Histone modifications and the HtrA-like serine protease Nma111p regulate apoptosis in budding yeast

Histone modifications and the HtrA-like serine protease Nma111p regulate apoptosis in budding yeast
David Walter, 2010, PhD Thesis, University of Basel
Apoptosis is a form of programmed cell death that plays a central role in development and cellular homeostasis in higher eukaryotes. Knowledge about apoptotic regulation is particularly important for medical research, since apoptotic misregulation is implicated in many human diseases, such as Alzheimer’s and Huntington’s disease, immunodeficiency and cancer. Recent studies have established yeast as model to study the mechanisms of apoptotic regulation. Changes in chromatin configuration are implicated in apoptotic regulation both in yeast and in higher eukaryotes. One mechanism that alters chromatin configuration is the covalent modification of histones, which associate with DNA to form the nucleosome, the fundamental unit of chromatin. In my thesis work, I have identified and characterized distinct interrelated histone modifications on histone H2B and histone H3 as regulators of apoptosis in yeast (Chapter 2 and 3). Histone H2B ubiquitination at lysine K123 by the E3 ligase BRE1 is required in promoting methylation of histone H3 at lysine K4 and K79. These methylations are brought about by the conserved methyltransferases Set1p and Dot1p, respectively. We found that disruption of the E3 ligase BRE1 or the methyltransferase SET1, which causes a lack of histone H2B K123 ubiquitination and histone H3 K4 methylation, respectively, causes metacaspase Yca1p-dependent apoptosis (Chapter 2 and 3). In contrast, we found that disruption of DOT1, which causes a lack of histone H3 K79 methylation confers apoptosis resistance (Chapter 3). Moreover, we found that Dot1p is required for Yca1p-dependent cell death of ∆set1 cells (Chapter 3).
How does disruption of DOT1 confer apoptosis resistance? Yeast cells that fail to methylate histone H3 K79 due to DOT1 disruption exhibit defects in the DNA damage response. Particularly, Dot1p mediated histone H3 K79 methylation is required for Rad9p-dependent checkpoint activation after DNA damage. In higher eukaryotes, the evolutionarily conserved DNA-damage response is a signaling cascade that senses DNA damage and activates cellular responses including apoptosis. Strikingly, we found that Rad9p is required for cell death of ∆set1 similar to Dot1p (Chapter 5), suggesting that Dot1p mediates apoptosis through its function in the DNA-damage response. Thus, we suggest that apoptosis in budding yeast is linked to the DNA damage response similar to apoptosis in higher eukaryotes.
Together, these studies highlight the requirement of Dot1p-mediated histone H3 K79 methylation for an Yca1p-dependent cell death scenario and points to a novel role of the conserved histone H2B/H3 crosstalk in apoptosis regulation. Moreover, our results imply a requirement of the DNA damage response for apoptosis induction in budding yeast.
Another objective of this thesis was the characterization of the functional role of the HtrA1-like serine protease Nma111p in yeast apoptosis (Chapter 4). Nma111p functions as a nuclear serine protease that is necessary for apoptosis under cellular stress conditions. We have examined the role of nuclear protein import in the function of Nma111p in apoptosis. Nma111p contains two small clusters of basic residues toward its amino terminus, both of which are necessary for efficient translocation into the nucleus. Nma111p does not shuttle between the nucleus and cytoplasm during either normal growth conditions or under environmental stresses that induce apoptosis. The amino-terminal half of Nma111p is sufficient to provide the apoptosis-inducing activity of the protein, and both the NLS sequences and catalytic serine 235 are necessary for this function. Together, we provide compelling evidence that intranuclear Nma111p activity is necessary for apoptosis in yeast.