Role of cysteine protease cathepsin L in maintenance of epigenetic histone modifications and constitutive heterochromatin

by Bulynko, Yaroslava A.

iii Cathepsin L is a major lysosomal cysteine protease ubiquitously expressed in mammalian cells. It has been recently shown to proteolytically target several important chromatin remodeling factors, however its role in regulation of chromatin structure and function remains to be understood. One of the potential cathepsin L nuclear targets is a developmentally regulated chromatin-condensing factor MENT (myeloid and erythroid nuclear terminal stagespecific factor). MENT belongs to a serpin (serine and cysteine protease inhibitors) family of proteins. In addition to its ability to condense chromatin, MENT acts as a very potent and specific cathepsin L inhibitor in vitro. Here I examine the role of this inhibition in living cells, specifically in modulation of epigenetic histone modifications at constitutive heterochromatin. Using radioactive in vivo protease active site labeling and ectopic expression of tagged cathepsin L revealed that cathepsin L partially localizes to the nucleus and interacts with nuclear MENT in living cells. In the nucleus, cathepsin L alters MENT binding to chromatin, depending on the serpin-protease ratio. Mutagenesis studies of MENT and cathepsin L active sites show that this interaction involves classical serpinprotease interaction that also promotes MENT relocation to euchromatin. Inhibition of cathepsin L by MENT or complete removal of cathepsin L by the gene knockout (in the absence of MENT) causes destabilization of chromatin epigenetic factors, specifically the localization and level of histone H3K9 trimethylation and the histone isoform H2A.Z, as well as spatial distribution of Heterochromatin protein 1 iv (HP1). Histone H3K9 methylation together with HP1 and histone methyltransferases (HMT) are particularly important for maintenance of constitutive heterochromatin (silent chromatin). This work for the first time reveals the control of this key epigenetic regulatory circuit by a protease. Cathepsin L gene knockout, but not the partial cathepsin L inhibition by MENT, leads to a dramatic loss of H3K9 trimethylation on pericentromeric heterochromatin and Y chromosome. I further show that cathepsin L knockout specifically affects nuclear targeting of Suv39h1, the major HMT responsible for heterochromatic H3K9 trimethylation. Neither DNA methylation nor several other epigenetic markers previously known to be linked to heterochromatin maintenance and H3K9 methylation are affected by cathepsin L knockout. Thus, the H3me3K9 and Suv39h1 have been identified as specific epigenetic targets of cathepsin L in the cell nucleus. Based on these results, I conclude that cathepsin L is a specific modulator of major epigenetic histone modifications, particularly H3K9 methylation. Inactivation of cathepsin L by expression of a nuclear inhibitor or by gene targeting thus provides potential means for targeted regulation of chromatin epigenetics without damaging other vital biochemical pathways in mouse cells. v