Dale V. Onisk

Date of Award

8-1-1983

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biomedical Sciences

First Advisor

John A. Duerre

Abstract

The histone lysine methyltransferases catalyze the transfer of methyl groups from S-adenosyl-L-methionine to specific ε-N-lysyl residues in the N-terminal regions of histones H3 and H4. These enzymes are located exclusively within the nucleus and are firmly bound to chromatin. The chromosomal bound enzymes do not methylate free or loosely bound histones. However, histones H3 and H4 associated within the nucleosomes are methylated.

The enzymes were extracted by limited digestion (12-16%) of chromosomal DNA from rapidly proliferating rat brain chromatin with micrococcal nuclease. The enzymes were further purified by gel filtration, ammonium sulfate fractionation and DEAE-cellulose chromatography. The histone methyltransferases were resolved into two distinct fractions by Sepharose 6B-100 and DEAE-cellulose chromatography. One enzyme fractionated by DEAE-cellulose chromatograpy was specific for histone H3, while the other enzyme was specific for histone H4.

Histone H3 lysine methyltransferase was shown to methylate only the lysyl residues of chromosomal bound or soluble histone H3. The molar ratio of mono- to di- to trimethyllysine in the soluble system was 1.0:2.1:1.0, while the ratio with chromosomal bound histone H3 was 1.9:1.0:0.08.

The histone H4 lysine methyltransferase which was detectable in the crude nuclease digest, was extremely labile loosing all activity upon further purification. The enzyme specific for histone H4 after DEAE-cellulose chromatography methylated only arginyl residues in histone H4 and would not methylate lysyl residues in histone H4.

The pH optimum for histone H3 lysine methyltransferase with soluble rat brain histone H3 as substrate was 8.5 with little variation from pH 8.2 to 8.7. The pH optimum for histone H4 arginine methyltransferase with soluble histone H4 as the methyl acceptor was 7.5 with little variation from pH 7.3 to 7.8.

After DEAE-cellulose chromatography both enzymes were extremely unstable. Complete removal of DNA by DNAase I digestion resulted in the complete loss of enzyme activity. However, when the enzymes remained associated with DNA fragments they were quite stable. Indicating that the enzymes require DNA for stability and/or activity. The requirement for DNA may only be important to prevent hydrophobic interactions involving the enzyme with itself and/or other non-histone chromosomal proteins.

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