Abstract
GAGE proteins are highly similar, primate-specific molecules with unique primary structure and undefined cellular roles. They are restricted to cells of the germ line in adult healthy individuals, but are broadly expressed in a wide range of cancers. In a yeast two-hybrid screen we identified the metazoan transcriptional regulator, Germ cell-less (GCL), as an interaction partner of GAGE12I. GCL directly binds LEM-domain proteins (LAP2β, emerin, MAN1) at the nuclear envelope, and we found that GAGE proteins were recruited to the nuclear envelope inner membrane by GCL. Based on yeast two-hybrid analysis and pull-down experiments of GCL polypeptides, GCL residues 209–320 (which includes the BACK domain) were deduced sufficient for association with GAGE proteins. GAGE mRNAs and GCL mRNA were demonstrated in human testis and most types of cancers, and at the protein level GAGE members and GCL were co-expressed in cancer cell lines. Structural studies of GAGE proteins revealed no distinct secondary or tertiary structure, suggesting they are intrinsically disordered. Interestingly GAGE proteins formed stable complexes with dsDNA in vitro at physiological concentrations, and GAGE12I bound several different dsDNA fragments, suggesting sequence-nonspecific binding. Dual association of GAGE family members with GCL at the nuclear envelope inner membrane in cells, and with dsDNA in vitro, implicate GAGE proteins in chromatin regulation in germ cells and cancer cells.
Highlights
The GAGE family of highly identical, small oligomeric proteins is expressed from a locus containing 13–39 copies of nearly identical genes on the X-chromosome [1,2]
GAGE12I-Germ cell-less (GCL) pairs exhibited Z scores in the range of 3.4–5.3 (Fig. 1B), clearly indicating an interaction between these proteins. In this assay GCL associated with GAGE2B (Z scores: 1.8– 5.3; Fig. 1B), which represents the GAGE2-type (GAGE2A-E) family, all of which lack a tyrosine at position 11 that can be phosphorylated in other GAGE proteins [28]
We speculate that direct binding of GAGE and GCL might: (a) require a cofactor or posttranslational modification not provided during bacterial expression; (b) be sterically hindered by the His-tag on GAGE12I or the GST-tag on GCL
Summary
The GAGE family of highly identical, small oligomeric proteins is expressed from a locus containing 13–39 copies of nearly identical genes on the X-chromosome [1,2]. We report that GAGE proteins interact with GCL, a metazoan protein important for nuclear envelope integrity and germ cell development in Drosophila and mice [9,10]. In both species, GCL localizes at the inner nuclear membrane, and several lines of evidence suggest that GCL inhibits transcription: GCL is required to silence transcription in Drosophila germ cells [11], and in mammalian cells, GCL binds the heterodimeric transcription factor DP and thereby inhibits DP-E2F-dependent genes, which are required for entry into S-phase. LEM-domain proteins bind lamins (nuclear intermediate filaments) and are key components of nuclear ‘lamina’ structure
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