Clr4 specificity and catalytic activity beyond H3K9 methylation

Area of Science:

• Epigenetics and Gene Regulation
• Protein Biochemistry
• Yeast Molecular Biology

Background:

• The protein lysine methyltransferase (PKMT) Clr4 is crucial for heterochromatin formation in fission yeast.
• Clr4 catalyzes histone 3 lysine 9 (H3K9) di- and tri-methylation and also methylates Mlo3.
• Understanding Clr4's substrate specificity is key to elucidating its role in epigenetic regulation.

Purpose of the Study:

• To investigate the substrate specificity of the fission yeast PKMT Clr4.
• To identify novel methylation sites and substrates of Clr4 in *S. pombe*.
• To develop a quantitative method for comparing PKMT specificity profiles.

Main Methods:

• Custom-made mutational scanning peptide arrays were used to determine Clr4 substrate recognition.
• In vitro methyltransferase assays were employed to identify and validate novel methylation sites.
• Specificity profiles were quantitatively compared to human homologs.

Main Results:

• Clr4 recognizes an RK core motif with a preference for R8 and specific contacts at H3 positions S10, T11, G12, and G13.
• Eleven putative methylation sites were identified in *S. pombe* proteins, with Mlo3 and seven novel sites confirmed.
• Strongest methylation signals were observed on Spbc28F2.11 (lysine 292) and Hrp3 (lysine 89).
• The specificity profile of Clr4 closely resembles that of human SUV39H2.

Conclusions:

• Clr4 methylates additional substrates beyond H3 and Mlo3, expanding its known functions in heterochromatin.
• The identified novel methylation sites warrant further investigation into their biological significance.
• The developed method for comparing PKMT specificity profiles can be applied to other enzyme families.