Formation of centromeric heterochromatin in fission yeast requires the combined 
action of chromatin modifying enzymes and small RNAs derived from centromeric 
transcripts. Positive feedback mechanisms that link the RNAi pathway and the 
Clr4/Suv39h1 histone H3K9 methyltransferase complex (Clr-C) result in 
requirements for H3K9 methylation for full siRNA production and for siRNA 
production to achieve full histone methylation. Nonetheless, it has been 
proposed that the Argonaute protein, Ago1, is the key initial trigger for 
heterochromatin assembly via its association with Dicer-independent "priRNAs." 
The RITS complex physically links Ago1 and the H3-K9me binding protein Chp1. 
Here we exploit an assay for heterochromatin assembly in which loss of silencing 
by deletion of RNAi or Clr-C components can be reversed by re-introduction of 
the deleted gene. We showed previously that a mutant version of the RITS complex 
(Tas3(WG)) that biochemically separates Ago1 from Chp1 and Tas3 proteins permits 
maintenance of heterochromatin, but prevents its formation when Clr4 is removed 
and re-introduced. Here we show that the block occurs with mutants in Clr-C, but 
not mutants in the RNAi pathway. Thus, Clr-C components, but not RNAi factors, 
play a more critical role in assembly when the integrity of RITS is disrupted. 
Consistent with previous reports, cells lacking Clr-C components completely lack 
H3K9me2 on centromeric DNA repeats, whereas RNAi pathway mutants accumulate low 
levels of H3K9me2. Further supporting the existence of RNAi-independent 
mechanisms for establishment of centromeric heterochromatin, overexpression of 
clr4(+) in clr4Δago1Δ cells results in some de novo H3K9me2 accumulation at 
centromeres. These findings and our observation that ago1Δ and dcr1Δ mutants 
display indistinguishable low levels of H3K9me2 (in contrast to a previous 
report) challenge the model that priRNAs trigger heterochromatin formation. 
Instead, our results indicate that RNAi cooperates with RNAi-independent factors 
in the assembly of heterochromatin.