Completion of the DNA sequences of the human genome and that of the nematode Caenorhabditis elegans allows the large-scale identification and analysis of orthologs of human genes in an organism amenable to detailed genetic and molecular analyses. We are determining gene expression profiles in specific cells, tissues, and developmental stages in C. elegans. Our ultimate goal is not only to describe detailed gene expression profiles, but also to gain a greater understanding of the organization of gene regulatory networks and to determine how they control cell function during development and differentiation. The use of C. elegans as a platform to investigate the details of gene regulatory networks has several major advantages. Two key advantages are that it is the simplest multicellular organism for which there is a complete sequence (C. elegans Sequencing Consortium 1998), and it is the only multicellular organism for which there is a completely documented cell lineage (Sulston and Horvitz 1977; Sulston et al. 1983). C. elegans is amenable to both forward and reverse genetics (for review, see Riddle et al. 1997). A 2-week life span and generation time of just 3 days for C. elegans allows experimental procedures to be much shorter, more flexible, and more cost-effective compared to the use of mouse or zebrafish models for genomic analyses. Finally, the small size, transparency, and limited cell number of the worm make it possible to observe many complex cellular and developmental processes that cannot easily be observed in more complex organisms. Morphogenesis of organs and tissues can be observed at the level of a single cell (White et al. 1986). As events have shown, investigating the details of C. elegans biology can lead to fundamental observations about human health and biology (Sulston 1976; Hedgecock et al. 1983; Ellis and Horvitz 1986). We are using complementary approaches to examine gene expression in C. elegans. We are constructing transgenic animals containing promoter green fluorescent protein (GFP) fusions of nematode orthologs of human genes. These transgenic animals are examined to determine the time and tissue expression pattern of the promoter::GFP constructs. Concurrently, we are undertaking serial analysis of gene expression (SAGE) on all developmental stages of intact animals and on selected purified cells. Tissues and selected cells are isolated using a fluorescence activated cell sorter (FACS) to sort promoter::GFP marked cell populations. To date we have purified to near homogeneity cell populations for embryonic muscle, gut, and a subset of neurons. The SAGE and promoter::GFP expression data are publicly available at http://elegans.bcgsc.bc.ca.
In eukaryotes, the post-translational addition of methyl groups to histone H3 lysine 4 (H3K4) plays key roles in maintenance and establishment of appropriate gene expression patterns and chromatin states. We report here that an essential locus within chromosome 3L centric heterochromatin encodes the previously uncharacterized Drosophila melanogaster ortholog (dSet1, CG40351) of the Set1 H3K4 histone methyltransferase (HMT). Our results suggest that dSet1 acts as a "global" or general H3K4 diand trimethyl HMT in Drosophila. Levels of H3K4 di-and trimethylation are significantly reduced in dSet1 mutants during late larval and post-larval stages, but not in animals carrying mutations in genes encoding other well-characterized H3K4 HMTs such as trr, trx, and ash1. The latter results suggest that Trr, Trx, and Ash1 may play more specific roles in regulating key cellular targets and pathways and/ or act as global H3K4 HMTs earlier in development. In yeast and mammalian cells, the HMT activity of Set1 proteins is mediated through an evolutionarily conserved protein complex known as Complex of Proteins Associated with Set1 (COMPASS). We present biochemical evidence that dSet1 interacts with members of a putative Drosophila COMPASS complex and genetic evidence that these members are functionally required for H3K4 methylation. Taken together, our results suggest that dSet1 is responsible for the bulk of H3K4 di-and trimethylation throughout Drosophila development, thus providing a model system for better understanding the requirements for and functions of these modifications in metazoans.
Regulatory elements for the mouse growth hormone (GH) gene are located distally in a putative locus control region (LCR) in addition to key elements in the promoter proximal region. The role of promoter DNA methylation for GH gene regulation is not well understood. Pit-1 is a POU transcription factor required for normal pituitary development and obligatory for GH gene expression. In mammals, Pit-1 mutations eliminate GH production resulting in a dwarf phenotype. In this study, dwarf mice illustrated that Pit-1 function was obligatory for GH promoter hypomethylation. By monitoring promoter methylation levels during developmental GH expression we found that the GH promoter became hypomethylated coincident with gene expression. We identified a promoter differentially methylated region (DMR) that was used to characterize a methylation-dependent DNA binding activity. Upon DNA affinity purification using the DMR and nuclear extracts, we identified structural maintenance of chromosomes hinge domain containing -1 (SmcHD1). To better understand the role of SmcHD1 in genome-wide gene expression, we performed microarray analysis and compared changes in gene expression upon reduced levels of SmcHD1 in human cells. Knock-down of SmcHD1 in human embryonic kidney (HEK293) cells revealed a disproportionate number of up-regulated genes were located on the X-chromosome, but also suggested regulation of genes on non-sex chromosomes. Among those, we identified several genes located in the protocadherin β cluster. In addition, we found that imprinted genes in the H19/Igf2 cluster associated with Beckwith-Wiedemann and Silver-Russell syndromes (BWS & SRS) were dysregulated. For the first time using human cells, we showed that SmcHD1 is an important regulator of imprinted and clustered genes.
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