Date of Award
apoptosis, C. elegans, dre-1/ FBX011, tail-spike cell, lymphoma
In the course of metazoan embryonic and post-embryonic development, more cells are generated than exist in the mature organism, and these cells are deleted by the process of programmed cell death. In addition, cells can be pushed toward death when they accumulate genetic errors, are virally-infected or are otherwise deemed potentially-harmful to the overall organism. Caenorhabditis elegans has proved to be an excellent model system for elucidating the genetic underpinnings of cell death, and research has shown that the core machinery, made up of the egl-1, ced-9, ced-4 and ced-3 genes, is conserved across metazoans, and their homologues are crucial for such diseases as cancer, neurodegeneration and autoimmunity. We used the C. elegans tail-spike cell as a model to uncover dre-1/FBXO11 as a conserved apoptotic regulator that controls tail-spike cell death and also plays a role in human lymphoma. The tail-spike cell is unique among cells fated for programmed cell death in two ways. First, unlike most dying C. elegans cells, the tail-spike cell lives for several hours before its demise, and during this time differentiates. In comparison, most cells die minutes after birth as undifferentiated cells. Second, while tail-spike cell death requires both the CED-3 caspase and CED-4/Apaf-1 adaptor proteins, the BH3-only protein EGL-1 is dispensable. Thus, other gene(s) substitute for egl-1's role as a regulator of caspase activation in this cell, and we set out to identify the relevant gene or genes. A screen for mutants in which the tail-spike cell survives inappropriately yielded a mutant, ns39, in which the ced-3 caspase is transcribed as normal, but fails to become activated. We mapped and cloned this mutant, and found that dre-1 (daf-12-redundant-1) is required for caspase activation in the tail-spike cell. Expression of the dre-1 cDNA in the tail-spike cell rescues the dre-1(ns39) defect in a cell autonomous manner, and expression studies show that dre-1 is expressed in the tail-spike cell. Partial loss-of-function alleles of dre-1, when combined with weak loss-of-function alleles of ced-3 and ced-4, a null allele of egl-1, or a weak gain-of-function allele of ced-9 exhibit a synergistic loss of tailspike cell death. A null allele of ced-9, however, when combined with a strong loss-of-function dre-1 allele, suppresses the tail-spike cell death phenotype of the dre-1(ns39). This epistatic relationship shows that dre-1 acts upstream of, or in parallel to ced-9. These results show that dre-1 has a central role in tail-spike cell death, and are consistent with it acting in place of egl-1 to promote tail-spike cell death. DRE-1 is an F box protein, and we showed via RNAi and genetic experiments that DRE-1 acts in an SCF complex with CUL-1 and SKR-1 to regulate tail-spike cell death, and present evidence that DRE-1 and CED-9 bind to each other in vitro. These results suggest a model in which the dre-1 SCF complex ubiquitinates CED-9 to eliminate its anti-apoptotic function and open the way for the cell to die. A collaboration with Louis Staudt and Lixin Rui at the National Cancer Institute revealed that dre-1Ê¼s human homologue, FBXO11 is mutated or deleted in 5% of germinal center-like diffuse large B cell lymphomas, and that reintroduction of the gene into lymphoma cell lines that have deleted it induces apoptosis. In addition, FBXO11 binds to BCL2 in lymphoma cell lines and induces the degradation of BCL2, and expression of BCL2 rescues the toxicity of FBXO11. Taken together, our results establish dre-1/FBXO11 as a regulator of apoptosis in C. elegans and human lymphoma, and suggest a model in which DRE-1/FBXO11 ubiquitinates and degrades BCL2, a major anti-apoptotic protein.
Chiorazzi, Michael, "DRE-1/FBXO11, A Conserved F Box Protein, Regulates Apoptosis in C. elegans and is Mutated in Human Lymphoma" (2011). Student Theses and Dissertations. 139.