A rapidly growing literature demonstrates that cells undergo dramatic developmentally-regulated changes in gene expression and cellular morphology upon transition from undifferentiated stem cells to specific lineages (Giadrossi et al., 2007
; Kim et al., 2008
; Murry and Keller, 2008
). Despite remarkable progress made in documenting epigenetic signatures such as “bivalent domains” i.e. an H3 tail bearing both H3K4me3 and H3K27me3 marks (Bernstein et al., 2006a
), little is known as to what mechanisms may function to bring about the above changes at a chromatin level. Our data suggest that mouse ESCs employ a novel, regulated histone H3 proteolysis mechanism that may serve to alter epigenetic signatures upon differentiation. Other means of actively removing histone methyl marks have been well documented, including enzymatic demethylation (Anand and Marmorstein, 2007
; Shi et al., 2004
) and selective histone variant replacement (Ahmad and Henikoff, 2002
). However, considerably less evidence documenting endogenous histone proteolysis has been reported (Allis et al., 1980
; Falk et al., 1990
). The identification and characterization of developmentally-regulated H3 cleavage by Cathepsin L during ESC differentiation is an important step in understanding limited nuclear histone proteolysis as a potential mode of transcriptional regulation.
Our finding that Cathepsin L cleaves histone H3 in mouse ES cells was unexpected as this enzyme was originally described as a lysosomal protease (Barrett et al., 2003
). However, Cathepsin L has been shown to localize to nuclei where it plays a role in the proteolytic processing of transcription factor CDP/Cux (Goulet et al., 2004
); biochemical studies have also shown that pro-Cathepsin L is localized to the nucleus in ras
-transformed mouse fibroblasts (Hiwasa and Sakiyama, 1996
). In addition, evidence of endogenous, nuclear serpin inhibitors with the ability to inhibit Cathepsin L activity, e.g. MENT (Bulynko et al., 2006
) and Cystatin B (Riccio et al., 2001
), further supports the notion that Cathepsin L, and potentially other cysteine proteases, play important but poorly understood roles in regulated nuclear proteolysis. As far as we are aware, transcription factor CDP/Cux is the only nuclear substrate of Cathepsin L identified to date, and histones have not yet been identified as substrates of this class of proteases in mammalian cells. Interestingly, however, recent studies in sea urchin have suggested that a Cathepsin L-like cysteine protease may be responsible for the degradation of sperm histones during a key chromatin remodeling event after fertlization (Morin et al., 2008
). Our data support other studies that demonstrate the nuclear localization of Cathepsin L and provide the first indication that cellular histones, H3 in particular, are key substrates of this family of proteases in mammalian cells.
Our finding that Cathepsin L is an H3 protease is interesting when considering the phenotype common to the Cathepsin L knockout mouse (Nakagawa et al., 1998
) and the Cathepsin L mutant mouse, furless
(Roth et al., 2000
). These mice exhibit periodic hair loss due to the improper cycling and morphogenesis of their hair follicles, suggesting a defect in stem cell renewal and/or differentiation. Cathepsin L knockout mice are viable and fertile, however, indicating that its functions are nonessential and/or redundant. Interestingly, Cathepsin L/Cathepsin B double knockout mice exhibit severe brain atrophy and die two to four weeks after birth (Felbor et al., 2002
). The severity and selectivity of this phenotype suggests that these two enzymes overlap in their specific functions. Although we do not observe significant inhibition of Cathepsin B upon in vivo
chemical inhibition of Cathepsin L, nor do we observe the same pattern of H3 cleavage with recombinant Cathepsin B compared to Cathepsin L in vitro
, we cannot exclude the possibility that redundancy in H3 cleavage function may exist between these or other related enzymes at other stages or lineages of differentiation.
Limited proteolysis of nuclear proteins is an important means of regulating transcription and other cell processes (Goulet and Nepveu, 2004
; Vogel and Kristie, 2006
). Here we show that limited proteolysis of histone H3 by Cathepsin L occurs during the differentiation of ESCs and may be regulated both positively and negatively by covalent modifications on the H3 tail itself. These findings require a revaluation as to the function of this family of enzymes in transcriptional and epigenetic regulation; many important questions remain. First, how is the protease cleavage activity regulated? Along this line, we show that covalent modifications (such as acetylation and/or methylation of nearby lysines) serve to regulate, positively and negatively, the H3 proteolytic processing event. We note that proteolytic processing of H3 in the ciliate model occurs selectively in a hypoacetylated, transcriptionally silent (micronuclear) genome, while processing of H3 fails to occur in hyperacetylated, transcriptionally active macronuclei (Allis et al., 1980
). These data raise the question as to whether other chromatin-modifying enzyme complexes, such as HATs, HDACs, or ubiquitinating enzymes, play a role in regulating histone proteolysis.
Second, by what mechanism is the cleaved H3 replaced, and is DNA replication and chromatin assembly required? Our findings that the histone variant H3.2 is preferably cleaved as compared to H3.3 suggests that S-phase/replication-coupled replacement may be involved (Loyola and Almouzni, 2007
). Along this line, we note that proteolytic processing of transcription factor CDP/Cux by Cathepsin L occurs during the G1/S-phase transition and is coupled to cell cycle progression (Goulet et al., 2004
). Whether proteolytic processing of H3 is cell cycle dependent remains unclear, although it is interesting to note that our studies were done in ESCs, which cycle rapidly and spend a high percentage of time in S-phase as measured by flow cytometry (data not shown).
Third, and more broadly, does this proteolytic mechanism apply to other histone substrates or to other cathepsin family members or cathepsin-like proteases, and is the general mechanism conserved in other organisms or operating in other stages of development or differentiation? Although Cathepsin L is the only member of this family shown to localize to the nucleus, the conservation in genomic structure and sequence between this protease and its related family members suggest that other cathepsins may function similarly.
Finally, with other mechanisms of histone demethylation well documented (i.e. enzymatic demethylation, variant replacement, etc.), does histone proteolysis serve other purposes that are not appreciated, such as the generation of new N-termini? Alternatively, histone cleavage may remove critical recognition elements and thereby block the binding of downstream effectors (see ). Such questions will be the aims of future studies.