Dual regulation of chemical stress-induced DDI2/3 expression by a transcription factor Fzf1 and nucleosome in Saccharomyces cerevisiae

Reviewer #1 (Public review):

Summary:

In this manuscript, Du et al. identify a putative URS (nucleotides −709 to −229) that contributes to CY/MMS-induced DDI2/3 transcription in the promoter of S. cerevisiae DDI2 through promoter mapping. They further showed that CY/MMS leads to histone loss, and that genetic depletion of histone triggers DDI2/3 expression in an Fzf1-dependent manner. Using MNase-seq, the authors demonstrate that CY treatment leads to nucleosome loss at the DDI2/3 promoter and coding sequences, and that this chromatin remodeling process requires Fzf1. Based on these results, the authors propose that Fzf1 promotes CY-induced DDI2/3 expression through two distinct mechanisms: as a transcription factor and as a regulator of nucleosome occupancy. This dual mode of regulation may contribute to the exceptionally high induction of DDI2/3 relative to other Fzf1 target genes in response to CY.

Strengths:

This manuscript identifies the URS region at the DDI2 promoter that regulates CY/MMS-induced DDI2 expression. In addition, the authors revealed an important role of nucleosome occupancy in regulating DDI2/3 transcription, proposing the intriguing dual regulation model of Fzf1. Overall, this work furthers our understanding of how Fzf1 mediates the increase of DDI2/3 expression in response to CY/MMS treatment.

Weaknesses:

Overall, the study is of interest, and the data are generally convincing; however, several conclusions would benefit from further experimental validation. Certain controls are necessary for several experiments to improve the strength of evidence. Several major points are listed below:

(1) For Figure 6A and Figure 7A, the authors concluded that there are 'joint effects' of CY treatment and histone depletion. However, it is unclear whether CY treatment acts dependently or independently of histone depletion. As shown in Figure 2, both CY and MMS can cause histone reduction. In addition, the depletion system in the RMY102 strain only depletes about half of the H3 (based on the western blots in Figure 5D, E). It would be necessary to test whether H3 abundance is further depleted in CY-treated RMY102 by Western blot.

(2) Proper controls are missing in Figure 6A and Figure 7A. The authors compare gene expression levels in RMY102 + histone depletion + CY/MMS treatment with RMY102 + non-histone depletion. There are two variables here: histone depletion and CY/MMS treatment. It would be more convincing to include RMY102 YPGal+ CY/MMS treatment (5-40 mM), so that the impact of histone depletion and CY/MMS treatment on Fzf1 target gene expression levels would be clearer.

(3) In lines 242-249 and 269-272, the authors compared RMY102 versus BY4741 to conclude that histone depletion affects dose dependency of CY/MMS treatment. However, RMY102 and BY4741 might have different responses to CY/MMS due to strain background differences. Thus, in line with point 2, showing the expression level curves for non-histone depleting RMY102 treated with different doses of CY/MMS would be necessary.

(4) Figure 4 shows that CY and MMS have differential impacts on cell growth, which is intriguing. However, the rest of the data did not provide further insights in regard to this observation. It might be helpful to speculate possible underlying mechanisms in the Discussion session.

Reviewer #2 (Public review):

Summary:

Previous work established that FZF1 is both necessary and sufficient for activation of FZF1 target genes through the CS2 sequence motif, which is present upstream of FZF1-responsive targets. This study extends that model by demonstrating that, in addition to direct binding of FZF1 to CS2 elements, FZF1 can also promote reduced nucleosome-mediated repression, thereby contributing an additional layer of transcriptional regulation.

The authors investigate why FZF1-dependent transcriptional responses exhibit different magnitudes despite FZF1 binding to CS2 elements with similar affinity. Using promoter constructs derived from the DDI2-3 gene, the authors identify a region upstream of the CS2 element that functions as a repressive regulatory element. Based on this observation and publicly available datasets, the authors propose that this repression may be mediated through nucleosome occupancy.

Strengths:

The authors demonstrate that the DDI2-3 promoter contains positioned nucleosomes and show that chemical stress results in decreased histone protein levels and reduced histone-associated transcripts. They further examine whether histone depletion alone is sufficient to activate the DDI2-3 response and find that reduced histone levels increase expression, although chemical treatment produces an additional increase that remains dependent on FZF1. These findings suggest that FZF1 contributes to reductions in nucleosome occupancy at DDI2-3 and SSU1, revealing a second, potentially independent mechanism by which FZF1 regulates transcriptional responses to chemical stress.

Overall, the authors provide strong evidence that nucleosome occupancy influences the magnitude of FZF1-mediated DDI2-3 responses to chemical stress. This work has important implications for understanding how transcriptional networks evolve to generate highly tuned responses by combining multiple regulatory mechanisms acting on shared molecular components.

Weaknesses:

However, several additional considerations should be addressed. While histone depletion may contribute to differential FZF1-mediated responses, alternative mechanisms may also influence the observed transcriptional differences. For example, YHB1 exhibits basal expression that is independent of FZF1, and SSU1 contains the CS1 regulatory element, which can promote increased expression independently of FZF1 responsiveness. Therefore, differences in promoter architecture and the presence of alternative regulatory sequences may also contribute to differential FZF1 responses and should be discussed.

Additionally, the authors should clarify whether nucleosome depletion is directly mediated by the FZF1 ZF5 domain or occurs indirectly as a consequence of RNA polymerase II (Pol II) recruitment. Although the data presented in Figure 9 are consistent with a direct interaction model, the current evidence does not fully exclude the possibility that Pol II recruitment contributes to subsequent nucleosome/histone depletion. Unless there is direct experimental evidence demonstrating that FZF1 ZF5 independently promotes nucleosome remodeling, this alternative mechanism should be acknowledged and considered in the discussion.

Reviewer #3 (Public review):

Summary:

In the manuscript titled "Dual regulation of chemical stress-induced DDI2/1 3 expression by a transcription factor Fzf1 and nucleosome in Saccharomyces cerevisiae" Du et al have discovered a dual role of Fzf1 in transcriptional control of DDI2/3 during cyanamide (CY) or MMS treatment. While previous literature established that Fzf1 regulates multiple targets (DDI2/3, SSU1, YHB1, and YNR064C) by binding the CS2 consensus sequence, it remained unclear why DDI2/3 uniquely undergoes a massive 1,000-fold induction under cyanamide (CY) stress, whereas the others show only a 20- to 40-fold induction. In this work, the authors showed that Fzf1 functions beyond standard transcriptional activation. Using MNase-seq and a series of promoter truncation mutants, the authors mapped Upstream Repressing Sequences (URS) in the DDI2/3 promoter that are heavily occupied by nucleosomes. The authors showed that Fzf1 is essential for chromatin remodelling and nucleosome eviction (specifically at the -2 nucleosome position) to de-repress the DDI2/DDI3 expression.

Strengths:

The two-tier mechanism of action of Fzf1 in controlling the DDI2/3 expression during CY/MMS stress is compelling and novel.

Weaknesses:

While the authors presented the in vivo MNase-seq data that show Fzf1 is necessary for nucleosome displacement, the current study lacks any in vitro mechanistic proof. As the authors acknowledge, it remains unknown whether Fzf1 directly displaces nucleosomes on its own (perhaps through unmapped post-translational modifications induced by chemical stress) or whether its ZF5 activation domain merely acts as a scaffold to recruit separate chromatin remodelling complexes.

To test nucleosome repression, the authors utilised extreme global interventions, such as deleting the SPT10 gene or halting de novo histone synthesis using a galactose-to-glucose medium shift in the RMY102 strain. While these methods effectively deplete histones and induce DDI2/3 up to 30- to 350-fold, completely depleting cellular histones causes massive, pleiotropic secondary effects across the entire genome, which can obscure specific regulatory relationships.