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Nuclear membrane protein LAP2ß mediates transcriptional repression alone and together with its binding partner GCL (germ-cell-less)

Einav Nili1, Gady S. Cojocaru1, Yael Kalma2, Doron Ginsberg2, Neal G. Copeland3, Debra J. Gilbert3, Nancy A. Jenkins3, Raanan Berger1, Sigal Shaklai1, Ninette Amariglio1, Frida Brok-Simoni1, Amos J. Simon1,* and Gideon Rechavi1

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



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  		Fig. 1. Yeast two-hybrid screening and the cloning of mGCL. (A) A diagrammatic representation of the LAP2ß bait used in the screen. (B) Summary of the results from the interaction studies performed in yeast of the indicated baits and preys. DBD, DNA binding domain; SR, specific region; TM, trans-membrane; TA, transactivation. (C) The mouse GCL (mGCL) predicted amino acid sequence and homology to Drosophila GCL. The BTB/POZ domain is boxed (amino acids 89-198). The LAP2ß interaction region is shaded grey (amino acids 138-524). The putative NLS sequences are in bold type (amino acids 48-53 and 82-87). The conserved putative tyrosine phosphorylation site is indicated by a bold asterisk (amino acid 405); the asterisk at amino acid 525 indicates the stop codon. The mGCL sequence has been submitted to the GenBank database under the accession number AF282322.

 


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  		Fig. 2. The full-length mGCL and LAP2ß bind in vitro. The mGCL and LAP2ß proteins were expressed as GST fusions in bacteria. Both proteins, together with LAP2{zeta}, were translated in vitro in the presence of [35S]-labelled methionine (10% of input, lanes 1, 4 and 7). Mixtures of [35S]-labelled LAP2ß and GST or GST-mGCL (lanes 2 and 3, respectively), [35S]-labelled LAP2{zeta} and GST or GST-mGCL (lanes 5 and 6, respectively) and [35S]-labelled mGCL and GST or GST-LAP2ß (lanes 8 and 9, respectively) were incubated with glutathione-Sepharose beads (Pharmacia Biotech). The bound proteins were eluted, separated on SDS-PAGE and identified by autoradiography and western blot analysis using anti-GST (lower panel, lanes 2, 5, 8), anti-mGCL (lower panel, lanes 3 and 6) or anti-LAP2ß (lane 9) antibodies.

 


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  		Fig. 3. Mgcl maps to the central region of mouse chromosome 6. Mgcl was placed on mouse chromosome 6 by interspecific backcross analysis. The segregation patterns of Mgcl and flanking genes in 88 backcross animals that were typed for all loci are shown at the top of the figure. For individual pairs of loci, more than 88 animals were typed (see text). Each column represents the chromosome identified in the backcross progeny that was inherited from the (C57BL/6JxM. spretus) F1 parent. The shaded boxes represent the presence of a C57BL/6J allele and white boxes represent the presence of a M. spretus allele. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. A partial chromosome 6 linkage map showing the location of Mgcl in relation to linked genes is shown at the bottom of the figure. Recombination distances between loci in centiMorgans are shown to the left of the chromosome and the positions of loci in human chromosomes, where known, are shown to the right. References for the human map positions of loci cited in this study can be obtained from GDB (Genome Data Base, http://gdbwww.gdb.org/), a computerized database of human linkage information maintained by The William H. Welch Medical Library of The Johns Hopkins University (Baltimore, MD).

 


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  		Fig. 4. Endogenous mGCL is expressed in pancreas and rat insulinoma cells and co-localizes with LAP2ß to the NE. (A) Whole-cell lysate of H1299 cells, transfected and untransfected with recombinant full-length mGCL (lanes1 and 2, respectively), whole-cell lysates of RIN cells (lanes 3 and 5), and mouse pancreatic tissue extracts (lanes 4 and 6) were separated on SDS-PAGE and analysed by western blot using either anti-mGCL (lanes 1-4) or anti-LAP2ß (lanes 5, 6) antibodies. (B) RIN cells were stained with anti-mGCL antibodies and analysed by confocal microscopy. Progressive serial sections of a nucleus are shown. (C) RIN cells were stained with both anti-mGCL and anti-LAP2ß antibodies and analysed by confocal microscopy.

 


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  		Fig. 5. LAP2ß and GCL have distinct biochemical fractionation properties. Cytosolic and nuclear fractions of RIN cells expressing endogenous GCL were prepared by lysis of transfected cells in hypotonic buffer. Nuclei were further extracted using either 8 M urea or a combination of Triton X-100 plus 250 mM or 500 mM NaCl, as indicated. The various extracts were separated by SDS-PAGE and analysed by western blot using anti-mGCL or anti-LAP2ß antibodies, as indicated. Cyt, cytosol; NP, nuclear pellet; NS, nuclear supernatant.

 


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  		Fig. 6. The mGCL and LAP2ß proteins regulate the transcriptional activity of the E2F5-DP3{alpha} heterodimer. The E2F reporter (0.5 µg) and pCMV-ß-gal (0.5 µg), together with expression vectors for E2F5 (150 ng), DP3{alpha} (300 ng), Rb (1 µg), mGCL (1 µg), LAP2ß (1 µg) and LAP2{zeta} (1 µg) were transfected into H1299 cells as indicated. The values shown represent the average of duplicate readings and represent the level of luciferase relative to the ß-galactosidase derived from the internal control. The values are presented as activation compared with the mock control.

 

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