Primordial germ cells (PGCs) are founder cells of all gametes. A number of genes that control PGCs development have been identified in invertebrates, whereas such genes are by and large unelucidated in mammals. Described here is the cloning, genomic structure and expression of the mouse homolog of germ cell-less gene which is required for PGCs formation in Drosophila. The mouse gcl shows 34% identity compared with Drosophila Gcl protein and contains BTB/POZ domain. The gcl gene consists of 14 exons and spans more than 50 kb. The CpG islands are found around exon 1 of the gene. Putative promoter region contains potential binding sites for various transcription factors. Northern blot analysis shows that its mRNA is highly expressed in adult testis with lower expression in ovary, ES (embryonic stem) cells, and various other organs. In situ hybridization analysis reveals strong expression of the gcl gene in the pachytene stage spermatocytes. The expression is also observed in post-migratory PGCs, but was not apparent in migratory and pre-migratory PGCs. Further studies including gene disruption analysis would provide an important insight into mammalian germ lineage development (Kimura, 1999).
Transcription factor E2F plays an important role in orchestrating early cell cycle progression through its ability to co-ordinate and integrate the cell cycle with the transcription apparatus. Physiological E2F arises when members of two distinct families of proteins interact as E2F-DP heterodimers, in which the E2F component mediates transcriptional activation and the physical interaction with pocket proteins, such as the tumour suppressor protein pRb. In contrast, a discrete role for the DP subunit has not been defined. DIP, a novel mammalian protein that can interact with the DP component of E2F, has been identified and characterized. DIP contains a BTB/POZ domain and shows significant identity with the Drosophila melanogaster germ cell-less gene product. In mammalian cells, DIP is distributed in a speckled pattern at the nuclear envelope region, and can direct certain DP subunits and the associated heterodimeric E2F partner into a similar pattern. DIP-dependent growth arrest is modulated by the expression of DP proteins, and mutant derivatives of DIP that are compromised in cell cycle arrest exhibit reduced binding to the DP subunit. This study defines a new pathway of growth control that is integrated with the E2F pathway through the DP subunit of the heterodimer (de la Luna, 1999).
LAP2beta is an integral membrane protein of the nuclear envelope involved in chromatin and nuclear architecture. Using the yeast two-hybrid system, a novel LAP2beta-binding protein, mGCL, has been cloned that contains a BTB/POZ domain and is the mouse homolog of the Drosophila Germ-cell-less (Gcl) protein. In Drosophila embryos, Gcl is essential for germ cell formation and is localized to the nuclear envelope. In mammalian cells, Gcl co-localizes with LAP2beta to the nuclear envelope. Nuclear fractionation studies reveal that mGCL acts as a nuclear matrix component and not as an integral protein of the nuclear envelope. mGCL has been found to interact with the DP3alpha component of the E2F transcription factor. This interaction reduces the transcriptional activity of the E2F-DP heterodimer, probably by anchoring the complex to the nuclear envelope. LAP2beta is also capable of reducing the transcriptional activity of the E2F-DP complex and it is more potent than mGCL in doing so. Co-expression of both LAP2beta and mGCL with the E2F-DP complex results in a reduced transcriptional activity equal to that exerted by the pRb protein (Nili, 2001).
Drosophila Germ cell-less is important in early events during the formation of pole cells, the germ cell precursors in the fly. A 524 amino acid mouse gene with 32% identity and 49% similarity to Drosophila gcl, termed mgcl-1, has been isolated. Like Drosophila Gcl, mGcl-1 localizes to the nuclear envelope. Ectopic expression of mgcl-1 in Drosophila rescues the gcl-null phenotype, indicating that mGcl-1 is a functional homolog of Gcl. mgcl-1 maps to chromosome 6 at 47.3 cM, and is expressed at low levels at all embryonic stages examined from 8.5 to 18.5 d.p.c. as well as in many adult tissues. Different from Drosophila gcl, mgcl-1 is not highly expressed at the time the primordial germ cells appear in the mouse, but high mgcl-1 expression is found in selected mouse adult male germ cells. The differences in these expression patterns in light of conserved activity between the two genes is discussed (Leatherman, 2000).
Emerin belongs to the 'LEM domain' family of nuclear proteins, which contain a characteristic approximately 40-residue LEM motif. The LEM domain mediates direct binding to barrier to autointegration factor (BAF), a conserved 10-kDa chromatin protein essential for embryogenesis in Caenorhabditis elegans. In mammalian cells, BAF recruits emerin to chromatin during nuclear assembly. BAF also mediates chromatin decondensation during nuclear assembly. The LEM domain and central region of emerin are essential for binding to BAF and lamin A, respectively (see Drosophila Lamin. However, two other conserved regions of emerin lacked ascribed functions, suggesting that emerin could have additional partners. These 'unascribed' domains of emerin mediate direct binding to a transcriptional repressor, germ cell-less (GCL). GCL co-immunoprecipitates with emerin from HeLa cells. The binding affinities of emerin for GCL, BAF, and lamin A have been determined and their oligomeric interactions analyzed. Emerin forms stable complexes with either lamin A plus GCL or lamin A plus BAF. Importantly, BAF competes with GCL for binding to emerin in vitro, predicting that emerin can form at least two distinct types of complexes in vivo. Loss of emerin causes Emery-Dreifuss muscular dystrophy, a tissue-specific inherited disease that affects skeletal muscles, major tendons, and the cardiac conduction system. Although GCL alone cannot explain the disease mechanism, these results strongly support gene expression models for Emery-Dreifuss muscular dystrophy by showing that emerin binds directly to a transcriptional repressor, GCL, and by suggesting that emerin-repressor complexes might be regulated by BAF. Biochemical roles for emerin in gene expression are discussed (Holaska, 2003).
A mouse homolog of the Drosophila germ cell-less (mgcl-1) gene is expressed ubiquitously, and its gene product is localized to the nuclear envelope based on its binding to LAP2ß (lamina-associated polypeptide 2ß). To elucidate the role of mgcl-1, two mutant mouse lines were analyzed that lacked mgcl-1 gene expression. Abnormal nuclear morphologies that are probably due to impaired nuclear envelope integrity are observed in the liver, exocrine pancreas, and testis. In particular, functional abnormalities are observed in testis in which the highest expression of mgcl-1 is detected. Fertility is significantly impaired in mgcl-1-null male mice, probably as a result of severe morphological abnormalities in the sperm. Electron microscopic observations show insufficient chromatin condensation and abnormal acrosome structures in mgcl-1-null sperm. In addition, the expression patterns of transition proteins and protamines, both of which are essential for chromatin remodeling during spermatogenesis, are aberrant. Considering that the first abnormality during the process of spermatogenesis is abnormal nuclear envelope structure in spermatocytes, the mgcl-1 gene product appears to be essential for appropriate nuclear-lamina organization, which in turn is essential for normal sperm morphogenesis and chromatin remodeling (Kimura, 2003).
The nuclear envelope, which separates the chromosomes from the cytoplasm and organizes the nuclear architecture, is composed of inner and outer membranes, NPC, and nuclear lamina. The inner nuclear membrane contains a unique set of integral membrane proteins, which includes the lamin B receptor (LBR), LAP1, LAP2, emerin, MAN1, and nurim. Most of these proteins bind to the nuclear lamina, which is a network of polymers formed by lamins. Mammals have three lamin genes (LMNA, LMNB, and LMNB2), which encode seven alternatively spliced lamin isoforms. Lamins and other nuclear envelope proteins are involved in the organization of the nuclear architecture. From the analyses of human diseases and gene targeting mice, three genes, LMNA, emerin and LBR have been revealed to be essential for the maintenance of normal nuclear envelope integrity (Kimura, 2003).
The cells of mgcl-1-null mice show abnormal nuclear structures in several organs. Although mGCL-1 was reported to bind LAP2ß, the functions of these two proteins in nuclear envelope formation remain unknown. LBR and emerin are integral proteins of the nuclear inner membrane, and lamins A and C, which are A-type lamins and gene products of the LMNA gene, are major components of the nuclear lamin. Compared to these proteins, mGCL-1 appears to be a relatively minor component of the nuclear lamina, since mGCL-1 does not bind directly to lamins and is not an integral protein. An essential role for mGCL1 in nuclear integrity suggests that the nuclear lamina forms a more complicated structure than was previously believed; i.e., more proteins may participate as essential members. An alternative, though not mutually exclusive, interpretation is that the Drosophila GCL and mGCL-1 proteins have unidentified molecular properties in common. Although there are no genes that code for LAP proteins in Drosophila, GCL is localized to the nuclear envelope. Furthermore, mGCL-1 can rescue the Drosophila gcl mutant phenotype. In addition, the full-length mGCL-1 and various deletion mutants of mGCL-1 have been localized to the nuclear lamina. It is conceivable that mGCL-1 binds to some other structural components of the nuclear envelope in order to maintain the structure (Kimura, 2003).
Nuclear envelopes break down at the onset of mitosis and are reconstructed around the chromosomes during telophase. Similar to other nuclear lamina proteins, mGCL-1 diffuses into cytoplasm and/or ER during mitosis and reassembles around chromosomes at the end of mitosis. Immunostaining with antibodies against LAP2 and NPC shows that the nuclear envelope components assemble around chromosomes at the end of mitosis in mgcl-1-/- embryonic fibroblasts, suggesting that reformation of nuclear envelopes is not affected by the absence of mGCL-1 (Kimura, 2003).
The most striking feature of the mgcl-1-/- mouse phenotype is abnormal spermatogenesis. The abnormalities can be divided into three categories: defects in nuclear architecture, sperm morphology, and proteins that were involved in chromatin remodeling. Given that mGCL-1 binds directly to LAP2ß, and that mGCL-1 is expressed abundantly in spermatocytes, the abnormal nuclear envelope structure is probably due to the primary effect of the null mutation in mgcl-1. However, it is uncertain whether the other abnormalities are direct sequels of the null mutation or are due to the indirect effects of the abnormal nuclear envelope structure (Kimura, 2003).
Although the mgcl-1 gene is expressed ubiquitously, abnormal nuclear morphology was detected only in the restricted organs. The testes show very high levels of mgcl-1 mRNA. However, the mRNA levels in the exocrine pancreas and liver, both of which showed abnormal nuclear structures, are comparable to those in other organs. Thus, the abundance of mgcl-1 transcripts alone does not explain why mGCL-1 is important for the normal nuclear structure. Differential expression of lamins may be one reason for the tissue-specific nuclear architecture abnormalities. The nuclear envelope of spermatocytes contains the short meiosis-specific lamin isoforms C2 and B3, which are A- and B-type lamins, respectively. Due to the presence of these spermatocyte-specific lamins, the stability of the nuclear envelope of spermatocytes is considered to be lower than that of somatic cells. Abnormal nuclear morphology was found not only in spermatocytes but also in the later stages of spermiogenesis. It seems reasonable to consider that the vulnerable nuclear structure originates in spermatocytes and persists until the later phases of spermatogenesis (Kimura, 2003).
Experimental and genetic studies suggest that the nuclear lamina is involved in a number of nuclear functions, such as nuclear envelope assembly, DNA synthesis, transcription, and apoptosis. The roles of the nuclear lamina in replication and transcription are putatively related to interactions between the nuclear lamina and chromatin. From this point of view, an intriguing phenotype of the mgcl-1-null mouse is the abnormal expression of transition proteins and protamines, both of which are involved in chromatin remodeling during spermatogenesis. It is unclear whether this abnormality is a direct consequence or an indirect downstream effect of nuclear envelope abnormality. In any case, this abnormality leads to insufficient chromatin condensation in the sperm heads of mgcl-1-/- mice. Perturbed chromatin remodeling caused by the impaired expression of transition proteins and protamines may be one of the reasons why mature mgcl-1mutant sperm have abnormal morphologies (Kimura, 2003).
mGCL-1 has been shown to interfer with the transcriptional functions of the E2F-DP complex. An independent explanation for the abnormality in mgcl-1-/- testis is that abnormal gene expression arose from the altered transcriptional activities of E2F-DP. However, this mechanism is unlikely to be true, since the expression levels of genes whose transcription was driven by E2F-DP are not altered significantly in the mgcl-1-/- testis (Kimura, 2003).
Interestingly, perturbation of the nuclear envelope structure is known to cause laminopathy diseases, such as Emery-Dreifuss muscular dystrophy, in which the lamin A/C or the emerin genes for nuclear-lamina components are mutated. Mice that lacked the A-type lamins had abnormal nuclear envelope integrity and suffered from muscular dystrophy. Mutations in the LBR-encoding gene alter the nuclear morphology of granulocytes. These laminopathies are defined as diseases that are caused by mutations in genes that encode either lamins or proteins that bind to lamins. The loss of mGCL-1 may not in itself constitute a laminopathy, since mGCL-1 does not bind directly to lamins. However, mGCL-1 deficiency may be considered a laminopathy in a broader interpretation of this disease. In terms of both the human diseases and the mutant mouse strains, it has been proposed that defects in the nuclear lamina eliminate the specific interaction between the nuclear matrix and chromatin, which leads to disorganized chromatin architectures and abnormal gene expression. However, the molecular mechanisms that link abnormal nuclear envelope integrity and these diseases have not been elucidated. It remains to be determined whether the pathophysiology reflects the primary molecular defect in the nuclear envelope, or is caused by downstream effects on chromatin structure and gene expression (Kimura, 2003).
Accordingly, it is difficult to ascertain how the mgcl-1-null mutation causes abnormal spermatogenesis. However, it is speculated that the LAP2-BAF association may provide a clue to the pathogenesis. Chromatin regions appear to be anchored to the nuclear lamina, and LAP2 is an important component for this binding, since two isoforms of LAP2, LAP2alpha and LAP2ß, bind to chromatin. Stable binding requires both the chromatin-binding domain and the LEM domain, which is a conserved region of ~43 amino acids that is also found in emerin and MAN1. In addition, LAP2 isoforms bind to the DNA-binding protein BAF via their LEM domains. BAF, which is a ubiquitous and highly conserved protein, colocalizes with chromatin during interphase and mitosis and probably plays a fundamental role in chromosome architecture. A null mutation in the mgcl-1 gene may affect chromatin organization and subsequent gene expression by inducing an abnormal LAP2ß-BAF association (Kimura, 2003).
The Drosophila gcl gene is required for the formation of germ cell precursors but not for germ cell development within the gonads. In contrast, mgcl-1 is dispensable for PGC formation but is important for spermatogenesis. Thus, despite the substitutable molecular function of mouse gcl in Drosophila, the physiological functions of the Drosophila and mouse gcl genes are distinct during germ cell development. One of the abnormal features of the Drosophila gcl mutant is the disordered budding of germ lineage cells. The gigantic sperm in mgcl-1-/- mice, which were presumably caused by unsuccessful cell separation at the final stage of spermiogenesis, are reminiscent of abnormal budding in Drosophila. It is believed that comparative studies in different taxa of the functions of the Drosophila GCL and its mouse homolog will provide phylogenetic clues as to germ cell specification and differentiation (Kimura, 2003).
The gene of germ cell-less (gcl) has been shown to be important in early differentiation of germ cells in Drosophila. Although the gcl homologue genes have been identified in some organisms, there is little data on the expression pattern and functional analysis of the gcl gene in zebrafish. In this research, real-time quantitative RTPCR showed that the level of gcl mRNA expression rapidly decreases from the 4-cell stage to the sphere stage at which it reaches a minimum, gradually increases from the 50%-epiboly stage, and then remains stable during the posterior stages. Results of in situ hybridization indicated that the transcripts of zebrafish gcl are evenly distributed in all blastomeres from the 2-cell stage to the blastula period, different from that of vasa, nonas1 and dead end mRNA, and condense into some clusters of cells located along the blastoderm margin from the gastrulation period. During subsequent development, the transcripts are segregated as subcellular clumps to a small number of cells that would migrate to the position of the gonad in the dorsal side. In the adult, gcl mRNA is widely expressed in developing germ cells of both ovary and testis. These data suggest that zebrafish gcl has potentially important roles in the formation of primordial germ cells (Li, 2006).
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