The 19S proteasome is directly involved in the regulation of heterochromatin spreading in fission yeast

Abstract

Cumulative evidence suggests that non-proteolytic functions of the proteasome are involved in transcriptional regulation, mRNA export, and ubiquitin-dependent histone modification and thereby modulate the intracellular levels of regulatory proteins implicated in controlling key cellular functions. To date, the non-proteolytic roles of the proteasome have been mainly investigated in euchromatin; their effects on heterochromatin are largely unknown. Here, using fission yeast as a model, we randomly mutagenized the subunits of the 19S proteasome subcomplex and sought to uncover a direct role of the proteasome in heterochromatin regulation. We identified a mutant allele, rpt4-1, that disrupts a non-proteolytic function of the proteasome, also known as a non-proteolytic allele. Experiments performed using rpt4-1 cells revealed that the proteasome is involved in the regulation of heterochromatin spreading to prevent its uncontrolled invasion into neighboring euchromatin regions. Intriguingly, the phenotype of the non-proteolytic rpt4-1 mutant resembled that of epe1⌬ cells, which lack the Epe1 protein that counteracts heterochromatin spreading. Both mutants exhibited variegated gene-silencing phenotypes across yeast colonies, spreading of heterochromatin, bypassing of the requirement for RNAi in heterochromatin formation at the outer repeat region (otr), and up-regulation of RNA polymerase II. Further analysis revealed Mst2, another factor that antagonizes heterochromatin spreading, may function redundantly with Rpt4. These observations suggest that the 19S proteasome may be involved in modulating the activities of Epe1 and Mst2. In conclusion, our findings indicate that the proteasome appears to have a heterochromatin-regulating function that is independent of its canonical function in proteolysis.

Proteasomes beyond proteolysis: Roles in heterochromatin maintenance

“…It is likely the 19S regulatory particle may alter the conformation of these proteins in order to regulate its activity. The current study by Seo et al (1) opens a whole new perspective in the 26S proteasome regulation of chromatin structure. Not only can the 26S proteasome promote changes in chromatin by degrading or displacing histones, but it also may have yet another role in regulating the activity of the enzymes responsible for putting on such modifications as lysine acetylation or removing others like lysine methylation.…”

“…The paper by Seo et al (1) demonstrates yet another nonproteolytic function for the 26S proteasome and that proteasomes may work in conjunction with two different histonemodifying enzymes. It is not clear as to their exact interplay, but localization data from ChIP-seq experiments suggest that the proteasome does not promote recruitment of Epe1.…”

“…These transcripts become double-stranded RNAs, which are involved in recruiting histone methylation machinery that leads to the formation of heterochromatin. At pericentric regions like those investigated in Seo et al (1), the RNAi machinery is the primary mechanism for both heterochromatin formation and maintenance. However, alternative pathways exist that can compensate for loss of the RNAi machinery, one involving Mst2, a histone acetyltransferase (3,4), and another involving Epe1, a H3K9 demethylase.…”

“…Most reports to date regarding the non-proteolytic function of the proteasome have shown its role in promoting transcription by regulating RNA polymerase II activity. In a new paper, Seo et al (1) delineate a role for the proteasome in controlling heterochromatin formation and regulating the borders between euchromatin and heterochromatin. This finding implies that the proteasome has a much broader role in regulating DNA-related processes than just transcription or degradation of histones.…”

“…Each ATPase subunit has its unique targets with which it interacts, and one subunit may be involved in more than one process (7). To explore the roles of these ATPases further, Seo et al (1) performed random mutagenesis on three and tested for effects using a heterochromatin formation reporter assay in which the ade6 gene is inserted into the outer repeat region of a centromere (Fig. 1A).…”