Reporting in Molecular Cell, Ezhkova and Tansey have taken our understanding of chromatin-modification patterns one step further, by showing that proteasomal ATPases connect histone ubiquitylation to histone methylation.

Covalent histone modifications, including acetylation, methylation, phosphorylation and ubiquitylation, represent a 'histone code' that controls the transcriptional status of genes. In Saccharomyces cerevisiae, the chromatin of transcriptionally active genes is methylated on histone H3 at lysine residues K4 (H3-K4) and K79 (H3-K79), which functions as a short-term 'memory' of recent transcription. H3 methylation, which, intriguingly, is also required for transcriptional silencing, depends on the ubiquitylation of histone H2B at K123 (H2B-K123).

Using chromatin immunoprecipitation (ChIP) analysis and real-time PCR, Ezhkova and Tansey investigated the interactions of the S. cerevisiae 19S proteasome subunits Rpt4 and Rpt6 with chromatin. By comparing constitutively active and inducible genes, they showed that Rpt6 associates with both active and uninduced promoters, but that its binding is enriched during active transcription. Furthermore, tagged Rpt4 was detected along the length of genes during active transcription, but was only found at the promoter and the 5′ end of an inducible gene under non-inducing conditions. This indicates that proteasomal ATPases associate with promoter sequences before gene activation occurs and then spread along the transcribed sequences during activation.

Temperature-sensitive mutations in the ATPase domain of Rpt6 (SUG1-25 and SUG1-3) and Rpt4 (SUG2-1) produced considerably lower levels of methylated H3-K4 and H3-K79 throughout the genome, although methylation of another lysine — H3-K36 — was not affected. A loss of gene silencing was also seen in these mutants. However, yeast that were mutant in different proteasome components — and therefore have compromised proteasome function — had relatively normal H3 methylation and showed no effect on gene silencing. So, Rpt4 and Rpt6 facilitate H3 methylation and gene silencing but these functions are independent of proteolysis.

The ubiquitin-conjugating enzyme Rad6, which ubiquitylates histone H2B, is required for methylation of H3-K4/K79, but not H3-K36. The authors found that the deletion mutant RAD6Δ and SUG1-25 yeast had similar defects in gene silencing, and, although the SUG1-25 mutation has no effect on H2B ubiquitylation, RAD6Δ yeast are unable to recruit Rpt6 to active promoters. Furthermore, ChIP analysis showed that a point mutation in the gene encoding H2B, which prevents ubiquitylation of H2B, also inhibits Rpt4 recruitment to chromatin.

So, Ezhkova and Tansey propose that H2B ubiquitylation by Rad6 recruits proteasomal ATPases to promoters. The proteasome components then move along the gene with RNA polymerase II, and use their ATP-dependent chaperone activity to reconfigure chromatin and allow access of histone methyltransferases to their target lysine residues — thereby coupling H2B ubiquitylation to transcription-dependent methylation of H3.