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Bruce M. Paterson, Ph.D.
Molecular Studies on Eukaryotic Gene Regulation
'As incredible as it seems, future research on flies and worms will quite often provide the shortest and most efficient path to curing human disease.'
Bruce Alberts, Science (editorial), 330:1724 (2010)
Many human diseases, including cancer, result from the misregulation of molecular mechanisms that control cellular specialization during development. Thus the study of gene regulation during development can provide insights into the molecular events that lead to disease, as well as suggest approaches for therapeutic intervention.
Embryonic muscle formation in vertebrates and Drosophila (the fruit fly) provide excellent systems in which to study the formation of striated muscle, one of the major tissues in higher organisms. The MyoD family of basic helix-loop-helix (bHLH) proteins, known as the muscle regulatory factors (MRFs), regulates the determination, proliferation, and differentiation of muscle cells during development in both vertebrates and invertebrates. In vertebrates, the determination of the first muscle precursor cells within the early somitic mesoderm requires the activation of MRFs. The committed myoblasts then migrate from the somite to the various areas of the embryo that will form the muscles of the trunk, limbs and thorax. Genes typically expressed in differentiated muscle remain repressed during migration. Terminal differentiation is marked by the withdrawal of the myoblast from the cell cycle. Cell cycle control during terminal differentiation also involves the MRFs in a pathway that regulates the phosphorylation status of the retinoblastoma protein, Rb, in response to extracellular cues. Exit from the cell cycle is followed by a change in the gene expression profile. More than 90% of the genes expressed in the dividing muscle cell are transcriptionally silenced, while the muscle-specific gene set is activated. This switch in the gene expression pattern is permanent and likely involves substantial changes in chromatin organization, mediated in part by the MRFs. My lab is interested in understanding the way the MRFs regulate the myogenic process, particularly their role in the the determination of the myogenic cell lineage, activation of the muscle-specific gene set, withdrawal from the cell cycle and the concommitant silencing of more thant 90% of the genes expressed in the dividing myoblast.
Regulation of growth and differentiation through MyoD-Cdk4 interaction
Within the nucleus, MyoD can bind to the G1 cyclin dependent kinase, cdk4. This interaction impacts the activity of both MyoD and cdk4. The binding of ectopically expressed MyoD to cdk4 inhibits the phosphorylation of Rb by cdk4, and blocks cell growth. The cdk4-MyoD interaction also disrupts the trans-activation functions of MyoD by interfering with the binding of the MyoD/E-protein heterodimer to DNA.
We have shown that the nuclear import of cdk4 is dependent upon cyclin D1, which itself is regulated by mitogen levels (4-6). Cyclin D1 is degraded in response to reduced levels of growth factors and mitogens, and consequently cdk4 remains in the cytoplasm, allowing MyoD to remain unbound and active within the nucleus. In contrast, in the presence of normal amounts of mitogens, cyclin D1 is stabilized and promotes the nuclear accumulation of cdk4, which binds to and inhibits MyoD function in dividing myoblasts.
We have identified a 15 amino acid domain on MyoD that is required for the interaction with cdk4. Expression of this domain either as a fusion protein with GST or GFP inhibits the kinase activity of cdk4 in vitro and in vivo, blocking its ability to phosphorylate the retinoblastoma protein, Rb. This results in the cessation of cell growth, and induces myoblast differentiation in the presence of mitogens. We have a patent pending on the inhibitory activity of the 15 amino acid MyoD cdk4-binding domain. Determining the structure of the interface formed by the MyoD-cdk4 binding domains will be an important next step. Structural data would aid in the design of G1 cdk inhibitors that could reduce uncontrolled cell growth in human disease.
Role of the nautilus protein in Drosophila myogenesis and muscle development
In Drosophila, the nautilus gene encodes the only member of the bHLH family of MRFs. We have previously shown that nautilus is required to convert S2 cells to a myogenic phenotype, and that ablation of nautilus-expressing cells in the Drosophila embryo completely abrogates muscle formation. We also found that nautilus in combination with the Drosophila E-protein, daughterless, can convert mammalian fibroblasts to a myogenic state. Finally, we have observed that nautilus can rescue the lethality of the C. elegans HLH-1 (MyoD) null (3,4). These results indicate that the nautilus gene product plays a critical role in Drosophila myogenesis.
In the absence of a null for the nautilus gene in Drosophila, we used RNAi to phenocopy a loss-of- gene function by injecting dsRNA into early embryos. Greater than 75% of the injected embryos did not develop normally, and in several instances showed a severe loss-of-muscle phenotype compared to embryos injected with beta-galactosidase dsRNA (7). A subsequent genetic study by another group reported that the loss-of-function phenotype in two EMS-induced mutant alleles differed from that determined using RNAi. In the case of the EMS alleles, embryos lacking nautilus were only missing a subset of muscles, and this loss was not lethal at any stage of development. However, the EMS alleles caused sterility
In order to resolve the phenotypic differences reported from using RNAi or EMS alleles, we disrupted the nautilus gene by introducing an armidillo-GFP expression cassette into the middle of the nautilus gene [in collaboration with Dr. Yikang Rong (LBMB NCI)]. We observe a range of muscle pattern phenotypes resulting from this targeted disruption, from severe loss of muscle organization to minor muscle pattern disruptions (2). The larval muscle pattern is also affected, and a majority of the resulting pupae are unable to undergo eclosion. The small percentage that hatch require significantly more time to leave the pupal case and show impaired mobility, often dying in the food. The surviving adult females display highly distended abdomens and do not lay eggs. Maturing eggs are not able to traverse the ovariole or exit the oviduct, suggestive of a muscle defect. The males are weak, but able to mate, and both sexes often perish within a few days after hatching. These results suggest that the loss of the nautilus gene is phenotypically similar to the loss of the C. elegans HLH-1 gene, CeMyoD: while not absolutely necessary for fiber formation both genes appear to be essential for muscle fiber integrity, and both nulls are essentially lethal. Normal levels of viability and fertility are restored to the targeted null by an HSP-nautilus transgene. In addition, the use of an inducible nautilus RNAi transgene phenocopies the targeted null. Thus, the nautilus gene is essential for normal embryonic myogenesis, muscle patterning and viability, but is not required for myogenic commitment.
We have also found that nautilus has a significant impact on the miRNA transcriptome. Differential miRNA expression studies, in which the nautilus null was compared to wild-type, revealed that the 8-miR (309-miR) locus is a target for nautilus regulation (8). The 8-mir locus is the largest polycistronic miRNA locus in Drosophila, and encodes nine miRNAs. miRNA output from this locus buffers the myogenic transcriptome and impacts key genes required for the regulation of homeobox proteins, chromatin function, and the Notch and Jak-Stat signaling pathways. Surprisingly, we have found that mir-3 from the 8-miR locus indirectly activates the miR-310 cluster, which encodes seven miRNAs. Four of the miRNAs from the miR-310 cluster target Dmef2, a key regulator of Drosophila myogenesis (9). We are performing transcriptome analyses to determine the impact on cells lacking or over-expressing mir-3, and to better define the mir-3 transcript targets.
We are also developing additional, more general ways to identify miRNA target genes and to decipher the miRNA regulatory networks required for normal development. Several miRNAs have been identified that are associated with cancer and human disease. Understanding this second layer of control on transcriptional output will be critical to determining the approaches taken to either eliminate or possibly restore the cancer cell to a normal developmental pathway.
Role of D-elp1 in transposon silencing
In addition to the standard protein components of the RNAi RISC, we have identified the elongator-1 subunit, or D-elp1, as a key component in the RNAi and transposon silencing pathways (1). Interestingly, D-elp1 is not involved miRNA targeting. At present, it appears D-elp1 plays an important role in the maintenance of heterochromatin while also acting as a scaffold for regulators of the RNAi pathway. Misregulation of transposons plays an important role in human disease, including certain types of cancer, and understanding transposon silencing could provide new strategies for disease treatment.
This page was last updated on 5/14/2014.