Role of Cysteine Protease Cathepsin L in Maintenance of Epigenetic Histone Modifications and Constitutive Heterochromatin
Open Access
- Author:
- Bulynko, Yaroslava
- Graduate Program:
- Biochemistry and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- September 06, 2006
- Committee Members:
- Sergei A Grigoryev, Committee Chair/Co-Chair
Laura Carrel, Committee Member
Momcilo Miljkovic, Committee Member
Vincent Chau, Committee Member
Judith S Bond, Committee Member - Keywords:
- histone modifications
protease
chromatin - Abstract:
- Cathepsin L (catL) is a lysosomal cysteine protease ubiquitously expressed in mammalian cells. It was recently shown to modulate several important chromatin-binding factors, however its role in regulation of chromatin function remains unclear. One of the potential catL nuclear targets is a developmentally-regulated chromatin-condensing factor MENT (myeloid and erythroid nuclear terminal stage-specific factor). MENT belongs to a serpin (serine protease inhibitors) protein family. In addition to condensing chromatin, MENT acts as a very potent, specific catL inhibitor in vitro. Here I examine the role of this inhibition in living cells, specifically in modulation of epigenetic histone modifications. Using radioactive in vivo protease labeling and ectopic expression of tagged catL, I demonstrate that catL partially localizes in the nucleus and interacts with MENT in living cells. CatL expression alters MENT binding to chromatin, depending on the serpin-protease ratio. Using mutagenesis of MENT and catL active centers, I show that this interaction involves classical serpin-protease binding, which promotes MENT relocation to euchromatin. I also show that inhibition of catL by MENT or complete catL removal by the gene knockout causes destabilization of chromatin epigenetic factors, specifically the localization and level of trimethyl-H3K9 and H2A.Z histone isoforms, as well as spatial distribution of Heterochromatin protein 1 (HP1). H3me3K9 together with HP1 and histone methyltransferases are particularly important for maintenance of constitutive heterochromatin. My work for the first time reveals the control of this key epigenetic circuit by a protease. CatL knockout leads to dramatic loss of H3me3K9 on pericentromeric heterochromatin and Y-chromosome. CatL knockout specifically affects nuclear targeting of Suv39h1, the major enzyme responsible for heterochromatic H3me3K9. Neither DNA methylation nor several other epigenetic markers previously linked to heterochromatin and H3K9 methylation are affected by catL knockout. Thus, I identify the H3me3K9 and Suv39h1 as specific epigenetic targets of catepsin L in the cell nucleus. Based on these results, I conclude that catL is a specific modulator of major epigenetic histone modifications, particularly H3me3K9. Inactivation of catL by expressing a nuclear inhibitor or by gene targeting thus provides potential means for a targeted regulation of chromatin epigenetics without damaging other vital biochemical pathways.