Mastermind-like transcriptional co-activators: emerging roles in regulating cross talk among multiple signaling pathways.

A family of Mastermind-like (MAML) genes encodes critical transcriptional co-activators for Notch signaling, an evolutionarily conserved pathway with numerous roles in both development and human diseases. Notch receptors are cleaved upon ligand engagement and the intracellular domain of Notch shuttles to the nucleus. MAMLs form a functional DNA-binding complex with the cleaved Notch receptor and the transcription factor CSL, thereby regulating transcriptional events that are specific to the Notch pathway. Here, we review recent studies that have utilized molecular, cellular and physiological model system strategies to reveal the pivotal roles of the MAML proteins in Notch signaling. Unexpectedly, however, emerging evidence implicate MAML proteins as exciting key transcriptional co-activators in other signal transduction pathways including: muscle differentiation and myopathies (MEF2C), tumor suppressor pathway (p53) and colon carcinoma survival (beta-catenin). Thus, the MAML family appears to function in transcriptional co-activation in a multitude of cellular processes. It is hypothesized that MAML proteins mediate cross-talk among the various signaling pathways and the diverse activities of the MAML proteins converge to impact normal biological processes and human diseases, including cancers.

Myopalladin, a novel 145-kilodalton sarcomeric protein with multiple roles in Z-disc and I-band protein assemblies.

We describe here a novel sarcomeric 145-kD protein, myopalladin, which tethers together the COOH-terminal Src homology 3 domains of nebulin and nebulette with the EF hand motifs of alpha-actinin in vertebrate Z-lines. Myopalladin's nebulin/nebulette and alpha-actinin-binding sites are contained in two distinct regions within its COOH-terminal 90-kD domain. Both sites are highly homologous with those found in palladin, a protein described recently required for actin cytoskeletal assembly (Parast, M.M., and C.A. Otey. 2000. J. Cell Biol. 150:643-656). This suggests that palladin and myopalladin may have conserved roles in stress fiber and Z-line assembly. The NH(2)-terminal region of myopalladin specifically binds to the cardiac ankyrin repeat protein (CARP), a nuclear protein involved in control of muscle gene expression. Immunofluorescence and immunoelectron microscopy studies revealed that myopalladin also colocalized with CARP in the central I-band of striated muscle sarcomeres. Overexpression of myopalladin's NH(2)-terminal CARP-binding region in live cardiac myocytes resulted in severe disruption of all sarcomeric components studied, suggesting that the myopalladin-CARP complex in the central I-band may have an important regulatory role in maintaining sarcomeric integrity. Our data also suggest that myopalladin may link regulatory mechanisms involved in Z-line structure (via alpha-actinin and nebulin/nebulette) to those involved in muscle gene expression (via CARP).

Identification of muscle specific ring finger proteins as potential regulators of the titin kinase domain.

The giant myofibrillar protein titin contains within its C-terminal region a serine-threonine kinase of unknown function. We have identified a novel muscle specific RING finger protein, referred to as MURF-1, that binds in vitro to the titin repeats A168/A169 adjacent to the titin kinase domain. In myofibrils, MURF-1 is present within the periphery of the M-line lattice in close proximity to titin's catalytic kinase domain, within the Z-line lattice, and also in soluble form within the cytoplasm. Yeast two-hybrid screens with MURF-1 as a bait identified two other highly homologous MURF proteins, MURF-2 and MURF-3. MURF-1,2,3 proteins are encoded by distinct genes, share highly conserved N-terminal RING domains and in vitro form dimers/heterodimers by shared coiled-coil motifs. Of the MURF family, only MURF-1 interacts with titin repeats A168/A169, whereas MURF-3 has been reported to affect microtubule stability. Association of MURF-1 with M-line titin may potentially modulate titin's kinase activity similar to other known kinase-associated proteins, whereas differential expression and heterodimerization of MURF1, 2 and 3 may link together titin kinase and microtubule-dependent signal pathways in striated muscles.

The N-terminal end of nebulin interacts with tropomodulin at the pointed ends of the thin filaments.

Strict regulation of actin thin filament length is critical for the proper functioning of sarcomeres, the basic contractile units of myofibrils. It has been hypothesized that a molecular template works with actin filament capping proteins to regulate thin filament lengths. Nebulin is a giant protein ( approximately 800 kDa) in skeletal muscle that has been proposed to act as a molecular ruler to specify the thin filament lengths characteristic of different muscles. Tropomodulin (Tmod), a pointed end thin filament capping protein, has been shown to maintain the final length of the thin filaments. Immunofluorescence microscopy revealed that the N-terminal end of nebulin colocalizes with Tmod at the pointed ends of thin filaments. The three extreme N-terminal modules (M1-M2-M3) of nebulin bind specifically to Tmod as demonstrated by blot overlay, bead binding, and solid phase binding assays. These data demonstrate that the N terminus of the nebulin molecule extends to the extreme end of the thin filament and also establish a novel biochemical function for this end. Two Tmod isoforms, erythrocyte Tmod (E-Tmod), expressed in embryonic and slow skeletal muscle, and skeletal Tmod (Sk-Tmod), expressed late in fast skeletal muscle differentiation, bind on overlapping sites to recombinant N-terminal nebulin fragments. Sk-Tmod binds nebulin with higher affinity than E-Tmod does, suggesting that the Tmod/nebulin interaction exhibits isoform specificity. These data provide evidence that Tmod and nebulin may work together as a linked mechanism to control thin filament lengths in skeletal muscle.

Probing the functional roles of titin ligands in cardiac myofibril assembly and maintenance.

Sarcomeres of cardiac muscle are comprised of numerous proteins organized in an elegantly precise order. The exact mechanism of how these proteins are assembled into myofibrils during heart development is not yet understood, although existing in vitro and in vivo model systems have provided great insight into this complex process. It has been proposed by several groups that the giant elastic protein titin acts as a "molecular template" to orchestrate sarcomeric organization during myofibrillogenesis. Titin's highly modular structure, composed of both repeating and unique domains that interact with a wide spectrum of contractile and regulatory ligands, supports this hypothesis. Recent functional studies have provided clues to the physiological significance of the interaction of titin with several titin-binding proteins in the context of live cardiac cells. Improved models of cardiac myofibril assembly, along with the application of powerful functional studies in live cells, as well as the characterization of additional titin ligands, is likely to reveal surprising new functions for the titin third filament system.

Painting Qa-2 onto Ped slow preimplantation embryos increases the rate of cleavage.

PROBLEM: Qa-2 protein, the Ped gene product, is linked to the cell surface by a glycosylphosphatidylinositol (GPI) anchor. Some GPI-linked proteins can be spontaneously incorporated into the membranes of cells via a technique called "protein painting."We investigated whether Qa-2 could be painted onto T cells and embryos and whether the painted protein would be functional. METHOD OF STUDY: Incorporation of Qa-2 into the membranes of T cells and embryos was measured by FACScan and Immuno-PCR, respectively. Function of Qa-2 was measured by cell proliferation. RESULTS: Qa-2 was incorporated by T cells and embryos and was functional. CONCLUSION: GPI-linked Qa-2 protein "painted" onto both T cells and preimplantation embryos is functional, as shown by increased proliferation of T cells after cross-linking with anti-Qa-2 antibody, and increased rate of cleavage division of the embryos.

Cross-linking of Qa-2 protein, the Ped gene product, increases the cleavage rate of C57BL/6 preimplantation mouse embryos.

The Qa-2 protein, a glycosylphosphatidylinositol (GPI)-linked major histocompatibility complex (MHC) Class Ib molecule found on the surface of mouse T-cells and preimplantation embryos, is the product of the preimplantation embryo development (Ped) gene. The Ped gene regulates the rate of early embryonic development and subsequent embryo survival. T-cells treated with anti-Qa-2 monoclonal antibody (mAb) and cross-linked with a secondary antibody, in the presence of a co-stimulatory signal, undergo increased proliferation. The purpose of this study was to determine whether cross-linking of Qa-2 similarly affects preimplantation embryos. We cross-linked Qa-2 protein on the surface of C57BL/6 2-cell and 8-cell embryos, in the presence of 4/5-phorbol-12-myristate-13-acetate (PMA), and assessed the percentage of embryos reaching the blastocyst stage, the percentage hatching from the zona pellucida, [(3)H-thymidine] incorporation into DNA, and the total number of cells per embryo as measures of embryonic cleavage rate. Both 2-cell and 8-cell embryos increased their cleavage rates 48 h after cross-linking of Qa-2, compared with control embryos (P < 0.05). Our results indicate that a Qa-2 protein cross-linking mechanism may be one way by which this protein regulates the rate of preimplantation mouse embryo development.

Expression of caspase and BCL-2 apoptotic family members in mouse preimplantation embryos.

Apoptosis, as determined by blastomere and DNA fragmentation, occurs in many preimplantation mouse embryos. To investigate which genes contribute to apoptosis in preimplantation embryos, we used the reverse transcription-polymerase chain reaction to assess mRNA levels for seven genes in the caspase family and seven genes in the BCL-2 family. All caspase mRNAs were detectable in oocytes, while expression in preimplantation embryos varied in a stage-specific manner. An assay for group II caspase enzymatic activity showed that although transcripts for these caspases could not be detected in zygotes, proteolytic activity could be detected in polar bodies, fragmented zygotes, and zygotes treated with staurosporine. This suggests that maternal caspases are inherited during oogenesis. Transcripts for some members of the BCL-2 family could be detected at every stage of preimplantation development. Transcripts for other members were rarely detected. When BCL-2 and BAX protein levels were assessed using immunofluorescence, both proteins were detected in zygotes and in blastocysts. When fragmented blastocysts were compared to normal blastocysts, levels of BCL-2 immunofluorescence tended to be lower in fragmented blastocysts. This result supports a model in which the ratio of BCL-2 to BAX is altered in apoptotic embryos.

The expression pattern of the Qa-2 antigen in mouse preimplantation embryos and its correlation with the Ped gene phenotype.

The Qa-2 antigen, the product of the Ped (Preimplantation embryo development) gene, is a glycosylphosphatidylinositol-linked cell surface protein encoded in the Q region of the mouse major histocompatibility complex (MHC). Ped fast (Qa-2+) mouse strains have significantly higher preimplantation embryo cleavage rates both in vivo and in vitro than Ped slow (Qa-2-) mice. In this study, we determined whether the Ped fast phenotype of blastocysts is due to an increased number of blastomeres in the trophectoderm (TE), the inner cell mass (ICM), or both. We also analysed the Ped gene expression pattern, both at the mRNA and at the protein level, in these lineages. Blastocysts were collected from the congenic mouse strains B6.K2 (Qa-2 +) and B6.K1 (Qa-2-). We performed reverse transcription-polymerase chain reaction (PCR) and Immuno-PCR and found that the Ped gene is expressed at the mRNA and protein level in whole embryos and in isolated ICM cells. Lastly, we differentially stained embryos from these strains and found that B6.K2 blastocysts had significantly higher cell numbers (P < 0.05) in both the ICM and in the TE than B6.K1 blastocysts. These results suggest that Qa-2 expression in both the TE and the ICM of blastocysts directly contributes to the Ped phenotype.

Genetic regulation of egg and embryo survival.

In both mice and humans, 15-50% of embryos die during the preimplantation period from mechanisms that are largely unknown. Two major criteria predict preimplantation embryo quality, the rate of development and the degree of fragmentation. We review evidence that both of these criteria have a genetic basis. Rate of development and subsequent embryo survival are controlled by a gene, Ped, we discovered in the mouse. Although progress is being made in the search for the human homologue of the mouse Ped gene, it has not yet been identified. Fragmentation, observed in both mouse and human embryos, is probably the result of apoptosis. We analysed transcription of two genes that regulate apoptosis, bcl-2 and bax, and found that both are transcribed in mouse and human preimplantation embryos. Overall, the literature reviewed and new data presented in this paper support the concept that there is a genetic basis for preimplantation egg and embryo survival.

Genetic regulation of preimplantation mouse embryo survival.

The preimplantation period of mammalian development is characterized by cleavage of a one-cell embryo to a blastocyst stage embryo. During preimplantation development, 15%-50% of the embryos die as a result of factors that are largely unknown. Two parameters of preimplantation development, a fast rate of development and a low degree of fragmentation, are indicative of good embryo quality. There is mounting evidence that genes control both rate of development and degree of fragmentation. We have discovered a gene, Ped (preimplantation embryo development), which controls the rate of preimplantation embryonic cleavage. The Ped gene is encoded by two similar genes, Q7 and Q9, in the Q region of the mouse major histocompatibility complex (MHC). The Ped gene product is an MHC class Ib protein, the Qa-2 antigen. The mechanisms by which the Ped gene controls rate of embryonic cleavage division are being explored. In order to understand genetic mechanisms underlying the second criterion of embryo quality, degree of fragmentation, we have begun to assess expression of the genes that could potentially regulate apoptosis in preimplantation embryos. We have shown that staurosporine can induce apoptosis in mouse blastocysts. By using RT-PCR, we have shown that genes encoding protein in the two major gene families that regulate apoptosis, the Bcl-2 and caspase gene families, are present in preimplantation embryos. We hypothesize that there is a homeostatic mechanism by which genes that regulate cell survival and those that regulate cell death determine the overall viability of preimplantation embryos.

Role of the Ped gene and apoptosis genes in control of preimplantation development.

PURPOSE: The properties of the mouse Ped gene and the genes that mediate apoptosis in mediating preimplantation embryonic survival were reviewed. METHODS: Preimplantation mouse oocytes and embryos were evaluated microscopically and biochemically for rate of development, degree of fragmentation, and gene expression to correlate these characteristics with embryo mortality, Biochemical assays included PCR for DNA analysis, RT-PCR for mRNA analysis, immuno-PCR for protein analysis, and TUNEL assay for assessment of apoptosis. RESULTS: Using the mouse as a model system we have identified a gene that controls the rate of development, the Ped gene. The Ped gene product is a class Ib major histocompatibility complex protein called the Qa-2 antigen. Research to understand the molecular mechanisms of Ped gene action and to identify the human homologue of the Ped gene is under way. We have also shown using the mouse model, that fragmented embryos show the morphological and biochemical characteristics of apoptosis. Genes in the two major gene families that regulate apoptosis, the caspase and Bcl-2 families, are expressed in mouse oocytes and preimplantation embryos. CONCLUSIONS: Preimplantation embryonic survival depends on two major morphological parameters: rate of development and degree of fragmentation. A fast rate of development and a low degree of fragmentation lead to a better chance of producing live offspring. Both rate of development and degree of fragmentation are genetically controlled, the former by the Ped gene and the latter most likely by genes that mediate apoptosis. It seems probable that regulation of apoptosis will prove to be a major mechanism that mediates oocyte and preimplantation embryonic survival.

The effect of melatonin on cleavage rate of C57BL/6 and CBA/Ca preimplantation embryos cultured in vitro.

There is growing interest in using melatonin as a therapeutic agent for the treatment of a variety of medical conditions, including cancer, heart disease, glaucoma, stress, jet lag, and sleep disorders. In addition, melatonin is being evaluated in a clinical trial to test its efficacy as an oral contraceptive. In order to test any possible adverse effects of melatonin on preimplantation embryos, we used the mouse as a model system. Two strains of mice, a Ped fast, melatonin-deficient strain, C57BL/6, and a Ped slow strain previously found to have detectable melatonin levels at nighttime, CBA/Ca, were studied. Two cell embryos were incubated with melatonin concentrations from 10(-5) M to 10(-13) M for 48 or 72 hours and the number of cells per embryo assessed quantitatively at the end of the incubation period. We used sufficiently high levels of melatonin to mimic the pharmacological concentration used in the oral contraceptive. It was found that there was no effect of melatonin on embryos from either mouse strain at any of the concentrations tested. Our results suggest that if conception occurs while melatonin is being administered to treat a range of conditions, it would not adversely affect the embryo.