The signaling mechanisms that transmit environmental information to regulate chromatin structure and elicit changes in gene transcription and chromatin states are poorly understood but critical as they may impact gene-environment interactions and epigenetic processes (1
). We have utilized the TORC1 pathway to begin identifying mechanisms by which environmental (i.e. nutrient) signals influence chromatin regulation and gene transcription. Our rapamycin screens determined that TORC1-regulated cell growth requires the H3K56ac pathway and its disruption sensitizes cells to rapamycin-induced growth inhibition. Furthermore, genetic disruption of TORC1 (tco89Δ
) or nutrient-dependent TORC1 activators (ego3Δ
), or pharmacological inhibition of TORC1 in wild-type cells (rapamycin treatment), negatively regulates global H3K56ac thus implicating active TORC1 signaling in the control of this chromatin modification pathway. Tco89 provides an essential function within TORC1 for H3K56ac regulation since neither a hyperactive TORC1 mutant nor a TORC1-independent Sch9 mutant rescued H3K56ac. However, the exact mechanism by which Tco89 regulates H3K56ac remains to be determined. Our results demonstrating that H3K56ac is rescued in tco89Δ
when the Sirtuin deacetylases Hst3 or Hst4 are deleted suggest one possible mechanism could be that TORC1 signaling negatively regulates the activity of these enzymes. In the absence of normal TORC1 signaling, as in tco89Δ
, these deacetylases may be more active and thus reduce the steady-state levels of H3K56ac (outlined in D). This regulation could be direct through TORC1-dependent modification of these enzymes or indirect through TORC1 regulation of metabolism since these deacetylases are regulated by cellular metabolic state (58
). Another possibility could be that TORC1 directly phosphorylates H3K56ac regulators to control H3K56ac and Tco89 provides an essential role in substrate recognition. To our knowledge, phosphorylation of yeast Asf1 or Rtt109 has not been demonstrated, although Asf1 in animal cells is phosphorylated by Tousled-like kinases (59
) suggesting that H3K56ac may be regulated by upstream kinases in yeast as well. Because Tco89 is a newly identified TORC1 subunit (60
), its functions within the complex are largely unknown and so how it functions in TORC1 signaling, and in particular H3K56ac regulation, will need to be determined in future studies. Regardless of the specific mechanisms involved, TORC1 regulated H3K56ac likely impacts many aspects of genome regulation since this histone modification functions in transcription, DNA repair and replication.
alone failed to decrease H3K56ac on the TORC1-regulated rDNA. This effect is most likely explained by the retention in tco89Δ
cells of enough TORC1-signaling activity to maintain rDNA H3K56ac and normal rRNA synthesis. Our results demonstrating that an inhibitory concentration of rapamycin could significantly decrease H3K56ac on the rDNA further supports this idea. The observation of the slight synthetic sick phenotype in the tco89Δ asf1Δ
double mutant, also detected by other groups (39
), further suggests there is likely significant, but not necessarily complete, overlap between TORC1 and H3K56ac in regulating cell growth. This overlap would be consistent with the biochemical data demonstrating a significant reduction, but not complete ablation, of H3K56ac in TORC1 pathway mutants. Our analysis of the housekeeping genes ACT1
suggest that TORC1 disruption may have differential impacts on H3K56ac that are gene specific. Whether H3K56ac is decreased elsewhere in the genome in TORC1 mutants, especially on other TORC1 regulated genes, will need to be determined in future studies. Our results due provide evidence that the H3K56ac pathway contributes directly to rDNA transcription since Asf1 coassociates with RNA Pol I, both Asf1 and H3K56ac localize to the rDNA, and their disruption significantly decreases RNA Pol I levels. These results are in contrast to a previous study that did not detect Asf1 recruitment to the rDNA (61
), which may reflect that the rDNA ChIP signal from the epitope-tagged Asf1 IP in this study was normalized to an internal control genomic region but a similar no-tag control was not performed. While we normalized our Asf1 ChIP signal to a control genomic region as well, we also compared Asf1 enrichment to a no tag control strain analyzed identically to confirm specific enrichment. Most importantly, our study provides several lines of independent evidence that the H3K56ac pathway functions directly in RNA Pol I transcription.
Besides affecting RNA Pol I levels on the rDNA, we also provide direct evidence that H3K56ac is critical for rDNA binding by Hmo1 and so it likely participates in forming the specialized rDNA chromatin structure necessary for high level RNA Pol I transcription. The rDNA determinants necessary for mediating Hmo1 incorporation are not defined so our data implicating H3K56ac in this process provides new insight into rDNA transcriptional mechanisms and the chromatin pathways essential for Hmo1 rDNA binding. Exactly how H3K56ac regulates Hmo1 incorporation is still not clear but is the subject of ongoing investigation. One possibility is that histone H3, perhaps within the tetrasome or other sub-nucleosomal particle, blocks access to the DNA and prevents Hmo1 binding. H3K56ac could then facilitate the disruption of histone H3–DNA contacts (22
) that would allow greater fluidity for histone repositioning or eviction that permits Hmo1 binding. While we demonstrate H3K56ac is crucial for Hmo1 incorporation and RNA Pol I transcription, we do not detect significant alterations in 18S or 25S rRNA expression in H3K56ac pathway mutants, unlike the decrease in 18S and 25S rRNAs detected in hmo1Δ
. These results suggest that the amount of Hmo1 bound in the H3K56A mutant is sufficient to maintain steady-state rRNA levels. This result is not surprising given that we did not detect significant changes in rRNA expression in the uaf30Δ
mutant which is known to significantly decrease RNA Pol I rDNA binding (52
). Yeast cells can adjust their rates of rRNA synthesis such that cells containing only 42 rDNA repeats express equivalent levels of rRNA as control cells carrying a normal repeat (~143) number (62
). Therefore, the H3K56ac pathway mutants likely maintain normal rRNA expression by either increasing the number of transcriptionally active rDNA repeats or altering the turnover of the rRNAs at a post-transcriptional level.
rRNA cotranscriptional processing by the SSU processome occurs in the ITS1 sequence at the A2
cleavage site and SSU processome defects cause accumulation of non-processed rRNAs (56
). A sub-complex of SSU processome components, the t-Utps, are also essential for RNA Pol I transcription as depletion of t-Utps reduces the number of RNA Pol I complexes engaged on the rDNA and decreases rRNA synthesis (56
). How the SSU processome is recruited to the rDNA has yet to be determined but is known to be independent of RNA Pol I transcription (56
). Our data implicate H3K56ac as a critical histone post-translational modification pathway necessary for SSU processome rDNA binding, since a H3K56A mutation results in decreased SSU processome recruitment across the rDNA. These data also explain why H3K56ac pathway mutants have elevated levels of ITS1-containing rRNA since decreased SSU processome binding presumably leads to decreased cotranscriptional rRNA processing within ITS1. However, whether the lower SSU processome association in H3K56A mutants also causes the reduction in rDNA-bound RNA Pol I remains to be determined. A previous study demonstrated that hmo1Δ
mutants also accumulate non-processed rRNAs (50
) suggesting that the specialized chromatin state existing at the rDNA is essential for correct rRNA processing. Although we only detected increased ITS1-containing rRNAs in H3K56ac pathway mutants and not hmo1Δ
, it is important to point out that this previous study examined different regions of the primary rRNA transcript in hmo1Δ
cells than we have analyzed.
We also demonstrate that deregulating rDNA transcription in tco89Δ
mutants significantly sensitizes cells to growth inhibition by lower rapamycin concentrations. While these results were unexpected they are not unprecedented. Two previous studies demonstrated that uncoupling rDNA transcription from its regulation by nutrient-signaling pathways (i.e. TORC1) causes cells to become significantly rapamycin hypersensitive (63
). Although the exact mechanisms underlying this effect are unknown, one explanation could be that TORC1 signaling also controls the expression of non-ribosomal factors essential for ribosome biogenesis, such as genes of the Ribi
regulon which may also include rRNA processing factors like the SSU processome (65
). As such, when pathways essential for SSU processome recruitment are compromised, such as H3K56ac, non-processed rRNAs accumulate. Under these conditions, forcing rDNA transcription using an inducible, RNA Pol II transcribed rDNA may generate high levels of non-processed rRNAs that cause significant ribotoxic stress in the cells, perhaps through the formation of non-functional ribosomal sub-complexes. This explanation could also account for why hmo1Δ
were not as sensitized as asf1Δ
under these same conditions. Since hmo1Δ
did not increase ITS1-containing rRNAs and had significantly lower levels of 18S and 25S rRNAs, the galactose-induced increase in rRNA likely could be compensated by the processing factors already expressed in these cells.
Overall, our studies demonstrate that nutrient signaling through TORC1 regulates global H3K56ac and that this histone modification regulates rDNA transcription and rRNA processing by the SSU processome. To our knowledge this is both the first demonstration that TORC1 signaling regulates the occurrence of a specific histone post-translational modification and that a single site of histone post-translational modification can control rDNA transcription and correct rRNA processing. These results implicate the chromatin environment as a regulator of the post-transcriptional rRNA processing necessary for TORC1-regulated cell growth.