PRB1 is required for clipping of the histone H3 N terminal tail in Saccharomyces cerevisiae

Abstract

Cathepsin L, a lysosomal protein in mouse embryonic stem cells has been shown to clip the histone H3 N- terminus, an activity associated with gene activity during mouse cell development. Glutamate dehydrogenase (GDH) was also identified as histone H3 specific protease in chicken liver, which has been connected to gene expression during aging. In baker's yeast, Saccharomyces cerevisiae, clipping the histone H3 N-terminus has been associated with gene activation in stationary phase but the protease responsible for the yeast histone H3 endopeptidase activity had not been identified. In searching for a yeast histone H3 endopeptidase, we found that yeast vacuolar protein Prb1 is present in the cellular fraction enriched for the H3 N-terminus endopeptidase activity and this endopeptidase activity is lost in the PRB1 deletion mutant (prb1Δ). In addition, like Cathepsin L and GDH, purified Prb1 from yeast cleaves H3 between Lys23 and Ala24 in the N-terminus in vitro as shown by Edman degradation. In conclusion, our data argue that PRB1 is required for clipping of the histone H3 N-terminal tail in Saccharomyces cerevisiae.

Figure 1. Identifying the histone H3 endopeptidase.

(a) Schematic of histone H3 endopeptidase enrichment. Whole cell extract from early stationary phase cells was used to purify the histone H3 endopeptidase. Endopeptidases were purified by chromatography by Anion exchange, Gel filtration and Hydrophobic Interaction. The cleavage activity was measured by western blot with antibody against the H3 C-terminus. (b) H3 endopeptidase assay of the fractions separated by Hydrophobic Interaction Chromatography. The red numbers represent the peaks of cleavage activity (analyzed by Image J). (c) Yeast histone H3 endopeptidases are serine proteases: The H3 endopeptidase activity of Fractions 19, 25 and 35 was assayed with and without protease inhibitors and clipping was assayed by western blot with antibody against the H3 C-terminus. (d) Mass spectrometry analysis of serine protease candidates (Prb1, Ysp3, Prc1) in fractions that include the peaks of endopeptidase activity.

Figure 2. PRB1 is required to cleave histone H3 at its N-terminus in vivo.

(a) PRB1 is essential for the H3 endopeptidase activity in whole cell extracts of early stationary phase cells. Whole cell extracts from WT, prb1Δ, prc1Δ and ysp3Δ were assayed for endopeptidase activity. Clipping was assayed by western blot with antibody against the H3 C-terminus. (b) PRB1 is required for H3 endopeptidase activity in nuclei. Nuclei from WT and prb1Δ cells were assayed for endopeptidase activity. Clipping was assayed by western blot with antibody against the H3 C-terminus. The nuclei gradient is represented by 4 fold dilutions. (c) Endogenous truncated histone H3 level was decreased in the prb1Δ strain. Nuclear fractions were purified from WT, prb1Δ and H3 Q19A, L20A mutant strains in early stationary phase and truncated H3 levels were measured by western blot with antibody against the HA tag.

Figure 3. Purified Prb1 can cleave recombinant histone H3 at its N-terminus in vitro.

(a) Purification of Prb1 from yeast demonstrated by silver staining. Endopeptidase was purified from yeast with or without the PRB1 expression plasmid as described in the Materials and Methods. The protease was separated by SDS-PAGE and stained with silver. (b) Prb1 cleaves recombinant X. laevis histone H3 at its N-terminus in vitro. Purified Prb1 was assayed for endopeptidase activity with recombinant X. laevis histone H3. Purified protease from host strain (without plasmid) was used as control. (c) and (d) Prb1cleaves recombinant S. cerevisiae histone H3 between Lys23/Ala24 and Lys27/Ser28. (c) Recombinant S. cerevisiae histone H3 was incubated with purified Prb1, then H3 was separated by SDS-PAGE, transferred to PVDF and stained with Coomassie blue. The bands labeled with asterisk were excised and subjected to Edman degradation. (d) Results of Edman degradation sequencing. Cycle 1 is a blank run. Cycle 2 is the amino acid standard. Cycles 3–8 represent the six cycles of the clipped H3 corresponding to the N-terminal amino acid. X/Y/Z means that 2 or more residues were identified in each cycle. (X/Y) means that the identification of some residues was uncertain.