of gene products that have been functionally characterized in any way. One systematic approach to ascribing possible function is to determine the sub-cellular location of proteins. Here we have shown that a visual gene trap screen is a powerful way to identify large numbers of proteins located in sub-compartments of the mammalian nucleus. It is complementary to other approaches, such as biochemical purification of complexes, and to other visual screens that rely on the over-expression of tagged cDNAs (10–14). A number of the known proteins that we trapped have a recognized or suspected role in human disease. These include Atrx (F9/18D6), Hmgi-c (ES446), Nibrin (F9/29A6), All-1 (F9/1D1), Pasg (F9/28A5), Wt1 (F9/23A3), Ewsh (ES361) and PRDM10 (ES34). In many cases, the site of gene trap integration suggests that the fusion protein is unlikely to be functional (Table 1). Gene trap cell lines bearing such mutations are available to the research community and may be useful for investigating molecular mechanisms, and the ES lines can be used to study the mutant phenotypes in whole animals.
It is clear from the large number of proteins that we and others have assigned to domains within the nucleus that we are far from understanding the full complexity of nuclear organization. However, emerging patterns of protein motifs married with sub-nuclear protein location promise that bioinformatics may have a prominent role to play in achieving this goal.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cell culture and transfection
The gene trap screen was performed with pGT1, 2 and 3 as previously described (15), but with the following modifications. The ES cell line E14 (53) was maintained in the presence of LIF and electroporated as described by Tate et al. (15). NeoR E14 colonies were selected with 250 µg/ml Geneticin/G418 (Gibco BRL) for 10 days. F9 embryonic carcinoma cells were selected with 400 µg/ml G418 (17). A subset of F9 clones, prefaced with F9HIS/ in Table 1, were derived using pGT1-3 constructs into which a 6x HIS tag was inserted between the splice acceptor and ßgeo.
All cells were exposed to 5 µg/ml ActD for 2 h to identify fusion proteins that alter their sub-cellular distribution in response to the cessation of transcription (15). To identify fusion proteins that change their level of expression, or their sub-cellular distribution during differentiation, all E14 lines were grown on gelatinized slides both in the presence and absence of LIF for 2–3 days prior to staining. Some cell lines were also stained after a 48 h incubation with 10–6 M RA in differentiating medium for ES cells and 10–7 M RA in normal medium for F9 cells (Fig. 1).
Cytological examination of fusion proteins
NeoR colonies were first screened for ßgal activity with X-Gal. Lines with nuclear X-Gal staining were then analysed by immunofluorescence with antibodies specific for ßgal (15). To co-localize fusion proteins with known components of nuclear compartments immunofluorescence using ßgal-specific antibody was combined with antibodies; D77 recognizing fibrillarin (54), Y12 recognizing Sm antigens in splicing speckles (15) and Lamin B (Santa Cruz) at the nuclear periphery. Slides were counterstained with 0.5 µg/ml DAPI and examined on a Zeiss Axioplan fluorescence microscope fitted with a Chroma #83000 filter set. Images were captured with a Photometrics cooled charged coupled device (CCD) camera and Smartcapture software (Digital Scientific).
Determining the sequence of trapped genes
Total RNA was prepared using Bio/RNA-X-cellTM (Bio/Gene Limited) and 5' RACE products were generated as described previously (15), except that P456 (5'-CCGTGCATCTGCCAGTTTGAGGGGA) [lac3 in (55)] was used to prime cDNA synthesis instead of primer 78 of Tate et al. (15). After dialysis on 0.1 µm nitrocellulose disks (Millipore), 5' RACE products were sequenced directly (Fig. 1) using Big DyeTM terminator cycle sequencing and –40 lacZ primer (USB/Amersham). Databases were searched for sequence matches using the BLAST algorithm (http://www.ncbi.nlm.nih.gov/BLAST/). Domains, repeats or motifs, and other parameters/features of the trapped proteins, were examined using ProtParam (http://expasy.cbr.nrc.ca/cgi-bin/protparam), PSORTII (http://psort.nibb.ac.jp/), InterProScan, which is an integrated search of PROSITE, Pfam and PRINTS databases (http://www.ebi.ac.uk/interpro/scan.html) and the Simple Modular Architecture Research Tool (SMART) (http://smart.embl-heidelberg.de/).
A database of nuclear protein localization
To facilitate data storing and analysis an NPD was created in Microsoft Access 97 (to be described in detail elsewhere). The nuclear protein database stores details about the DNA and protein sequences of trapped genes and related genes, together with protein function and subnuclear localization. Gene Ontology (GO) Project annotations were used to describe function and biological process (56) (http://www.geneontology.org). A world wide web interface is being developed to provide public access to data from this screen. Details about gene and protein sequence, sub-nuclear protein location, and molecular and biological function are linked to supporting references, images and external databases.
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ACKNOWLEDGEMENTS |
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H.G.E.S. was supported by an EMBO fellowship and G.K.M. and L.V.F. are supported by a grant from the James S. McDonnell Foundation. G.D. is supported by a fellowship from MRC Canada. W.A.B. is a Centennial Fellow of the James S. McDonnell Foundation. We thank our colleagues at the MRC Human Genetics Unit for reading and criticism of this manuscript.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +44 131 332 2472; Fax: +44 131 343 2620; Email:w.bickmore@hgu.mrc.ac.uk
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