Michael R. Green received his MD and PhD degrees from Washington University School of Medicine in 1981. He was awarded a Helen Hay Whitney Postdoctoral Fellowship to perform postdoctoral work at Harvard University in the Department of Biochemistry and Molecular Biology. He became a faculty member in that department at Harvard in 1984, where he remained until he joined the Program in Molecular Medicine at the University of Massachusetts Medical School in 1990. He has been the recipient of the Searle Scholar Award, the Presidential Young Investigators Award, the McKnight Neuroscience Award, and in 1993 was invited to deliver a Harvey Lecture. In 1994 Dr. Green was made an Investigator of the Howard Hughes Medical Institute.
Eukaryotic Gene Regulation and Cancer Molecular Biology
We are interested in the mechanisms that regulate gene expression in eukaryotes, and the role of gene expression in various human disease states. To pursue these interests we use transcription-based approaches and functional screens to identify new genes and regulatory pathways involved in cancer. These studies are intended to enhance our understanding of how normal cells become cancerous and reveal potential new targets for therapeutic intervention.
Much of eukaryotic gene expression is regulated at the transcriptional level through interactions between promoter-specific activator proteins (activators) and the general transcription machinery. 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 used 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 results in a developmental defect strikingly similar to that induced by Trf3 depletion. Injection of mespa mRNA restores 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 fail 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.
Transcriptional regulation also plays a key role in modulating expression of genes involved in tumorigenesis. For example, in many instances, inactivation of genes critical for cancer development (tumor suppressor genes) 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 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 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.
In higher eukaryotes gene expression is also regulated at the post-transcriptional level. We have a long-standing interest in the mechanisms involved in splicing of messenger RNA precursors (pre-mRNAs). Splicing occurs in a large multi-subunit complex, the spliceosome, the formation of which is dependent upon multiple proteins and small nuclear ribonucleoprotein proteins (snRNPs). We are particularly interested in splicing factors that act early during spliceosome assembly; these factors play a critical role in defining splice sites and are targets for splicing regulators. One such factor that we originally identified and continue to study is U2 snRNP Auxiliary Factor (U2AF), a heterodimer, comprised of large (U2AF65) and small (U2AF35) subunits, which binds to the pre-mRNA and initiates the process of spliceosome assembly.
Using a yeast two-hybrid screen, we identified a human 56 kDa DExD/H-box protein that interacts with U2AF65 [U2AF65-Associated Protein (hUAP56)]. Recently, we have used a series of hUAP56 mutants that are defective for ATP-binding, ATP hydrolysis or double-stranded RNA (dsRNA) unwindase/helicase activity, to assess the relative contributions of these biochemical functions to pre-mRNA splicing. We found that pre-spliceosome assembly requires hUAP56's ATP-binding and ATPase activities, which, unexpectedly, are required for hUAP56 to interact with U2AF65 and be recruited into splicing complexes. Surprisingly, hUAP56 is also required for mature spliceosome assembly, which requires, in addition to the ATP-binding and ATPase activities, hUAP56's dsRNA unwindase/helicase activity. We demonstrated that hUAP56 directly contacts U4 and U6 snRNAs and can promote unwinding of the U4/U6 duplex, and that both these activities are dependent upon U2AF65. Our results indicate that hUAP56 first interacts with U2AF65 in an ATP-dependent manner, and subsequently with U4/U6 snRNAs to facilitate stepwise assembly of the spliceosome.
Another group of proteins that are required for spliceosome assembly are serine-arginine (SR) proteins, a family of 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 analyzed the role of SR proteins in splicing of U12-type introns and in the second step of U2-type intron splicing. We found that RS domains also contact the branchpoint and 5' splice site of a U12-type intron. On a U2-type intron, 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.
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. We used expression cloning to isolate 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 found 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 acquisition of apoptotic resistance is one of the hallmarks of cancer. Paradoxically, however, expression of an oncogene in a primary cell can induce apoptosis or senescence, and thus block cellular proliferation through pathways that remain to be elucidated. We have performed genome-wide RNAi screening to identify 17 genes required for an activated BRAF oncogene (BRAFV600E) to block proliferation of human primary fibroblasts and melanocytes. Surprisingly, we found that a secreted protein, IGFBP7, has a central role in BRAFV600E-mediated senescence and apoptosis. Expression of BRAFV600E in primary cells leads to synthesis and secretion of IGFBP7, which acts through autocrine/paracrine pathways to inhibit BRAF-MEK-ERK signaling and induce senescence and apoptosis. Apoptosis results from IGFBP7-mediated up-regulation of BNIP3L, a pro-apoptotic BCL2 family protein. Recombinant IGFBP7 (rIGFBP7) induces apoptosis in BRAFV600E-positive human melanoma cell lines, and systemically administered rIGFBP7 markedly suppresses growth of BRAFV600E-positive tumors in xenografted mice. Immunohistochemical analysis of human skin, nevi and melanoma samples implicates loss of IGFBP7 expression as a critical step in melanoma genesis.
We have also used genome-wide RNAi screens to identify new metastasis suppressors genes that inhibit one or more steps required for metastasis without affecting primary tumor formation. Following expression in weakly metastatic B16-F0 mouse melanoma cells, small hairpin RNAs (shRNAs) were selected based upon enhanced satellite colony formation in a three-dimensional cell culture system and confirmed in a mouse experimental metastasis assay. Using this approach we discovered 22 genes whose knockdown increases metastasis without affecting primary tumor growth. We focused on one of these genes, Gas1, because we found that it is substantially down-regulated in highly metastatic B16-F10 melanoma cells, which contributes to the high metastatic potential of this mouse cell line. We further demonstrated that Gas1 has all the expected properties of a melanoma tumor suppressor including: suppression of metastasis in a spontaneous metastasis assay, promotion of apoptosis following dissemination of cells to secondary sites, and frequent down-regulation in human melanoma metastasis-derived cell lines and metastatic tumor samples. Thus, we have developed a genome-wide shRNA screening strategy that enables the discovery of new metastasis suppressor genes. to identify factors involved in oncogene-induced senescence and apoptosis, factors required for cancer cell survival, factors that mediate the induction of apoptosis by chemotherapeutic agents, new tumor suppressor genes and genes that regulate metastasis.
The lipocalin mouse 24p3 has been implicated in diverse physiological processes, including apoptosis due to interleukin-3 (IL-3) deprivation and iron transport. 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 either iron uptake or apoptosis, dependent upon the iron content of the ligand: iron-loaded 24p3 (holo-24p3) increases intracellular iron concentration withough promoting apoptosis; iron-lacking 24p3 (apo-24p3) decreases intracellular iron levels, which induces expression of the proapoptotic protein Bim, resulting in apoptosis.