Section: Research

Postdoctoral Position Available

Michael Green, Ph.D.,M.D.

Academic Role: Professor

Faculty Appointment(s) In:
   Program in Gene Function and Expression
   Program in Molecular Medicine

Joint Faculty In:
   Biochemistry and Molecular Pharmacology

Other Affiliation(s):
   Center for AIDS Research
   Interdisciplinary Graduate Program

Eukaryotic Gene Regulation and Cancer Molecular Biology

We have a broad interest in the mechanisms that regulate gene expression in eukaryotes, and the role of gene expression in various human disease states, in particular cancer. We pursue this interest through the use of molecular biology, molecular genetic, and biochemical approaches involving diverse experimental systems that bear on different aspects of gene regulation. Thus, investigators in this laboratory are exposed to a wide range of scientific questions, systems and experimental approaches.

Gene Regulation

Much of eukaryotic gene expression is regulated at the transcriptional level. A central question in the field is how do promoter-specific activator proteins (activators) communicate with the general transcription machinery to stimulate transcription? We study transcription activation both in vitro and in various in vivo systems, including yeast and mammalian cells.

Recently, we have identified a new, vertebrate-specific TATA-box–binding protein-related factor (TRF), which we have named TRF3.  To elucidate TRF3 function we have been using zebrafish as an experimental system (in collaboration with Nathan Lawson). We find that Trf3-depleted zebrafish embryos exhibit multiple developmental defects and fail to undergo hematopoiesis. Using a combination of expression profiling, chromatin immunoprecipitation and epistasis analysis, we have elucidated the pathway by which Trf3 directs hematopoiesis.  We have identified a single Trf3 target gene, mespa, which encodes a transcription factor whose murine orthologue is required for mesoderm specification.  Mespa then binds to the promoter of cdx4, a caudal-type homeobox transcription factor required for hematopoiesis.  Thus, in vertebrates commitment of mesoderm to the hematopoietic lineage occurs through a transcription factor pathway initiated by a TBP-related factor.

In higher eukaryotes gene expression is also regulated at the post-transcriptional level. For example, alternative processing of an mRNA precursor (pre-mRNA) can generate multiple polypeptides from a single gene. 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), which binds to the pre-mRNA and initiates the process of spliceosome assembly. We are also very interested in serine-arginine (SR) proteins, which are general metazoan splicing factors that contain an essential arginine-serine rich (RS) domain. We have found that mammalian spliceosome assembly involves a series of sequential interactions between RS domains and two splicing signals, the branchpoint and 5’ splice site.  The RS domain-splicing signal interaction promotes (or stabilizes) base-pairing between the U snRNA and pre-mRNA substrate, thereby enhancing splicing.

Cancer Molecular Biology

We use transcription-based approaches and functional screens to identify new genes and regulatory pathways involved in cancer, and to address fundamental questions in cancer molecular biology.  We hope these studies will both enrich our understanding of how normal cells become cancerous and reveal potential new targets for therapeutic intervention.  Some of our ongoing projects are described below.

Transcriptional Regulation of Apoptosis.   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 have elucidated a new transcription-based pathway by which deprivation of the growth factor IL-3 results in apoptosis. 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 that is involved in intracellular iron trafficking. 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 apoptosis.  Following intracellular uptake, 24p3 sequesters iron thereby decreasing intracellular iron levels and inducing apoptosis (see Figure). Unexpectedly, we find that the BCR-ABL oncoprotein activates expression of 24p3 and represses expression of 24p3R. Intracellular iron delivery blocks apoptosis resulting from 24p3 addition, IL-3 deprivation or imatinib (Gleevec) 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.

Antiangiogenesis Directed by the p53 Tumor Suppressor Gene.   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 thealpha(II) collagen prolyl-4-hydroxylase [alpha(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 alpha(II)PH selectively inhibits growth of primary human endothelial cells.  When expressed intracellularly or exogenously delivered, alpha(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.

Epigenetic Silencing of Tumor Suppressor Genes.   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 pro-apoptotic 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.  By performing a genome-wide RNA interference (RNAi) screen we have identified 28 genes required for Ras-mediated silencing of Fas that encode cell signalling molecules, chromatin modifiers, transcription factors, components of transcriptional repression complexes, and the DNA methyltransferase DNMT1. 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.

Finally, we are currently performing genome-wide RNAi screens 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.

Office: LRB-628
Phone: 508-856-5330
E-mail: Michael.Green@umassmed.edu
Keywords: RNA Splicing, Cancer Biology, Apoptosis, Gene Expression, Gene Regulation

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Postdoctoral Position Available A postdoctoral position is available to study in this laboratory. Contact Dr. Green for additional details.