Angela J. DiBenedetto (Dr.
Molecular Biology and Genetics
Programmed cell death, or "cell suicide", plays a central role in the development of the nervous system and in the regulation of cell number in adult tissues. It is also a fail-safe cellular response to oncogenic transformation, viral invasion, and damage-inducing agents. To better understand the regulation of cell death, we are characterizing several primary response genes transcriptionally induced in the programmed death of rat PC12 cells and sympathetic neurons deprived of nerve growth factor (NGF), mud1-8 (message upregulated during death 1-8;; Dev.Biol. 188, 322-36). Since the processes of cell death, differentiation, and proliferation are now known to share common regulatory molecules, with crosstalk between control pathways of each, it is likely that the mud genes have in vivo regulatory roles other than a predicted one in cell death. Therefore, we take a three-pronged, longterm approach to the study of mud genes and their potential in vivo functions that includes:
1) Molecular characterization of selected mud genes and test for effects on neuronal cell death. This includes cloning and characterizing all cDNAs for mud genes; analyzing in vivo developmental and tissue RNA and protein expression; and testing the direct involvement and potential signaling pathways of muds in neuronal cell death in neuronal PC12 cells. Although some muds may not be necessary or sufficient for cell death, we believe they nevertheless reflect, directly or indirectly, intracellular events and mechanisms that may be occurring during the apoptotic process in neurons. As such, their functions are informative in their own right. Corollary study: Examining effects of cell cycle regulatory genes and muds on cell death. One hypothesis about cell death is that it results from inappropriate proliferative signals. Several mud genes are known to be induced in cells undergoing active proliferation, and to interact with pro-mitotic transcription factor E2F and possibly anti-mitotic regulator Rb. We are investigating whether similar or contrasting interactions occur in the context of cell death.
2) Survey of cell death paradigms using cloned genes. mud genes are expressed during cell death in vivo in rat sympathetic ganglia. Any muds expressed in other cell death paradigms may be involved in a general mechanism of programmed death and may aid our understanding of how the process is regulated. We superimpose RNA in situ and immunohistochemical studies of mud genes with TUNEL and BrdU studies of apoptotic and mitotic nuclei in various non-neuronal model systems to identify general control factors. The involvement of some mud genes in non-neuronal apoptosis may give us a window into how the death process differs or not in mitotic vs post-mitotic cell contexts.
3) Cloning of zebrafish homolog to rat cell death gene. mud6 is the rat homolog of Drosophila female sterile homeotic (fsh-1), a maternal effect gene involved in segment formation and identity that activates homeotic regulatory loci via chromatin-modifying genes; the human version of this gene is Ring3/brd2, encoding a mitogen activated nuclear kinase that facilitates E2F-mediated gene expression of pro-proliferative genes, presumably by its ability to bind chromatin via conserved bromodomain motifs, and perhaps bring in some modifying functions. The known activities of these species homologs provide clues as to rat mud6 function in vertebrate development in general, and in neuronal cell death in particular. To study the various implicated roles of this sequence in a vertebrate system amenable to both genetic and early embryonic analysis, we cloned the zebrafish homolog of mud6/fsh/Ring3/brd2, zbrd2, and established wild type RNA expression patterns in zfish embryos. Currently, we are examining the expression pattern of zBRD2 protein, and testing the effects of knockout and overexpression in zebrafish embryos. We are also looking for protein interactors by yeast two hybrid analysis. By analogy with species homologs, we will look for zbrd2 interaction with zebrafish homologs of Drosophila genes known to interact with fsh, and for mutant effects on cell death, proliferation, or homeotic transformation in developing zebrafish. Such genetic and biochemical analyses of gene orthologs provide the parallel studies across several species necessary to distinguish primary conserved functions from later, specific and derived functions, and help illustrate the adaptive changes of particular gene sequences through evolutionary time.
I. Refereed journals:
DiBenedetto, A.J., Lakich, D., Kruger, W., Belote, J., Baker, B.S., and Wolfner, M.F. 1987. Sequences expressed sex-specifically in Drosophila melanogaster adults. Developmental Biology 119: 242-251.
DiBenedetto, A.J., Harada, H.A., and Wolfner, M.F. 1990. Structure, cell-specific expression, and mating-induced regulation of a Drosophila melanogaster male accessory gland gene. Developmental Biology 139: 134-148.
Kalb, J.M., DiBenedetto, A.J. and Wolfner, M.F. 1993. Probing the function of Drosophila melanogaster accessory glands by directed cell ablation. Proceedings of the National Academy of Science, USA 90(17): 8093-8097.
Pittman, R,N,, Wang, S., DiBenedetto, A.J. and Mills, J.C. 1993. A system for characterizing cellular and molecular events in programmed neuronal cell death. Journal of Neuroscience 13: 3669-3680.
Wang S, DiBenedetto AJ, Pittman RN. 1997. Genes induced in programmed cell death of neuronal PC12 cells and developing sympathetic neurons in vivo. Developmental Biology 188: 322-36.
Erhardt, J.A., Hynicka, W., DiBenedetto, A., Shen, N., Stone, N., Paulson, H., and Pittman, R.N. 1998. A novel F box protein, NFB42, is highly enriched in neurons and induces growth arrest. Journal of Biological Chemistry 273: 35222-7.
DiBenedetto, A.J., Klick Stoddard, J., and Glavan, B.J. 2001. Cloning and molecular characterization of a novel gene encoding a WD-repeat protein epxressed in restricted areas of adult rat brain. Gene 271: 21-31.
DiBenedetto, A.J., Guinto, J.B., Ebert, T.D., and Bee, K.J. Cloning and characterization of zebrafish homologs of brd2, a member of the highly conserved BET family of transcriptional regulators.
DiBenedetto, A.J., St. John, A., and Pittman, R.N. BWD, a brain-enriched WD-40 repeat protein, intereacts with STAT3.
II. Reviews, Book Chapters.
DiBenedetto, A.J. and Pittman, R.N. 1996. Death in the Balance. Perspectives in Developmental Neurobiology 3(2): 109-117.
Pittman, R.N. and DiBenedetto, A.J. 1996. "Apoptosis of undifferentiated and terminally differentiated PC12 cells." In: Cellular Aging and Cell Death. (Holbrook, J.J., Martin, G.R. and Lockshin, R.A., eds.). John Wiley and Sons, Inc. New York, NY, pp. 255-265.
Bowden, T.S and DiBenedetto, A.J. 2001. Information literacy in a biology laboratory session: an example of librarian-faculty collaboration. Research Strategies 18: 143-149.