Control of chromatin structure by spt6: different consequences in coding and regulatory regions.

Spt6 is a highly conserved factor required for normal transcription and chromatin structure. To gain new insights into the roles of Spt6, we measured nucleosome occupancy along Saccharomyces cerevisiae chromosome III in an spt6 mutant. We found that the level of nucleosomes is greatly reduced across some, but not all, coding regions in an spt6 mutant, with nucleosome loss preferentially occurring over highly transcribed genes. This result provides strong support for recent studies that have suggested that transcription at low levels does not displace nucleosomes, while transcription at high levels does, and adds the idea that Spt6 is required for restoration of nucleosomes at the highly transcribed genes. Unexpectedly, our studies have also suggested that the spt6 effects on nucleosome levels across coding regions do not cause the spt6 effects on mRNA levels, suggesting that the role of Spt6 across coding regions is separate from its role in transcriptional regulation. In the case of the CHA1 gene, regulation by Spt6 likely occurs by controlling the position of the +1 nucleosome. These results, along with previous studies, suggest that Spt6 regulates transcription by controlling chromatin structure over regulatory regions, and its effects on nucleosome levels over coding regions likely serve an independent function.

Anti-tumor therapeutic molecules that target the programmed cell death machinery.

Apoptosis is a process that governs the elimination of unwanted, damaged, or infected cells in most organisms. Defects in its execution are associated with several diseases, including cancer. Herein, we discuss novel molecules with potential anti-tumor activity that target components of the apoptotic machinery, specifically Bcl-2 proteins, IAPs and caspases.

Viruses activate a genetically conserved cell death pathway in a unicellular organism.

Given the importance of apoptosis in the pathogenesis of virus infections in mammals, we investigated the possibility that unicellular organisms also respond to viral pathogens by activating programmed cell death. The M1 and M2 killer viruses of Saccharomyces cerevisiae encode pore-forming toxins that were assumed to kill uninfected yeast cells by a nonprogrammed assault. However, we found that yeast persistently infected with these killer viruses induce a programmed suicide pathway in uninfected (nonself) yeast. The M1 virus-encoded K1 toxin is primarily but not solely responsible for triggering the death pathway. Cell death is mediated by the mitochondrial fission factor Dnm1/Drp1, the K+ channel Tok1, and the yeast metacaspase Yca1/Mca1 encoded by the target cell and conserved in mammals. In contrast, cell death is inhibited by yeast Fis1, a pore-forming outer mitochondrial membrane protein. This virus-host relationship in yeast resembles that of pathogenic human viruses that persist in their infected host cells but trigger programmed death of uninfected cells.

Proapoptotic N-truncated BCL-xL protein activates endogenous mitochondrial channels in living synaptic terminals.

Neuronal death is often preceded by functional alterations at nerve terminals. Anti- and proapoptotic BCL-2 family proteins not only regulate the neuronal death pathway but also affect excitability of healthy neurons. We found that exposure of squid stellate ganglia to hypoxia, a death stimulus for neurons, causes a cysteine protease-dependent loss of full-length antiapoptotic BCL-xL, similar to previous findings in mammalian cells. Therefore, to determine the direct effect of the naturally occurring proapoptotic cleavage product of BCL-xL on mitochondria, recombinant N-truncated BCL-xL was applied to mitochondria inside the squid presynaptic terminal and to purified mitochondria isolated from yeast. N-truncated BCL-xL rapidly induced large multi-conductance channels with a maximal conductance significantly larger than those produced by full-length BCL-xL. This activity required the hydrophobic C terminus and the BH3 domain of BCL-xL. Moreover, N-truncated BCL-xL failed to produce any channel activity when applied to plasma membranes, suggesting that a component of the mitochondrial membrane is necessary for its actions. Consistent with this idea, the large channels induced by N-truncated BCL-xL are inhibited by NADH and require the presence of VDAC, a voltage-dependent anion channel present in the outer mitochondrial membrane. These observations suggest that the mitochondrial channels specific to full-length and N-truncated BCL-xL contribute to their opposite effects on synaptic transmission, and are consistent with their opposite effects on the cell death pathway.

BAD is a pro-survival factor prior to activation of its pro-apoptotic function.

The mammalian BAD protein belongs to the BH3-only subgroup of the BCL-2 family. In contrast to its known pro-apoptotic function, we found that endogenous and overexpressed BAD(L) can inhibit cell death in neurons and other cell types. Several mechanisms regulate the conversion of BAD from an anti-death to a pro-death factor, including alternative splicing that produces the N-terminally truncated BAD(S). In addition, caspases convert BAD(L) into a pro-death fragment that resembles the short splice variant. The caspase site that is selectively cleaved during cell death following growth factor (interleukin-3) withdrawal is conserved between human and murine BAD. A second cleavage site that is required for murine BAD to promote death following Sindbis virus infection, gamma-irradiation, and staurosporine treatment is not conserved in human BAD, consistent with the inability of human BAD to promote death with these stimuli. However, loss of the BAD N terminus by any mechanism is not always sufficient to activate its pro-death activity, suggesting that the N terminus is a regulatory domain rather than an anti-death domain. These findings suggest that BAD is more than an inert death factor in healthy cells; it is also a pro-survival factor, prior to its role in promoting cell death.

Regulation of cell death in the lymphoid system by Bcl-2 family proteins.

Programmed cell death is an ordered process that is essential for the normal development and homeostasis of an organism. Dysregulation of this programmed pathway, resulting in either excess cell numbers or unscheduled cell death, underlies a number of disease states. Bcl-2 family proteins play a key role in regulating cell death and survival, and a number of studies have demonstrated their role as important regulators of cell fate in the lymphoid system. Mice that are genetically deficient or overexpress various Bcl-2 family proteins have provided important clues regarding their roles in lymphocyte development, progression of lymphoid tumors and analogous human disorders. In addition, lymphotropic viruses may trigger cell proliferation and inhibit cell death with the help of their own Bcl-2 homologues. Comparing the shared and distinct functions of viral and cellular Bcl-2-related proteins yields new insight into their fundamental mechanisms.