Umeå University's logo

Post-translational modifications of histones enable differential transcriptional control of the genome between cell types and developmental stages, and in response to environmental factors. The methylation of Histone 3 Lysine 36 (H₃K₃6) is one the most complex and well-studied histone modifications and is known to be involved in a wide range of molecular processes. Commonly associated with active genes and transcriptional elongation, H₃K₃6 methylation also plays a key role in DNA repair, repression of cryptic transcription, and guiding additional post-translational modifications to histones, genomic DNA, and RNA. In Drosophila melanogaster, trimethylated H₃K₃6 has also been linked to dosage compensation of the single male X chromosome as a binding substrate for the Male-Specific Lethal (MSL) complex. However, this model has been challenged by structural and biochemical studies demonstrating higher MSL complex affinity for other methylated lysines. There is an additional system of chromosome-specific gene regulation in D. melanogaster where transcription from the small heterochromatic fourth chromosome is increased by Painting of fourth (POF), a protein specifically binding nascent RNA on the fourth chromosome. The fourth chromosome is thought to have been an ancestral X chromosome that reverted into an autosome. POF mediating high transcription levels from an autosome is believed to be a remnant of an ancient sex-chromosome dosage compensation mechanism. 

Proximity ligation assays revealed no interaction between MSL complex components and methylated H₃K₃6. This finding was corroborated by RNA sequencing of H₃K₃6 methylation impaired mutants: the transcriptional output of the male X chromosome was unaffected in mutants where Lysine 36 on Histone 3 was replaced by an Arginine, abolishing methylation of this site. However, we found that knocking out Set2, which encodes the methyltransferase responsible for H₃K₃6 trimethylation, significantly reduced X-linked transcription relative to autosomal transcription. This strongly suggests the existence of previously unrecognized alternate Set2 substrates. Interestingly, we also found that Ash1- and NSD-mediated methylation of H₃K₃6 was required to maintain high expression from chromosome four. 

Recent studies have also implicated H₃K₃6 methylation in the silencing of transposon activity in somatic cells. By analyzing the transcription of transposable elements and Piwi-interacting RNAs (piRNAs), we identified dimethylation of H₃K₃6 by Set2 as the main methylation mark involved in this process and showed that dual-stranded piRNA clusters are preferentially activated upon disturbing the methylation machinery. These findings extends the long list of processes dependent on functional H₃K₃6 methylation.