1 Pediatric Hemato-Oncology Department, Division of Hematology, Chaim Sheba Medical Center, Tel-Hashomer and the Sackler School of Medicine, Tel-Aviv University, Israel
2 Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot, Israel
3 National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702, USA
*Author for correspondence (e-mail: amosimon{at}yahoo.com)
Accepted June 10, 2001
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SUMMARY |
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Key words: GCL, Germ-cell-less, LAP2, Nuclear envelope, Thymopoietin, Transcription regulation
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INTRODUCTION |
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LAP2ß belongs to the LAP2 (thymopoietin) family of nuclear proteins (Berger et al., 1996; Furukawa et al., 1995; Harris et al., 1994). These proteins are ubiquitously expressed and are highly conserved in mammals (Zevin-Sonkin et al., 1992; Harris et al., 1994; Berger et al., 1995; Theodor et al., 1997; Ishijima et al., 1996). To date, six mouse and human alternatively spliced LAP2 isoforms, designated , ß,
,
,
and
were isolated and characterized (Harris et al., 1994; Berger et al., 1996). All of them share an identical N-terminal 186 amino acid domain. The ß,
,
and
isoforms also share an identical C terminus that contains a transmembrane domain that enables their insertion into the inner NE and to the perinuclear space. LAP2ß has been isolated and characterized as an inner nuclear membrane protein that binds to lamin B and chromosomes in a phosphorylation dependent manner (Foisner and Gerace, 1993). Thus, it was assigned roles in linking chromatin to the NE during interphase and in NE breakdown and reassembly during mitosis (Foisner and Gerace, 1993; Furukawa et al., 1998; Furukawa et al., 1995). The microinjection of the recombinant lamin binding region of LAP2ß into mammalian cells inhibited nuclear volume increase and progression into S phase (Yang et al., 1997). In Xenopus laevis the addition of human recombinant LAP2ß truncation mutants to cell-free nuclear assembly reactions, severely affected nuclear envelope and lamin assembly (Gant et al., 1999). Both studies clearly indicate a role for LAP2ß in lamin dynamics, which are crucial to NE reassembly and nuclear growth. The latter is essential for cell cycle progression from G1 into S phase, possibly linking LAP2ß to cell cycle control.
Recently, a protein named BAF (barrier to autointegration factor) was identified as a chromatin binding LAP2ß-interacting partner (Furukawa, 1999). This DNA binding protein was initially discovered in retrovirus-infected cells, where it protected viral DNA from self-integration (Lee and Craigie, 1998). LAP2ß is suggested to bind BAF through its LEM box, thus predicting that all other LAP2 isoforms, including those in X. laevis (Gant and Wilson, 1997; Lang et al., 1999), as well as the LEM domain proteins emerin and MAN1, bind BAF (Wilson, 2000). Interestingly, a recombinant polypeptide that contains the chromatin-binding region of LAP2ß enhanced the efficiency of DNA replication in Xenopus extracts (Gant et al., 1999). This suggests that LAP2ß affects chromatin structure and organization, possibly through its binding to BAF or other, yet unidentified, chromatin proteins.
In four independent studies, including the one presented here, the mouse homologue of GCL was isolated and characterized (de La Luna et al., 1999; Kimura et al., 1999; Leatherman et al., 2000). In Drosophila, the maternal GCL protein is required for germ line specification. Drosophila females with reduced GCL function give rise to sterile adult progeny that lack germ cells but are otherwise normal. It was suggested that the Drosophila GCL protein is localized to the NE of those nuclei that later become the nuclei of the germ cell precursors (Jongens et al., 1994). All studies of mouse GCL demonstrated the high conservation, in structure, localization and function, between the mouse and Drosophila GCL proteins. This is best exemplified by the ability of mouse GCL to rescue the Drosophila GCL-null phenotype (Leatherman et al., 2000). However, because the primordial germ cell (PGC) specification process in mouse does not depend on the presence of a maternally inherited germ plasm, as it is in Drosophila, it seems that, in mammalian testis, GCL functions mainly in the process of spermatogenesis.
Both Drosophila and mouse GCL belong to the BTB/POZ domain containing proteins. This evolutionarily conserved protein-protein interaction domain is generally found at the N terminus of either actin binding or, more commonly, nuclear DNA binding proteins (Albagli et al., 1995). It is suggested that, by mediating protein binding in large aggregates, the BTB/POZ domain serves to organize higher order macromolecular complexes involved in nuclear events such as chromatin folding (Albagli et al., 1995). In Drosophila melanogaster this domain is found in transcription factors that are required for developmental processes such as pole cell formation in the embryo (Albagli et al., 1995). Nearly all mammalian proteins that contain the BTB/POZ domains are zinc finger proteins that are involved in transcriptional regulation. The two best known examples of these are BCL6 and PLZF. These two proteins were shown to act as transcriptional repressors and their aberrant expression is connected to the development of hematopoietic malignancies (Deweindt et al., 1995; Dong et al., 1996).
The mGCL protein was also isolated by de La Luna and colleagues (de La Luna et al., 1999) as a DP-interacting protein (DIP). In human osteosarcoma U20S cells, mGCL/DIP was suggested to cause the translocation of the E2F-DP heterodimer to the NE, the reduction of its transcriptional activity and the accumulation of cells in the G1 phase of the cell cycle. In this study, we describe the isolation of mGCL as a LAP2ß-binding protein, the co-localization of both proteins to the NE and their repression of the E2F-DP transcriptional activity. The possible mechanisms of this transcription regulation are discussed.
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MATERIALS AND METHODS |
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In order to clone the full-length cDNA of mGCL, adult mouse testis and mouse thymus cDNA libraries were screened using various cDNA segments of the 7.3 clone. Several positive clones were isolated. The sequence of the isolated clones was determined in both strands by automated sequencing. Two of the isolated clones, designated 6.1 and 1.8, containing the full-length mGCL and cloned in the pBluescript vector, were further used for subcloning. Database searches and sequence comparisons were done using the BLAST program (Altschul et al., 1990) provided by the National Center for Biotechnology Information.
Plasmids
Yeast two-hybrid system
PAS2 has been previously described (Bai and Elledge, 1997). pAS2-LAP2ß-SR was constructed by inserting a NdeI-BamHI digested PCR product corresponding to amino acids 219-328 of LAP2ß in frame with the Gal4 DNA binding domain.
Bacterial expression vectors
GST-mGCL and GST-LAP2ß were constructed by inserting EcoRI (mGCL) and SalI (LAP2ß) digested cDNA fragments corresponding to the full length proteins into the pGEX vector (Pharmacia Biotech).
Mammalian expression vectors
The following plasmids have been described previously: pcDNA3-HA-E2F5 (Lindeman et al., 1997), pCMV-Rb (Qin et al., 1992), E2F-Luciferase (Krek et al., 1993) and pcDNA3.1-HA (gift of M. Walker, Weizmann Institute, Israel). pcDNA-HA-LAP2ß and pcDNA-HA-LAP2 were constructed by inserting full-length mouse LAP2ß and LAP2
in frame into pcDNA3.1-HA. pcDNA3-mGCL was constructed by inserting a BamHI-EcoRV fragment containing the full length mGCL derived from the 6.1 pBluescript vector into pcDNA3 (invitrogen).
pcDNA3.HA-DP3 used in the luciferase assay was cloned by first performing PCR on cDNA derived from mouse kidneys using primer oligonucleotides derived from the pl-2 murine DP-3 cDNA sequence (Ormondroyd et al., 1995). Four different PCR products coding the various spliced products of DP3 were cloned into the pCR2.1 TA cloning vector (Invitrogen) and sequenced. The clone coding the DP3
sequence (Ormondroyd et al., 1995) was then subcloned in frame into the pcDNA3.1HA vector.
Tissue culture
Human lung carcinoma H1299 cells were grown in RPMI supplemented with 10% foetal calf serum (FCS), glutamine, penicillin and streptomycin. CHO cells were grown in DMEM supplemented with 10% FCS, glutamine, penicillin and streptomycin. RIN (rat insulinoma) cells were grown in M199 supplemented with 10% FCS, glutamine, penicillin and streptomycin.
Transfections
Transfections were performed using the calcium-phosphate method. Cells were plated 24 hours before transfection at 105 cells per well in a six-well plate (transcription assays) or 106 cells per 10 cm plate (protein expression). Glycerol shock, washes and refeeding were performed after 18 hours in the presence of the precipitate. The cells were harvested 40 hours after transfection. DNA amounts were kept constant by adding pcDNA3 when required. In the transcription assays pcDNA-ß-galactosidase was used as an internal control for the transfection efficiency. Luciferase and ß-galactosidase activities were measured in duplicate plates for each point.
Protein expression and immunoblots
RIN cells were harvested in PBS, washed twice, pelleted and resuspended in 1x SDS. Pancreatic tissue was derived from an adult male mouse. The tissue was washed with ice-cold PBS, minced in ice-cold lysis buffer (20 mM Tris pH 7.8, 1% NP-40, 150 mM NaCl, 50 mM NaF, 0.2%SDS, 2 mM EDTA, 10% glycerol, 0.5 mM DTT, protease inhibitor cocktail (complete; Roche)) and pelleted at 4°C for 15 minutes. Protein concentration of the supernatants was determined (BCA, PIERCE) and equal amounts of proteins were subjected to SDS-PAGE.
For the solubility assay, 107 RIN cells were harvested in ice-cold PBS, washed, pelleted and resuspended in 400 µl hypotonic buffer (10 mM HEPES pH 7.9, 10 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF and protease inhibitor cocktail (complete; Roche)) for 15 minutes on ice. After that, 25 µl of 10% NP-40 were added and the cells were strongly vortexed for 10 seconds and pelleted at 16,000 g for 30 seconds. The supernatant designated cyt. was resuspended in 5x SDS sample buffer. The pelleted nuclei were washed, resuspended in an ice-cold lysis buffer (20 mM HEPES pH 7.9, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF and protease inhibitor cocktail (complete; Roche)) and 8 M urea, or 250 mM NaCl and 1% Triton X-100, or 500 mM NaCl and 1% Triton X-100, vortexed vigorously for 15 minutes at 4°C and pelleted. Supernatants (designated NS) were resuspended in 5x SDS sample buffer. Pellets (designated NP) were resuspended in 1x SDS sample buffer.
Proteins were separated on 12% SDS-PAGE, transferred to nitrocellulose (Schleicher & Schuell) and detected using the Western Blot Chemiluminescence Reagent Plus (NEN). The following primary antibodies were used: rabbit anti-mGCL polyclonal antibodies raised against a 12 amino acid peptide derived from the C terminus of mGCL (dilution 1:500) and anti-LAP2ß monoclonal antibodies (clone 6G11, dilution 1:10000, generous gift of G. Goldstein, NJ, USA).
Immunostaining
Cells that were grown on coverslips were treated as follows: fixation in ice-cold methanol for 5 minutes and then ice-cold acetone for another 5 minutes. After the fixation, the cells were washed with TBS (100mM Tris-HCl pH 7.5, 150 mM NaCl). Blocking was done using 5% skim milk in TBS containing 0.1% Tween 20 (TBS-T) for 15 minutes. Incubations with primary and secondary antibodies were in the blocking solution for 30 minutes each. Between and after the incubation with the antibodies were washed using TBS-T. The coverslips were mounted in immunofluore (ICN) and cells photographed with a confocal microscope.
The primary antibodies were used as follows: anti-LAP2ß was used in a 1:100 dilution and anti-mGCL was used in a 1:50 dilution. Cy2- and Cy3-conjugated goat anti-mouse, donkey anti-rabbit and goat anti-rat antibodies (Jackson Laboratories) were used as secondary antibodies in a 1:100 dilution.
In vitro protein interaction
Full length LAP2ß and mGCL were expressed as Glutathione-S-transferase (GST) fusion proteins in the pGEX bacterial expression system. Bacterial DH5 cells expressing GST or both GST fusion proteins were grown overnight at 37°C and diluted 1:100. The cells were then grown to O.D 0.6 at 30°C (LAP2ß and mGCL) or 37°C (GST). For GST expression, 0.1 mM IPTG was added for an additional 3 hours. Cells were harvested, sonicated and the recombinant proteins were extracted from the bacteria at 4°C using 1% Triton X-100 and 50 mM EDTA in the presence of protease inhibitor cocktail (complete; Roche). The final volume of cell lysate that contained the recombinant proteins was 1/25 of the starting culture. The expressed proteins were detected by western blot analysis using either specific anti LAP2ß and anti-mGCL antibodies or monoclonal antibodies against GST (clone B-11, Santa Cruz). The [35S]-labelled LAP2ß, LAP2
and mGCL proteins were synthesized in vitro using the TNT T7 quick system (Promega) in the presence of [35S]-labelled methionine.
For interaction with the in vitro translated products, GST or GST fusion proteins were diluted in 1 ml PBS and incubated by shaking for 5 hours at room temperature with 50 µl of reduced glutathione-Sepharose beads (Pharmacia). Protein-bound beads were washed three times with 1 ml PBS three times, diluted in 0.5 ml PBS and incubated with 10 µl of the in vitro translated products for 1 hour at room temperature. The beads were then washed five times with 1 ml PBS and pelleted. Bound proteins were eluted by boiling the beads in 50 µl of 2x SDS sample buffer, separated on SDS-PAGE and detected by autoradiography ([35S]-labelled proteins) or western blot analysis (GST or GST fusion proteins).
Interspecific mouse backcross mapping
Interspecific backcross progeny were generated by mating (C57BL/6JxMus spretus) F1 females and C57BL/6J males as described (Copeland and Jenkins, 1991). A total of 205 N2 mice were used to map the Mgcl locus (see text for details). DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, Southern blot transfer and hybridization were performed essentially as described (Jenkins et al., 1982). All blots were prepared with Hybond-N+ nylon membrane (Amersham). The probe, an 2.0 kb EcoRI fragment of mouse cDNA was labelled with [
32P] dCTP using a nick translation labelling kit (Boehringer Mannheim); washing was done to a final stringency of 1.0x SSCP, 0.1% SDS, 65°C. Fragments of 8.3 kb, 7.0 kb, 5.9 kb, 3.7 kb, 3.2 kb, 2.6 kb and 1.3 kb were detected in ScaI digested C57BL/6J DNA, and fragments of 10.5 kb, 7.0 kb, 5.4 kb, 3.7 kb, 2.8 kb and 1.3 kb were detected in ScaI digested M. spretus DNA. The presence or absence of the 10.5 kb, 5.4 kb and 2.8 kb ScaI M.-spretus-specific fragments, which co-segregated, was followed in backcross mice. A description of the probes and restriction-fragment-length polymorphisms (RFLPs) for the loci linked to Mgcl, including Catna2, Mad and Gpr73, has been reported previously (Uchida et al., 1994; Parker et al., 2000). Recombination distances were calculated using Map Manager version 2.6.5. Gene order was determined by minimizing the number of recombination events required to explain the allele distribution patterns.
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RESULTS |
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The main feature of the mGCL protein sequence is the existence of the evolutionarily conserved protein-protein interaction BTB/POZ domain in its N terminus (amino acids 89-198) (Fig. 1C). However, although nearly all BTB/POZ proteins isolated so far contain either a DNA or an actin binding domain in their C terminus, both the Drosophila and mouse GCL do not contain any known motif in their C terminus. Other features of mGCL include two putative nuclear localization signal (NLS) motifs at its N terminus (residues 48-52 and 82-87) (Kalderon et al., 1984) and a possible tyrosine phosphorylation site (amino acid 405) that are also conserved in the Drosophila GCL homologue (Fig. 1C). Hydrophobicity and motif analyses of mGCL do not reveal any hydrophobic region suggestive of a transmembrane domain as found in its binding partner LAP2ß (data not shown).
mGCL binds LAP2ß in vitro
Because the bait used in our two-hybrid assay consisted only of LAP2ß specific region and the prey contained only the C terminus of mGCL, we carried on to prove that the full length proteins physically interact with each other. In order to do so, we expressed the full-length mGCL and LAP2ß as GST fusion proteins in the pGEX bacterial expression system. Both fusion proteins were toxic, insoluble and expressed at very low levels in bacteria. Thus, the expressed GST-mGCL or GST-LAP2ß protein could be detected only with specific antibodies raised against mGCL (Fig. 2, western blot lanes 3 and 6), LAP2ß (lane 9) and GST (data not shown). We then performed GST pull down experiments using in vitro translated [35S]-labelled mGCL, LAP2ß and LAP2, which is an alternatively spliced product of LAP2ß, missing its lamin and mGCL binding region but containing its chromatin binding domain, thus serving as a natural deletion mutant of LAP2ß. As shown in Fig. 2, [35S]-labelled LAP2ß specifically bound GST-mGCL (lane 3) but did not bind GST alone. As expected, [35S]-labelled LAP2
did not bind either GST or GST-mGCL (lanes 5 and 6, respectively). In the opposite orientation, [35S]-labelled mGCL specifically bound GST-LAP2ß (lane 9) but did not bind GST alone (lane 8).
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These results support our hypothesis that, although GCL is specifically associated with LAP2ß at the NE, it is not an integral membrane protein but is rather a nuclear matrix protein, which might associate with complex nuclear structures containing lamins or chromatin, in addition to its previously reported binding to the E2F-DP transcription factor.
LAP2ß and mGCL can reduce the transcriptional activity of the E2F-DP complex
Recently, GCL was independently isolated as a specific DIP (de la Luna et al., 1999). It was shown that co-expression of mGCL with the E2F5-DP3a heterodimer caused the translocation of this transcription factor to the NE and a significant reduction in its transcriptional activity (there). We hypothesize that LAP2ß is the protein responsible for the anchoring of mGCL to the NE. We therefore examined whether LAP2ß also affected the transcriptional activity of the E2F-DP3 complex. This was performed by transfecting H1299 cells with various combinations of the E2F5, DP3, LAP2ß and mGCL proteins, and studying their effect on the activity of a luciferase reporter gene under the control of a minimal promoter containing three E2F binding sites in tandem. As can be seen in Fig. 6, both E2F5 and DP3
activated the promoter on their own (lanes 2 and 5), albeit at different potencies: E2F5 was usually two to three times more potent then DP3
. Co-expression of mGCL with either DP3
or E2F5 caused the reduction of DP3
s activity, as expected, but, interestingly, also reduced the activity of E2F5 (lanes 6 and 3, respectively). Similarly, co-expression of LAP2ß with either DP3
or E2F5 caused a reduction of their transcriptional activity (lanes 7 and 4, respectively). When E2F5 and DP3
were co-expressed, their activity as a complex was slightly more than additive (lane 8). Co-expression of the E2F5-DP3
heterodimer plus either mGCL (lane 9) or LAP2ß (lane 10), caused a reduction of 50-60% of the transcription activity, with LAP2ß reproducibly causing more repression than mGCL (lanes 9 and 10, respectively). By contrast, addition of LAP2
, which does not bind mGCL (Fig. 2), did not affect the transcriptional activity of the heterodimer (lane 11). Co-expression of the E2F5-DP3
heterodimer with both LAP2ß and mGCL resulted in the reduction of transcriptional activity to nearly background level (lane 12) and was equal to that of pRb (lane 13) transfected at the same DNA concentration. These results suggest that LAP2ß and mGCL might be potent inhibitors of gene expression for genes regulated by the E2F-DP complex and possibly other transcriptional regulators.
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DISCUSSION |
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Both mGCL and LAP2ß can affect transcription
mGCL is a BTB/POZ domain containing protein that was originally cloned as a DIP (de la Luna et al., 1999). DP3 belongs to the DP family of proteins, which are essential heterodimeric partners of the E2F proteins in creating the various functional E2F transcription factors (Ormondroyd et al., 1995; Lam and La Thangue, 1994). These factors have a major role in the co-ordination and integration of early cell-cycle progression and an aberrant regulation of E2F activity is found in most human tumour cells (Lam and La Thangue, 1994). mGCL was shown to interact with the E2F5-DP3 heterodimer, probably through the DP subunit, and to cause its localization to the region of the NE (de la Luna et al., 1999). Co-expression of mGCL with the E2F5-DP3 transcription factor caused a reduction in its transcriptional activity (de la Luna et al., 1999).
LAP2ß is an integral protein of the NE, which was shown to bind lamins and chromatin in a cell-cycle-dependent manner. Expression of LAP2ß deletion mutants revealed a role for this protein in the expansion of reforming nuclei and an effect on DNA replication efficiency in Xenopus extracts (Foisner and Gerace, 1993; Yang et al., 1997; Gant et al., 1999). This replication effect is hypothesized to be due to LAP2ß effect on chromatin structure. A direct role for LAP2ß in transcription regulation has, however, not yet been examined or demonstrated.
In our study, we show that, similar to its binding partner (mGCL), LAP2ß can also reduce the activity of the E25F-DP3 heterodimer. Although the nuclear lamina was shown to be involved in transcription regulation (Mancini et al, 1994; Imai et al, 1997; Rafiq et al, 1998), we demonstrate for the first time evidence for the direct involvement of an integral NE protein in transcriptional repression. We also show that the co-expression of both LAP2ß and mGCL causes a stronger transcriptional repression than that of either mGCL or LAP2ß alone, and that this repression equals the effect of the pRb protein, the best known inhibitor of the E2F complex. These results could reveal a novel spatial regulatory pathway for the E2F complex. In this pathway, under certain physiological circumstances, the complex is bound to the nuclear envelope through mGCL and LAP2ß and is thus rendered inactive. We propose two possible models for this repression of transcriptional activity.
In the first model, the spatial separation of the E2F-DP heterodimer from its target promoters, by its association to the NE, causes repression of transcriptional activity. This can then be viewed as part of an emerging pattern in which E2F-DP activity is controlled by regulating its intracellular location (de la Luna et al., 1996; Magae et al., 1996; Allen et al., 1997; Lindeman et al., 1997). The second model involves transcriptional repression through modulation of higher order chromatin structure. Several studies in mammals, Drosophila and yeast have demonstrated that the recruitment of euchromatin genes to heterochromatin regions at the nuclear periphery can cause position effect transcriptional repression of these genes (Andrulis et al., 1998; Henikoff et al., 1995; Brown et al., 1997). For example, the Ikaros transcriptional regulator, which usually activates lymphocyte specific expression, was found to associate with transcriptionally inactive genes in heterochromatin foci (Brown et al., 1997). To this effect LAP2ß and mGCL might act as heterochromatin recruiters. As such, they might cause the translocation of the E2F-DP heterodimer to the NE while still bound to chromatin through its target promoters. The chromatin is then modulated into a repressive form, thus inactivating transcription of E2F-DP regulated genes. A major group of proteins involved in transformation of chromatin into a repressive form is the histone deacetylases (HDACs) family (Cress and Seto, 2000). Because mGCL contains a BTB/POZ domain, it is interesting that this domain, found in several other proteins, was proposed to mediate transcriptional repression through recruitment of HDACs (Muto et al., 1997; Dhordain et al., 1997; David et al., 1998; Huynh and Bardwell, 1998). It would be interesting to check whether mGCL is indeed associated with HDACs. If demonstrated, this interaction could provide the actual mechanism for chromatin modulation discussed above.
Our results show that LAP2ß was able to reduce the transcriptional activity of the E2F5-DP3 heterodimer without the co-expression of mGCL (Fig. 6, lane 10). This effect is probably not exerted through the interaction of LAP2ß with endogenous mGCL because no expression of mGCL was revealed in these cells (data not shown). Moreover, LAP2ß reduced the activity of E2F5 without the co-expression of DP3 (Fig. 6, lane 4). Thus, we believe that LAP2ß might regulate the activity of the E2F-DP complex through interacting with other cellular E2F-associating proteins, or by a more general effect on chromatin structure. Such an effect is proposed to be connected to LAP2ßs recently reported association with BAF, a chromatin and DNA binding protein (Furukawa, 1999).
The mGCL protein was able to reduce the transcriptional activity of the E2F5-DP3 heterodimer without the co-expression of LAP2ß and also repressed E2F5 without co-expression of DP3 (Fig. 6, lanes 9 and 3, respectively). The mGCL protein was also shown to possess an intrinsic ability to inhibit transcription, provided that it was brought to DNA by a DNA binding protein such as the GAL4 DBD (de la Luna et al., 1999). This transcriptional regulation could be through the interaction of mGCL with endogenous LAP2ß, a ubiquitous protein found in nearly all cells (Zevin-Sonkin et al., 1992; Harris et al., 1994; Berger et al., 1995; Theodor et al., 1997; Ishijima et al., 1996). Alternatively, the activity of HDACs, mediated through the mGCL BTB/POZ domain, as discussed above, might be involved.
Finally, there is evidence that the regulatory pathway proposed here is not specific for the E2F-DP protein but might be relevant to other transcription factors. For example, a similar regulatory mechanism was found to involve the insulin transcription factor PDX/IPF1. In insulinoma cells, it was shown that the NE localization of PDX/IPF1 was connected with repression of its transcriptional activity (Rafiq et al., 1998). It is yet unknown what factors cause the translocation of PDX/IPF1 to the NE but, as we show in Fig. 4, GCL is highly expressed in pancreas and specifically in insulinoma cells. Thus, it would be interesting to check whether LAP2ß and GCL are also involved in the regulation of the transcriptional activity of PDX in these cells.
Possible involvement of LAP2ß and mGCL in pathological states
Recent studies from various groups strongly link the NE to human diseases. Loss or defects in the emerin protein cause the X-linked recessive form of Emery-Dreifuss muscular dystrophy (EDMD) (Bione et al., 1994). Mutations in LMNA gene, which encodes lamins A and C, cause three autosomal dominant diseases: a form of EDMD (Bonne et al., 1999), a form of dilated cardiomyopathy (CMDA1) (Fatkin et al., 2000) and Dunnigan-type familial partial lipodystrophy (FPLD) (Cao and Hegele, 2000; Shackleton et al., 2000). In this study, we have mapped the mGCL gene to the central region of chromosome 6 (Fig. 3), a region that is syntenic to human chromosome 2p13-14. Indeed, human GCL (HGCL), recently cloned and characterized by us, was mapped to this region (Nili et al., 2001). Linkage analysis studies mapped the gene responsible for the Alstrom syndrome, a rare autosomal recessive disorder (Alstrom et al., 1959) to the same region (Collin et al., 1997; Collin et al., 1999; Macari et al., 1998). Alstrom syndrome is characterized by retinitis pigmentosa, deafness, obesity, hyperlipidaemia and non-insulin-dependent diabetes mellitus (NIDDM). In some cases, acanthosis nigricans, cardiomyopathy, hepatic dysfunction, progressive chronic nephropathy and male hypogonadism are also observed. We have proposed that HGCL is a candidate gene that is responsible for the Alstrom syndrome (Nili et al., 2001). If further studies searching for GCL mutations in this syndrome do indeed verify this hypothesis, this will be yet another example of the connection of the NE and its related proteins to disease. Interestingly, some of the symptoms in this syndrome, such as the NIDDM and cardiomyopathy, overlap those of CMDA1 and FPLD, which are caused by LMNA mutations. This raises the possibility of a common defective mechanism in these diseases. Based on the ability of both mGCL and LAP2ß to regulate transcription, as presented in this study, this mechanism could be the regulation of gene expression, which is normally dependent on correct interactions and concerted interplay between transcription factors, chromatin and proteins of the NE.
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ACKNOWLEDGMENTS |
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