Functional Separation of the Requirements for Establishment and Maintenance of Centromeric Heterochromatin

Abstract

The establishment and maintenance of centromeric heterochromatin in fission yeast require the RITS complex. Comprised of centromeric siRNAs, the chromodomain protein Chp1, Argonaute (Ago1), and Tas3, RITS couples the cellular RNAi pathway with assembly of constitutive heterochromatin. However, the mechanisms governing RITS-dependent establishment versus maintenance of centromeric heterochromatin remain unresolved. Here, we report that a mutant Tas3 protein that cannot bind Ago1 supports the maintenance of centromeric heterochromatin but cannot mediate efficient de novo establishment from cells transiently depleted for the histone H3 lysine 9 methyltransferase Clr4. In contrast, centromeric heterochromatin efficiently assembles in mutant cells transiently depleted for dicer. This mutant therefore allows ordering of the events leading to establishment of centromeric heterochromatin and places lysine 9 methylation of histone H3 upstream of dicer function.

Common themes in siRNA-mediated epigenetic silencing pathways

“…As described above, a siRNA-nascent RNA base-pairing mechanism can play a central role in recruiting RITS to chromatin (Motamedi et al, 2004;Verdel and Moazed, 2005). However, RITS recruitment to chromatin also necessitates a chromodomain present on RITS subunit Chp1, which interacts with H3K9me2 in vitro (Partridge et al, 2007;Partridge et al, 2002) (Fig. 2).…”

“…RITS is formed of three proteins, Chp1, Ago1 and Tas3 , a protein that has been recently found to bridge Chp1 to Ago1 (Debeauchamp et al, 2008;Partridge et al, 2007). RITS also contains siRNAs that bind Ago1 .…”

“…The fact that RITS turns out to be devoid of siRNAs in a ∆clr4 strain, or when Chp1 chromodomain is inactivated, points to a strong interdependency between the Ago1-bound siRNA and the chromodomain, a situation that complicates the study of their respective role (Motamedi et al, 2004;Noma et al, 2004). Nonetheless, recent studies have highlighted important features of these two RITS chromatin-binding modules (Debeauchamp et al, 2008;Partridge et al, 2007). RITS subunit Tas3 interacts with Ago1 through a motif formed by the repetition of WG/GW amino acids (Partridge et al, 2007).…”

Post-transcriptional gene silencing, transcriptional gene silencing and human immunodeficiency virus

“…Furthermore, the interaction between GW182/TNRC6 and AGO-1 occurs through binding of the GW repeats of GW182/TNRC6 to the Piwi domain of AGO-1 [77] . This is intriguing because the fission yeast RITS member protein, Tas3, has a GWrepeat-containing motif and interacts with AGO-1 to promote TGS [132] . It is therefore possible that, the Tas3/ AGO-1 interaction in fission yeast could be analogous, not homologous, to the AGO-1 and GW182/TNRC6 interaction in humans.…”

Epigenetics: heterochromatin meets RNAi

“…A Tas3 mutation that abolishes binding of Ago1 can support maintenance but not re-establishement of centromeric heterochromatin. The protein will remain localized to heterochromatin due to the Chp1 interaction with H3K9me and Ago1 can independently localize to cognate loci guided by siRNA [95]. A Tas3 mutation that interferes with Chp1 binding, however, has a more severe phenotype.…”