Investigating a role for linker histone H1 in quiescent cells of Saccharomyces cerevisiae

Abstract

The aim of this study was to determine whether either Hho1p or Hmo1p can be considered the best candidate to function as linker histone H1 in Saccharomyces cerevisiae. The hypothesis was that yeast linker histone H1 would function to compact chromatin and repress gene transcription in the quiescent cell population that forms during yeast stationary phase. The hho1 and hmo1 deletion mutants were therefore characterised to understand the role of Hmo1p and Hho1p in chromatin function, cellular physiology, and transcription during quiescent cell development in stationary phase. Initial analysis revealed that the hho1 mutant did not show many growth defects during exponential phase. On the other hand, the hmo1 mutant had a longer lag phase and entered stationary phase at a low cell density. Microscopy also revealed that the hmo1 mutant cells had an elongated cell morphology suggesting a role for Hmo1p in cell structure regulation. Stress response tests indicated that during exponential growth, while the hho1 mutant was more sensitive to both acetic acid and hydrogen peroxide, the hmo1 mutant had improved growth on acetic acid. Thus, both mutants displayed distinct phenotypes during exponential phase and in early stationary phase. Chromatin sensitivity assays of exponentially growing cells showed an increase in micrococcal nuclease sensitivity in both mutants, pointing towards roles for both Hmo1p and Hho1p in chromatin compaction in actively growing cells. Interestingly, the level of phosphorylated H2A at serine 129 in the exponential hho1 mutant was higher than wt suggesting this mutant accrued greater DNA damage indicating a probable role for Hho1p in DNA repair. Neither deletion mutant had a large effect on thermotolerance of stationary phase cells. However, overexpression of both Hmo1p and Hho1p yielded cells in stationary phase with increased thermotolerance compared to wt, indicating a protective role for Hmo1p and Hho1p during stationary phase. Together, this supports a role for Hmo1p and Hho1p in influencing chromatin structure in exponential phase and stationary phase cells. Analysis of the quiescent and non-quiescent cell populations formed in stationary phase wt, hho1 and hmo1 mutant cells showed aberrant cell development occurred in both mutants. Less quiescent (Q) cells were formed in the hho1 mutant, and these cells lost viability over time. Furthermore, the viability of the hho1 mutant non-quiescent (NQ) population increased over time. This suggests Hho1p has an anti-apoptotic effect in Q cells, and is pro-apoptotic in NQ cells. In the hmo1 mutant, correct quiescent cell development was delayed with viability of the mutant Q cells taking longer to increase than that seen in wt. Similar to what was seen in hho1, the viability of hmo1 NQ cells increased as stationary phase developed. Thus, both Hho1p and Hmo1p are important for correct Q and NQ cell development during stationary phase, but may play different roles in this process. Global transcriptome analyses revealed different gene expression changes in hho1 and hmo1 mutants. HHO1 did not influence transcription widely in exponential phase cells, although the influence of HHO1 upon transcription increased during growth into SP. Gene repression by both HMO1 and HHO1 increased during quiescent cell development, and co-repression by HMO1 and HHO1 acting on the same genes also increased over time. However, the hmo1 mutant had the greatest defect in gene activation in quiescent cells suggesting the dominant role for Hmo1p in quiescent cells was to promote transcription as opposed to bringing about gene repression, as was the original hypothesis if Hmo1p acted as linker histone H1. The analysis of global occupancy of Hho1p and Hmo1p revealed that occupancy by Hho1p increased as cells grew into stationary phase and showed that genome-wide Hho1p occupancy was greater than Hmo1p occupancy in quiescent cells. By correlating Hmo1p and Hho1p occupancy with the gene transcription changes in the respective mutants, the data showed that the number of genes directly repressed by Hmo1p and Hho1p increased during quiescent cell development. Although the genes subject to direct repression by Hmo1p and Hho1p were largely independent of each other, a few genes were subject to direct co-repression by both Hmo1p and Hho1p, with the greatest number of these directly co-repressed genes evident in quiescent cells. Together, these data suggest Hmo1p and Hho1p have independent and overlapping roles in both the positive and negative regulation of gene transcription as quiescence develops. Thus, neither Hho1p of Hmo1p fulfilled our predicted role for linker histone in yeast which would be to definitively repress gene transcription and compact chromatin in the quiescent cell population. Instead, these data suggest that linker histone H1 function in yeast may be fulfilled by a combination of Hho1p and Hmo1p activity acting at all stages of yeast cell growth.