We show that Fun30 is required for silencing of reporter genes embedded within transcriptionally repressed domains, the silent HMR
, telomeres and rDNA repeats. The ATPase function appears essential for this activity, because mutating an essential lysine residue in the ATPase domain abrogates this function. A toxicity assay upon overexpression reinforces the importance of the ATPase for Fun30 activity. In budding yeast, ATP-dependent chromatin remodelling factors ISW1 and ISW2 have been linked to gene silencing within the HMR
and rDNA, but not telomeres, whereas the SWI/SNF complex has been shown to be required for rDNA and telomeric silencing, but not silencing within the HMR
. Therefore, silencing appears to be the result of the concerted action of several remodelling factors. Fun30 appears to be special in that it affects silencing in the HMR
, telomeres and rDNA loci. We can presently not exclude an indirect role of Fun30 in heterochromatin maintenance. However, we favour a direct role, because we find that Fun30 binds at the silent HMR
locus, and less so to the adjacent euchromatic region. Furthermore, we find that deletion of Fun30 results in an altered chromatin structure at the HMR
boundary element. Future work will establish if Fun30 promotes gene silencing in heterochromatin by assisting the establishment of specific histone modifications (e.g., histone hypoacetylation), by regulating histone variants, or heterochromatin-specific non-histone proteins.
How is the role of Fun30 in heterochromatin connected with its genetic interaction with ORC? Our data suggest that deletion of the ORC binding site of the silencer element does not completely abrogate a role of Fun30 in silencing within the HMR
. This is because there is some difference in the accumulation of red pigment (indicative of silencing) between cells containing single deletions of either the ORC binding site or FUN30
, or the double mutant in this sensitive assay (see , accumulation of red pigment is apparent even when growth is not compromised). However, an assay that monitors growth in the absence of adenine (and that maybe somewhat less sensitive) indicates at least some epistatic interaction between the role of Fun30 in HMR
silencing and the ORC binding site within the silencer, because there is almost no difference in growth between single deletion strains at the ORC site or FUN30
, or the double deletion strains (). Therefore, there may be an interaction between Fun30 and ORC at the heterochromatic regions, and this, in turn, may be important for the silencing-related function of Fun30 and the replication-related function of ORC at these sites: The ORC complex may be involved in recruiting Fun30 to establish a silent chromatin configuration within the HMR
or Fun30 may facilitate the binding of ORC within the silencer element and other sites within the genome. If the number of functional ORC complexes is diminished, as in the orc5-1
mutation, the supportive role of Fun30 for ORC establishment may become apparent. Alternatively, Fun30 may promote cell cycle progression in a parallel pathway to ORC function, e.g., by regulating histone metabolism or facilitating DNA polymerase progression through chromatin. However, it is noteworthy that the deletion of FUN30
does not affect viability in the presence of hydroxyurea (data not shown), a drug that inhibits ribonucleotide reductase, leading to diminished deoxyribonucleotide pools and DNA replication stress. If Fun30 has a role in facilitating replication through chromatin, one would expect that the deletion of it would aggravate the deleterious effect of hydroxyurea, as is the case with Ino80, a chromatin remodelling factor implicated in facilitating chromatin replication 
. It is interesting that when other genes involved in silencing are deleted, they cause synthetic lethality or sickness with ORC mutants 
. Additional genes involved in such synthetic interactions with ORC mutants are involved in cohesion 
. An interesting suggestion might be that silencing established by Fun30 and other factors such as ASF1 is linked to some aspect of cohesin function.
We attempted to obtain further insight into the biological function of Fun30 by identification of interaction partners using TAP (Tandem Affinity Purification) of endogenously tagged Fun30, but did not find any interaction partner using this approach (data not shown). This may indicate that Fun30 does not form a heteromeric complex as so many other chromatin remodelling factors 
. Another explanation may be that such a complex is only formed under specific circumstances or is not extractable from the chromatin under our conditions.
Several members of the FUN30/ETL1 family of remodellers are characterized by the presence of putative CUE motifs. CUE motifs of other proteins bind to polyubiquitin chains, but also to monoubiquitin in vitro 
. The binding to ubiquitin is required for the function of CUE motif-containing proteins, for example, in intracellular trafficking 
. Moreover, the CUE motif can mediate intramolecular monoubiquitination 
. Here, we provide evidence that the putative CUE motif of the budding yeast Fun30 is required for the full function of the protein. In chromatin, regulation by ubiquitin assures genomic stability and cellular maintenance. Relevant examples of ubiquitinated proteins include histones 
, PCNA 
, RNA PolII 
, numerous transcription factors 
and various components of the DNA repair machinery 
. It will be interesting to determine if the function of Fun30 is dependent of the recognition of ubiquitinated factors, e.g., ubiquitinated histones or if Fun30 is regulated by ubiquitin. However, one cannot presently exclude that CUE motifs and related motifs recognize other ubiquitin related molecules, such as SUMO. Future work will determine the mechanisms by which Fun30 affects gene silencing and if the FUN30/ETL1 putative chromatin remodellers share biological roles.