Ifh1 is not required for Rap1, Fhl1 and Hmo1 recruitment at RP gene promoters
The co-activator depletion analysis described in the first chapter of this Results section revealed that RP gene transcription is controlled by multiple co-activators. Nevertheless, how NuA4, SAGA and Mediator are recruited to RP gene promoters is still unknown. Some old studies showed that Esa1 binding on RP gene promoters is affected in a Rap1 temperature-sensitive strain at not permissive temperature, hinting to a primary role of Rap1 in NuA4 recruitment (Reid et al., 2000a; Rohde and Cardenas, 2003; Uprety et al., 2012). However, as shown in the first section of this chapter, more recent insights into the RP gene promoter architecture revealed a precise hierarchy in the TF binding at RP gene promoters, with Rap1 being the pioneer TF required for the proper binding of the other three TFs, Hmo1, Fhl1 and Ifh1 (Knight et al., 2014; Reja et al., 2015).
Therefore, we hypothesized that Ifh1 was the TF directly responsible for the recruitment of co-activator complexes on RP gene promoters. To test this hypothesis, we took advantage of the anchor-away technique and we carried out Ifh1 nuclear depletion. Importantly, Ifh1 nuclear depletion by anchor-away almost abolished Ifh1 recruitment (Figure 22A) without affecting the binding of Rap1, Fhl1 and Hmo1 (Figure 22B-D), confirming the Ifh1 is not required for the recruitment of the other TFs and indicating that the Ifh1 anchor-away strain is a good tool to study the specific role of Ifh1 in RP gene transcription.
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Figure 22. (A) Ifh1, (B) Rap1, (C) Fhl1 and (D) Hmo1 ChIP-qPCR in Ifh1-FRB strain (Ifh1 anchor-away strain) treated with rapamycin or vehicle for the indicated times.
Ifh1 nuclear depletion affects RNAPII binding and histone acetylation at RP promoters
In order to investigate the role of Ifh1 in RP gene transcription we performed RNAPII, H3, H3K9 acetylation and H4 acetylation ChIP-Seq upon 60 minutes of rapamycin treatment in Ifh1-FRB strain.
Ifh1 depletion led to a significant decrease in RNAPII recruitment at RP gene promoters (Figure 23A) associated with a drop in both H3K9 and H4 acetylation. Importantly, the effects of Ifh1 depletion are specific for RP genes consistently with Ifh1 binding as revealed by ChIP-Seq. Furthermore, the effects of Ifh1 depletion on the three categories of RP genes correlate well with the binding of Ifh1 on the categories, with the category I RP genes being the most affected (Figure 23B).
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Figure 23. (A) On the left, heatmap showing Ifh1 binding for all the yeast genes (5036 genes) sorting
according to Ifh1 signal (Knight et al., 2014). Then, heatmaps showing the change in H3K9ac, H4ac and RNAPII in Ifh1-FRB strain (measured as log2 of the ratio rapamycin treated vs untreated cells). (B)
On the left, box-plots showing Ifh1 binding for the three RP gene categories (Knight et al., 2014). On the right, box-plots showing H3K9ac, H4ac and RNAPII change in Ifh1-FRB for the three RP gene
categories. For H4ac and H3K9ac the ChIP-Seq signal has been normalized to H3 ChIP-Seq.
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Ifh1 interacts with Tra1 to recruit Gcn5 and Esa1 at RP gene promoters
The strong decrease in histone acetylation measured in Ifh1 nuclear depleted cells might suggest a direct involvement of Ifh1 in recruiting NuA4 and SAGA complexes at RP gene promoters.
Consistently with this hypothesis ChIP of Gcn5 and Esa1 showed reduced recruitment of these two proteins at RP gene promoters in Ifh1 nuclear depleted cells (Figure 24A and B). Interestingly, also the binding of Tra1 was strongly affected in Ifh1 nuclear-depleted cells (Figure 24C). Moreover, analysis of MNase-digested chromatin in Ifh1 nuclear-depleted cells did not show any major change in nucleosome positioning at RP gene promoters (Figure 24D), indicating that the decrease in HAT binding is not caused by re-organization of the nucleosomes. These results might suggest that Ifh1 directly interacts with different subunits of NuA4 and SAGA complexes. Notably, Ifh1 has been shown to be acetylated by Gcn5 (Cai et al., 2013; Downey et al., 2013) and the drastic decrease in Tra1 binding in Ifh1 nuclear-depleted cells might indicate a direct interaction between these two proteins.
Consistently with this scenario, unpublished mass spectrometry data from our lab identified Tra1 as an interactor of Ifh1 (data not shown) and co-immunoprecipitation experiments confirmed this interaction (Figure 24E). In the light of these observations we propose that Ifh1 directly interacts with Tra1 promoting Esa1 and Gcn5 recruitment at RP gene promoters and thus RP gene transcription.
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Figure 24. (A) Esa1, (B) Gcn5 and (C) Tra1 ChIP-qPCR in Ifh1-FRB strain. (D) Nucleosome profile of category I (62 genes) and category II (66 genes) RP gene measured by MNase-Seq in Ifh1-FRB strain.
(E) Western Blot showing Ifh1 and Tra1 co-immunoprecipitation.
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Tra1 target genes are mostly PAC and RRPE promoter-containing genes
The co-activator depletion analysis that we performed revealed that transcription of a group of about 700 genes is strongly dependent on Tra1. As already said, this group of genes includes vast majority of the RiBi genes and many other genes that similarly to RiBi genes are involved in RNA processing and metabolism. Furthermore, a main characteristic of these 700 genes is the presence of the PAC and RRPE motifs on their promoters. A detailed motif analysis of yeast gene promoters revealed indeed that Tra1 target genes are characterized by the presence of PAC and RRPE motifs in their promoters and that the presence of both motifs makes the genes more responsive to Tra1 depletion (Figure 25A). As already mentioned in the Introduction, PAC and RRPE motifs are the binding sites for the repressor proteins Tod6/Dot6 and Stb3 respectively. In order to see whether Tra1 action on its targets is modulated by these repressors proteins we measured RNAPII binding in yeast cells nuclear depleted of Tra1 and where Tod6, Dot6 and Stb3 genes were deleted. Notably, deletion of Tod6, Dot6 and Stb3 did not affect the response of Tra1 targets to Tra1 depletion (Figure 25B), indicating that the action of Tra1 on PAC and RRPE promoter-containing genes is not modulated by PAC and RRPE binding proteins.
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Figure 25. (A) Box-plots showing RNAPII change in Tra1 nuclear depleted cells for all the yeast genes (left) or all the yeast genes excluding the RiBi genes (right) classified according to the presence of PAC
and RRPE motifs in their promoters. (B) RNAPII ChIP-qPCR in FRB-Tra1 strain (left) and FRB-Tra1 dot6Δ, tod6Δ, stb3Δ strain.
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Disruption of PAC and RRPE motifs affects nucleosome positioning and impairs transcription
In order to investigate the connection between Tra1 and PAC and RRPE motifs, we introduced several point mutations in both PAC and RRPE motifs of the RiBi gene Rrp8 at its endogenous genomic locus (Figure 26A). Importantly, we intended to measure the effect of Tra1 depletion on RNAPII recruitment in the wild type and in the mutant strain. However, RNAPII ChIP in the wild type and mutant strains revealed that mutation of the PAC and RRPE motifs impairs transcription of the Rrp8 gene and, as expected, abolishes the rapamycin response (Figure 26B). Furthermore, MNase digestion of chromatin from the wild type and mutant strains revealed that mutation of both PAC and RRPE motifs leads to shift of the -1 nucleosome towards the TSS (Figure 26C). Mutation of the PAC and RRPE motifs of the endogenous promoter of the RiBi gene Imd4 also resulted in shift of the -1 nucleosome (data not shown). Importantly, the shift of --1 nuclesome reduces the size of the NDR and we concluded that this leads to the decreased recruitment of RNAPII observed in the mutant strains.
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Figure 26. (A) RRPE and PAC motifs on the Rrp8 promoter. (B) RNAPII-ChIP qPCR in Rrp8 wild type and
mutant strains treated with rapamyicin (200 ng/mL). Please note that these experiments are not performed in the anchor-away background. (C) Relative nucleosome occupancy measured by
MNase-qPCR for the Rrp8 promoter in wild type and mutant strains.
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First genome-wide investigations of Tra1 binding by ChIP-Seq
As we were unable to show that Tra1 action on RiBi genes is related to PAC and RRPE motifs, we decided to directly investigate Tra1 binding in the genome by ChIP-Seq. We thus performed ChIP-Seq of a myc-Tra1 tagged strain and of an untagged control strain. Unfortunately, comparison of the genome-wide profiles of the myc-Tra1 tagged strain with the untagged control strain showed high similarity between the two and MACS analysis identified very few specific binding sites for Tra1 (Figure 27A). Furthermore, the 83 specific binding sites identified by MACS analysis were prevalently in large promoters as RP gene promoters (67 out of 83) and no specific binding was measured on RiBi genes, the main Tra1 targets. This last observation indicates that Tra1 binding on many of its target genes is not detectable by standard ChIP-Seq. We hypothesized that the large size of Tra1 could represent an obstacle in the cross-linking step. To increase the cross-linking efficiency of Tra1, we combined formaldehyde with EGS, a cross-linker with a longer arm, and we then analyzed Tra1 binding by ChIP-qPCR. As shown in Figure 27B, addition of EGS did not improve the efficiency of the cross-linking step and did not allow detection of Tra1 on RiBi gene promoters.
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Figure 27. (A) Genome Browser tracks showing myc-Tra1 and untagged ChIP-Seq signal in a region of
400 kb. (B) ChIP-qPCR in myc-Tra1 and untagged strain (UT). EF: EGS and Formaldehyde. F:
Formaldehyde.
Tra1 ChEC-Seq detected Tra1 and Gcn5 binding on SAGA-dominated genes and Esa1 binding on TFIID-dominated ones
Whereas Tra1 ChIP-Seq was unable to detect Tra1 binding on many of its putative target genes, as the RiBi genes, we decided to use the ChEC-Seq method to investigate Tra1 binding in the genome.
We therefore tagged Tra1 with MNase and performed the ChEC as described in (Hafner et al., 2018).
We also performed the ChEC for Esa1 and Gcn5 the two HATs that are recruited by Tra1. A strain where MNase protein is under the control of the strong Reb1 promoter was used as control (hereafter referred to as free MNase control). The ChEC-Seq of Esa1, Gcn5 and Tra1 revealed specific binding of the three proteins on many promoters already at very early time points (1 minute calcium
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treatment, see comparison with 20 minutes free MNase control in Figure 28A). Notably, Gcn5 and Tra1 ChEC-Seq profiles were clearly different from the Esa1 one. Indeed, while Gcn5 and Tra1 binding on promoters correlated very well (R=0.70), it was not the case for Tra1 and Esa1 (R=0.02) (Figure 28B). Importantly, while Esa1 was mainly detectable on the promoters of the TFIID-dominated genes, Gcn5 and Tra1 binding was stronger on the promoters of the SAGA-dominated ones (Figure 28C).
Figure 28. (A) Genome Browser tracks showing Esa1, Gcn5, Tra1 and free MNase control ChEC-Seq signal. (B) Scatterplots showing correlation between Esa1 (left) or Gcn5 (right) and Tra1 ChEC signal (calculated as average signal in the UAS for all the 5036 yeast genes). R: Pearson coefficient. (C) Box plots showing Esa1, Gcn5 and Tra1 ChEC-Seq signal calculates as in (B) for the genes classified in
SAGA-dominated and TFIID-dominated genes as described in (Huisinga and Pugh, 2004).
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The co-activator depletion analysis described in the first section of these Results hinted to the conclusion that Esa1 controls transcription of the TFIID-dominated genes while Gcn5, though it is more required for transcription of the SAGA-dominated genes, is involved in transcription regulation of all yeast genes. Importantly, comparison of the ChEC-Seq profiles of Esa1 and Gcn5 with the change in RNAPII binding measured upon their nuclear depletion revealed a relatively good correlation between these two measurements (Figure 29A and B). Conversely, this was not the case for Tra1 (Figure 29C). Indeed, while Tra1 nuclear depletion affected mostly TFIID-dominated genes such as RiBi and RP genes (cluster 3 and 4 genes), Tra1 ChEC-Seq revealed Tra1 binding almost exclusively on SAGA-dominated genes (cluster 1 and 5, Figure 29D and E).
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Figure 29. (A) Scatterplot and heatmap showing correlation between Esa1 ChEC signal and RNAPII change in Esa1 nuclear depleted cells. (B) and (C) same as in (A) for Gcn5 and Tra1. (D) Heatmap showing Esa1, Gcn5 and Tra1 ChEC-Seq signal for the same clusters of genes as in the heatmap on the left taken from (Bruzzone et al. (under revision)). (E) Box-plots showing Esa1, Gcn5 and Tra1 ChEC-seq
signal in the five gene clusters as in (D).
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The ChEC protocol leads to activation of stress genes and down-regulation of growth-promoting genes
Tra1 ChEC-Seq revealed a strong binding of Tra1 on many SAGA-dominated stress responsive genes (cluster 1 genes) that are not transcribed in optimal growing conditions. This observation led us to think that the ChEC protocol could result in activation of stress genes. Indeed, the protocol for ChEC involves many steps that could represent an important stress for the cells. Specifically, the cells are first centrifuged and then resuspended in a small volume of a buffer (buffer A) that does not contain any nutrient source. The cells in buffer A are then incubated for 5 minutes with digitonin in order to permeabilize the cell membrane to calcium. In order to evaluate the stress response during the ChEC protocol, we collected the cells at the three main steps of the protocol (after cell centrifugation, resuspension in buffer A and incubation with digitonin) and performed RNAPII ChIP-Seq. Importantly, RNAPII ChIP-Seq showed that the ChEC protocol results in transcription activation of many stress genes and downregulation of RiBi and RP gene transcription (Figure 30). Notably, resuspension of the cells in buffer A represents the most stressful step of the protocol, perhaps due to the absence of nutrients in the buffer that mimics starvation.
Figure 30. Heatmap showing RNAPII change in cells collected during three steps of the ChEC procedure.
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Development of a new protocol for ChEC that does not involve nutrient starvation
In the light of the observation that the standard ChEC protocol leads to stress response activation probably due to nutrient starvation, we aimed to optimize the protocol in order to reduce the stress.
Specifically, we decided to replace the buffer A with rich-medium (YPAD) and to reduce the incubation time with digitonin prior to calcium addition from 5 to 3 minutes. We thus performed Tra1 ChEC-Seq in these conditions. As a control for the new protocol, together with the usual free MNase control, we carried out also the Seq of the TF Sfp1. Importantly, standard Sfp1 ChEC-Seq, contrary to Sfp1 ChIP-ChEC-Seq, revealed strong Sfp1 binding on RiBi gene promoters indicating that binding of proteins on RiBi gene promoters can be identified by the ChEC-Seq technique (Albert et al.
manuscript in preparation). Furthermore, similarly to Tra1, Sfp1 nuclear depletion by anchor-away leads to strong downregulation of RiBi gene transcription (Figure 31A and (Albert et al. manuscript in preparation)). Indeed, we measured a high correlation between the change in RNAPII binding in Sfp1 and Tra1 nuclear depleted cells (R=0.62, Figure 31B), suggesting a possible role of Sfp1 in promoting Tra1 recruitment at many of its targets. Notably, this correlation was much stronger for RiBi genes than for RP genes (Figure 31C), consistently with the observation that also Ifh1 is involved in Tra1 recruitment at RP gene promoters.
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Figure 31. (A) Heatmap showing RNAPII change in Tra1 and Sfp1 nuclear depleted cells. (B)
Scatterplot showing correlation between RNAPII change in Tra1 and Sfp1 nuclear depleted cells. (C) Same as in (B) for RiBi and RP genes. RNAPII-ChIP-Seq in Sfp1-FRB strain and Sfp1 ChEC have been
performed by Benjamin Albert (Albert et al. manuscript in preparation).
Remarkably, the new ChEC protocol successfully detected Sfp1 binding on RiBi genes showing that MNase cleavage occurs also in growing medium and upon short digitonin exposure. However, quite unexpectedly, Tra1 binding on RiBi gene promoters was not detectable also with the new protocol.
Furthermore, the new ChEC protocol revealed a strong binding of Tra1 on RP gene promoters and on some SAGA dependent genes (Figure 32A and B). Regardless of the fact that Sfp1 and Tra1 control the transcription of the same groups of genes, RiBi genes first and then RP genes, comparison of the ChEC-Seq profiles of Tra1 and Sfp1 revealed a quite bizarre scenario. Indeed, while Sfp1 binding, as anticipated, was detectable mainly at RiBi gene promoters, Tra1 was detected almost exclusively at RP promoters (Figure 32C).
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Figure 32. (A) Genome Browser tracks showing Tra1, free MNase control and Sfp1 ChEC-Seq signal.
(B) Heatmaps showing Tra1 and Sfp1 ChEC-Seq signal on all yeast genes sorted according to Tra1 ChEC-Seq signal. (C) Average plots of Tra1 and Sfp1 ChEC-Seq signal at RiBi and RP genes.