Posttranslational histone modifications and histone variants form a unique 
epigenetic landscape on mammalian chromosomes where the principal epigenetic 
heterochromatin markers, trimethylated histone H3(K9) and the histone H2A.Z, are 
inversely localized in relation to each other. Trimethylated H3(K9) marks 
pericentromeric constitutive heterochromatin and the male Y chromosome, while 
H2A.Z is dramatically reduced at these chromosomal locations. Inactivation of a 
lysosomal and nuclear protease, cathepsin L, causes a global redistribution of 
epigenetic markers. In cathepsin L knockout cells, the levels of trimethylated 
H3(K9) decrease dramatically, concomitant with its relocation away from 
heterochromatin, and H2A.Z becomes enriched at pericentromeric heterochromatin 
and the Y chromosome. This change is also associated with global relocation of 
heterochromatin protein HP1 and histone H3 methyltransferase Suv39h1 away from 
constitutive heterochromatin; however, it does not affect DNA methylation or 
chromosome segregation, phenotypes commonly associated with impaired histone 
H3(K9) methylation. Therefore, the key constitutive heterochromatin determinants 
can dynamically redistribute depending on physiological context but still 
maintain the essential function(s) of chromosomes. Thus, our data show that 
cathepsin L stabilizes epigenetic heterochromatin markers on pericentromeric 
heterochromatin and the Y chromosome through a novel mechanism that does not 
involve DNA methylation or affect heterochromatin structure and operates on both 
somatic and sex chromosomes.