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Eukaryotic Gene Regulation
Summary: Michael Green is interested in the mechanisms that regulate gene expression in eukaryotes, and the role of gene expression in various human disease states. To pursue his interests, he uses molecular biology, molecular genetic, and biochemical approaches. His studies involve diverse experimental systems that bear on different aspects of gene regulation.
Transcriptional Regulation
The general transcription factor TFIID comprises the TATA-box–binding protein (TBP) and a set of highly conserved associated factors (TAFs). We have identified a new, vertebrate-specific TBP-related factor (TRF) that we have named TRF3. To elucidate TRF3 function we have been using zebrafish embryos as an experimental system (in collaboration with Nathan Lawson, University of Massachusetts Medical School). Zebrafish embryos depleted of Trf3 exhibit multiple developmental defects and fail to undergo hematopoiesis. Expression profiling for Trf3-dependent genes identified mespa, which encodes a transcription factor whose murine ortholog is required for mesoderm specification; chromatin immunoprecipitation verified that Trf3 binds to the mespa promoter. Depletion of Mespa resulted in a developmental defect strikingly similar to that induced by Trf3 depletion. Injection of mespa mRNA restored normal development to a Trf3-depleted embryo, indicating mespa is the single Trf3 target gene required for zebrafish embryogenesis. Zebrafish embryos depleted of Trf3 or Mespa also failed to express cdx4, a caudal-related gene required for hematopoiesis. MespA binds to the cdx4 promoter, and epistasis analysis revealed an ordered trf3-mespa-cdx4 pathway. Thus, in vertebrates, commitment of mesoderm to the hematopoietic lineage occurs through a transcription factor pathway initiated by a TBP-related factor.
RNA Processing and Gene Regulation
We have a long-standing interest in the mechanisms involved in splicing of messenger RNA precursors (pre-mRNAs). Serine-arginine (SR) proteins are general metazoan splicing factors that contain an essential arginine-serine–rich (RS) domain. We have previously found that on typical U2-type introns, mammalian spliceosome assembly involves a series of sequential interactions between RS domains and two splicing signals, the branchpoint and 5' splice site, which promote base-pairing with U small nuclear RNAs (snRNAs).
More recently, we have analyzed the role of SR proteins in splicing of U12-type introns and in the second step of U2-type intron splicing. We find that RS domains also contact the branchpoint and 5' splice site of a U12-type intron. On a U2-type intron, we find that the RS domain contacts the pre-mRNA at the site of the U6 snRNA–5' splice site interaction during the first step of splicing and, unexpectedly, then shifts to contact the pre-mRNA at the site of the U5 snRNA–exon 1 interaction during the second step. Our results reveal alternative interactions between the RS domain and the 5' splice site region that facilitate remodeling of the spliceosome between the two steps of splicing.
Gene Regulation and Cancer Molecular Biology
Programmed cell death (apoptosis) is a critical aspect of both the genesis and treatment of cancer. There is substantial evidence that certain types of apoptosis may be transcriptionally regulated and that there are transcriptionally activated genes whose products induce cell death. We are using a variety of experimental systems to identify transcriptionally regulated death-inducing genes and new apoptotic pathways.
Using DNA microarrays to analyze interleukin-3 (IL-3)-dependent murine FL5.12 pro–B cells, we found that the gene undergoing maximal transcriptional induction following cytokine withdrawal is 24p3, which encodes a secreted lipocalin. Addition of 24p3 induces apoptosis in a variety of lymphoid cells. 24p3 has also been implicated in other physiological responses, including iron uptake and differentiation. The cell surface receptor for 24p3 has not been identified, nor is it known whether the different responses elicited by 24p3 result from a single or multiple receptors. We have isolated by expression cloning a complementary DNA encoding a 24p3 cell surface receptor (24p3R). Ectopic 24p3R expression confers on cells the ability to undergo 24p3-dependent iron uptake or apoptosis. The differential response is controlled by the iron status of 24p3: iron-loaded 24p3 increases intracellular iron concentration without promoting apoptosis; iron-lacking 24p3 decreases intracellular iron levels, which induces Bim, a proapoptotic BCL-2 protein, resulting in apoptosis. Unexpectedly, we find that the BCR-ABL oncoprotein activates expression of 24p3 and represses expression of 24p3R. The down-regulation of 24p3R renders BCR-ABL+ cells refractory to the secreted 24p3. Intracellular iron delivery blocks apoptosis resulting from 24p3 addition, IL-3 deprivation, or imatinib treatment of BCR-ABL–transformed cells. Our results reveal an unanticipated role of intracellular iron regulation in an apoptotic pathway relevant to BCR-ABL–induced myeloproliferative disease and its treatment.
The conversion of a normal cell to a cancer cell is a stepwise process that typically involves the activation of oncogenes and inactivation of tumor-suppressor and proapoptotic genes. In many instances, inactivation of genes critical for cancer development occurs by epigenetic silencing that often involves hypermethylation of CpG-rich promoter regions. Whether silencing occurs by random acquisition of epigenetic marks that confer a selective growth advantage, or through a specific pathway initiated by an oncogene, remains to be determined. To address this question, we have performed a genome-wide RNA interference (RNAi) screen to identify genes required for Ras-mediated epigenetic silencing of the proapoptotic Fas gene. Using K-ras–transformed NIH 3T3 cells, we have identified 28 genes required for Ras-mediated silencing of Fas that encode cell signaling molecules, chromatin modifiers, transcription factors, components of transcriptional repression complexes, and the DNA methyltransferase DNMT1. At least nine of these Ras epigenetic silencing effectors (RESEs), including DNMT1, are directly associated with specific regions of the Fas promoter in K-ras–transformed NIH 3T3 cells but not in untransformed NIH 3T3 cells. RNAi-mediated knockdown of any of the 28 RESEs results in failure to recruit DNMT1 to the Fas promoter, loss of Fas promoter hypermethylation, and derepression of Fas expression. Analysis of five other epigenetically repressed genes indicates that Ras directs silencing of multiple, unrelated genes through a largely common pathway. Finally, we have shown that nine RESEs are required for anchorage-independent growth and tumorigenicity of K-ras–transformed NIH 3T3 cells; these nine genes have not been previously implicated in transformation by Ras. Our results demonstrate that Ras-mediated epigenetic silencing occurs through a specific, unexpectedly complex pathway involving components that are required for maintenance of a fully transformed phenotype.
Recent evidence suggests that antiangiogenic therapy is sensitive to p53 status in tumors, implicating a role for p53 in the regulation of angiogenesis. We have found that p53 transcriptionally activates the α(II) collagen prolyl-4-hydroxylase [α(II)PH] gene, resulting in the extracellular release of antiangiogenic fragments of collagen type 4 and 18. Conditioned media from cells ectopically expressing either p53 or α(II)PH selectively inhibits growth of primary human endothelial cells. When expressed intracellularly or exogenously delivered, α(II)PH significantly inhibits tumor growth in mice. Our results reveal a genetic and biochemical linkage between the p53 tumor-suppressor pathway and the biosynthesis of antiangiogenic collagen fragments.
Metastasis-suppressor genes encode proteins that inhibit one or more steps required for metastasis without affecting primary tumor formation. We have developed a genome-wide RNAi screening strategy to identify new metastasis-suppressor genes. Following expression in poorly metastatic B16-F0 mouse melanoma cells, candidate shRNAs were selected based upon enhanced formation of satellite colonies in a three-dimensional cell culture system. Individual B16-F0 knockdown cell lines were then tested in a mouse tail vein injection assay for their ability to promote lung metastasis. Using this approach, we discovered 22 genes that suppress metastasis without affecting primary tumor growth. Cancer-profiling database mining reveals that five of these genes are significantly down-regulated in metastatic melanoma, and 15 are down-regulated in metastases of multiple tumor types. Thus, the genome-wide shRNA screen we have devised reveals genes that, on the basis of both experimental and clinical evidence, are new metastasis suppressors.
Grants from the National Institutes of Health provided partial support for the projects described above.
Last updated: November 3, 2008
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