The "MAN antigens" are polypeptides recognized by autoantibodies from a patient with a collagen vascular disease and localized to the nuclear envelope. We now show that one of the human MAN antigens termed MAN1 is a 82.3-kDa protein with an amino-terminal domain followed by two hydrophobic segments and a carboxylterminal tail. The MAN1 gene contains seven proteincoding exons and is assigned to human chromosome 12q14. Its mRNA is approximately 5.5 kilobases and is detected in several different cell types that were examined. Cell extraction experiments show that MAN1 is an integral membrane protein. When expressed in transfected cells, MAN1 is exclusively targeted to the nuclear envelope, consistent with an inner nuclear membrane localization. Protein sequence analysis reveals that MAN1 shares a conserved globular domain of approximately 40 amino acids, which we term the LEM module, with inner nuclear membrane proteins lamina-associated polypeptide 2 and emerin. The LEM module is also present in two proteins of Caenorhabditis elegans. These results show that MAN1 is an integral protein of the inner nuclear membrane that shares the LEM module with other proteins of this subcellular localization.Only a few proteins other than the nuclear lamins, some with various isoforms, have been localized to the inner nuclear membrane during interphase. The first to be identified was avian lamin B receptor (LBR) 1 (1, 2). LBR was subsequently characterized in mammals (3-6), and an immunochemically crossreactive protein has been identified in sea urchins (7). LBR has a nucleoplasmic, amino-terminal domain of approximately 200 amino acids that binds to B-type lamins and chromatin proteins and confers inner nuclear membrane retention (2, 4, 8 -12). The amino-terminal domain of LBR is followed by a hydrophobic domain with eight putative transmembrane segments that is similar in sequence to sterol reductases, including two human proteins of the endoplasmic reticulum, one of which is a 7-dehydrocholesterol reductase (13).Two other integral proteins of the inner nuclear membrane have been termed lamina-associated polypeptides (LAPs). Three related isoforms of rat LAP1 (LAP1A, LAP1B, and LAP1C) were identified by reaction with a single monoclonal antibody and shown to be integral membrane proteins associated with the nuclear lamina (14). LAP1C has a nucleoplasmic amino-terminal domain followed by one transmembrane segment, and the other LAP1 isoforms are probably of similar overall structure, arising from the same gene by alternative RNA splicing (15). LAP2 was also first identified by reaction with a monoclonal antibody and shown to be an integral membrane protein that binds to nuclear lamins and chromatin (16). Several isoforms of LAP2, which have also been called thymopoietins because they were thought to possibly be thymocyte growth factors, are generated by alternative RNA splicing (17-21). Some of the LAP2 isoforms are integral proteins of the inner nuclear membrane, with nucleoplasmic amino-terminal domains and single transmembr...

SummaryCell therapies treating pathological muscle atrophy or damage requires an adequate quantity of muscle progenitor cells (MPCs) not currently attainable from adult donors. Here, we generate cultures of approximately 90% skeletal myogenic cells by treating human embryonic stem cells (ESCs) with the GSK3 inhibitor CHIR99021 followed by FGF2 and N2 supplements. Gene expression analysis identified progressive expression of mesoderm, somite, dermomyotome, and myotome markers, following patterns of embryonic myogenesis. CHIR99021 enhanced transcript levels of the pan-mesoderm gene T and paraxial-mesoderm genes MSGN1 and TBX6; immunofluorescence confirmed that 91% ± 6% of cells expressed T immediately following treatment. By 7 weeks, 47% ± 3% of cells were MYH+ve myocytes/myotubes surrounded by a 43% ± 4% population of PAX7+ve MPCs, indicating 90% of cells had achieved myogenic identity without any cell sorting. Treatment of mouse ESCs with these factors resulted in similar enhancements of myogenesis. These studies establish a foundation for serum-free and chemically defined monolayer skeletal myogenesis of ESCs.

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The Saccharomyces cerevisiae NuA4 histone acetyltransferase complex catalyzes the acetylation of histone H4 and the histone variant Htz1 to regulate key cellular events, including transcription, DNA repair, and faithful chromosome segregation. To further investigate the cellular processes impacted by NuA4, we exploited the nonessential subunits of the complex to build an extensive NuA4 genetic-interaction network map. The map reveals that NuA4 is a genetic hub whose function buffers a diverse range of cellular processes, many not previously linked to the complex, including Golgi complex-to-vacuole vesicle-mediated transport. Further, we probe the role that nonessential subunits play in NuA4 complex integrity. We find that most nonessential subunits have little impact on NuA4 complex integrity and display between 12 and 42 genetic interactions. In contrast, the deletion of EAF1 causes the collapse of the NuA4 complex and displays 148 genetic interactions. Our study indicates that Eaf1 plays a crucial function in NuA4 complex integrity. Further, we determine that Eaf5 and Eaf7 form a subcomplex, which reflects their similar genetic interaction profiles and phenotypes. Our integrative study demonstrates that genetic interaction maps are valuable in dissecting complex structure and provides insight into why the human NuA4 complex, Tip60, has been associated with a diverse range of pathologies.

Wnt1 and Wnt3a are signaling factors known to play a role in the induction of myogenesis in the myotome of the differentiating somite. Both factors may transduce their signal by a conserved pathway that leads to transcriptional regulation by ␤-catenin/Lef1. ␤-Catenin and Lef1 are found in the myotome prior to MyoD expression. We have utilized the P19 cell system to study the mechanisms by which Wnt3a may activate MyoD expression and subsequent skeletal muscle development. We have isolated P19 cell lines that stably express either Wnt3a or activated ␤-catenin and found that aggregation of these cells results in the induction of myogenesis compared with control cells. Pax3, Gli2, Mox1, and Six1 were expressed during Wnt3a and ␤-catenin-induced differentiation prior to MyoD expression. Furthermore, we have shown that the nuclear function of ␤-catenin was essential for skeletal myogenesis in P19 cells by overexpression of a dominant negative ␤-catenin/engrailed chimera. Primitive streak factors were present, but expression of Pax3, Mox1, Gli2, and Six1 was lost in these cells, indicating that nuclear ␤-catenin is essential for specification of mesodermal precursors to the myogenic lineage. Therefore, Wnt signaling, acting via ␤-catenin, is necessary and sufficient for skeletal myogenesis in P19 cells.

Pax3 is a paired box transcription factor expressed during somitogenesis that has been implicated in initiating the expression of the myogenic regulatory factors during myogenesis. We find that Pax3 is necessary and sufficient to induce myogenesis in pluripotent stem cells. Pax3 induced the expression of the transcription factor Six1, its cofactor Eya2, and the transcription factor Mox1 prior to inducing the expression of MyoD and myogenin. Overexpression of a dominant negative Pax3, engineered by fusing the active transcriptional repression domain of mouse EN-2 in place of the Pax3 transcriptional activation domain, completely abolished skeletal myogenesis without inhibiting cardiogenesis. Expression of the dominant negative Pax3 resulted in a loss of expression of Six1, Eya2, and endogenous Pax3 as well as a down-regulation in the expression of Mox1. No effect was found on the expression of Gli2. These results indicate that Pax3 activity is essential for skeletal muscle development, the expression of Six1 and Eya2, and is involved in regulating its own expression. In summary, the combined approach of expressing both a wild type and dominant negative transcription factor in stem cells has identified a cascade of transcriptional events controlled by Pax3 that are necessary and sufficient for skeletal myogenesis.

The transcriptional mechanisms underlying lineage specification and differentiation of embryonic stem (ES) cells remain elusive. Oct-3/4 (POU5f1) is one of the earliest transcription factors expressed in the embryo. Both the pluripotency and the fate of ES cells depend upon a tight control of Oct-3/4 expression. We report that transgene- or TGFbeta-induced increase in Oct-3/4 mRNA and protein levels in undifferentiated ES cells and at early stages of differentiation triggers expression of mesodermal and cardiac specific genes through Smad2/4. cDNA antisense- and siRNA-mediated inhibition of upregulation of Oct-3/4 in ES cells prevent their specification toward the mesoderm and their differentiation into cardiomyocytes. Similarly, Oct-3/4 siRNA injected in the inner cell mass of blastocysts impairs cardiogenesis in early embryos. Thus, quantitative Oct-3/4 expression is regulated by a morphogen, pointing to a pivotal and physiological function of the POU factor in mesodermal and cardiac commitments of ES cells and of the epiblast.

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