Mutation of the Rpd3S complex results in the appearance of promoter-associated histone modifications across the STE11 gene ORF
In addition to the full-length transcript, mutants in components of the Set2-Rpd3S pathway have cryptic internally initiated transcripts 
. While cryptic transcripts originate from the same sites, they show different levels of transcript between different mutants or under different growth conditions (, 
). These data suggest that cryptic promoters are regulated independently of the full-length promoter, and that the factors involved in transcription activation differ from one cryptic promoter to the next.
Variable transcript levels in mutants of proteins involved in the Set2-Rpd3S pathway.
The location of histone modifications associated with gene promoters is tightly regulated. Specifically, acetylated histone H3 (AcH3) and H4 (AcH4), and tri-methylated histone H3, lysine four (H3K4me3), are associated with the promoter and 5′ ORF region of actively transcribed genes (Reviewed in 
). We wanted to determine what defines a cryptic promoter. Do these promoters show characteristics similar to canonical promoters? In order to address this question, we used a mutant of a subunit unique to the Rpd3S histone deacetylase complex, rco1
Δ, which does not have HDAC function 
. Previous studies have shown that about 30% of genes in rco1
Δ mutants have hyperacetylated ORFs 
. We chose one of these genes, STE11
, which has two cryptic transcripts (, top panel, lane 2). The location of the start site for each cryptic transcript was mapped by 5′-RACE (data not shown). We performed chromatin immunoprecipitation (ChIP), to determine which promoter-associated modifications were present in the gene ORF in rco1
Δ mutants using primers tiling the STE11
locus (). Not only was acetylated H4 increased across the ORF as previously described 
, but Acetylated H3, lysine 14 (H3AcK14), dimethylated H3 lysine 4 (H3K4me2), and trimethylated H3 lysine 4 (H3K4me3) were also increased in rco1
Δ mutants when compared to the wild type strain (). There was no change in trimethylated H3 lysine 36 (H3K36me3), which was expected since Rpd3S HDAC activity is downstream of co-transcriptional methylation activity by the Set2 histone methyltransferase 
. Thus, loss of functional Rpd3S results in the appearance of promoter-associated histone modifications in the ORF of the STE11
gene. These data show that the region surrounding the transcription start site for each cryptic transcript resembles that of a canonical promoter.
Mutants of the Rpd3S subunit, Rco1, show an increase in promoter-associated histone modifications in the ORF of the STE11 gene.
Deletion of the bromodomain-containing protein, Rsc1, partially suppresses the small STE11 cryptic transcript
Bromodomain-containing proteins are important for recruitment of chromatin remodeling complexes to acetylated histones (reviewed in 
). Subunits of the RSC complex contain multiple bromodomains, which recognize acetyl lysine residues on histones and other proteins. In vitro
, purified RSC complex was shown to stimulate Pol II transcription through a nucleosome template; an event that was enhanced by NuA4 or SAGA-mediated histone acetylation 
. In vivo
, RSC activity has been implicated in nucleosome repositioning and maintenance of the nucleosome free region (NFR) at Pol II-transcribed promoters 
. We wanted to determine if RSC activity was important for the formation of cryptic promoters in gene ORFs that showed the hyperacetylation phenotype in Rpd3S mutants. We deleted Rsc1 and Rsc2, which are present in two distinct RSC subcomplexes 
, and show a very similar genome-wide occupancy profile 
. Both proteins contain tandem bromodomains that are essential for RSC function, but not complex assembly 
. Deletion of these proteins in combination is lethal, but deleting RSC1
individually results in cells that are viable, but show growth defects due to the loss of transcription at sporulation-specific genes 
We first examined the genome-wide effect of deleting RSC1
on acetylated H4 at genes that show hyperacetylation in rco1
Δ mutants (). ChIP samples were amplified using a double T7 linear amplification protocol 
, followed by hybridization to yeast high-resolution tiling microarrays. The log2
ratios of immunoprecipitated (IP) AcH4 versus input were subjected to a modified average gene analysis 
, which allowed us to examine the average AcH4 signal genome-wide at any given gene and surrounding intergenic region (). Using the dataset comparing enrichment of AcH4 in rco1
Δ mutants versus wild type, we identified genes that grouped into three clusters based on their enrichment patterns ().
Genome-wide occupancy of acetylated H4 in rco1Δ, rsc1Δ, and rsc2Δ mutant strains.
Earlier work from our group showed that the cluster of hyperacetylated ORFs (Cluster 2) was composed mainly of longer, less frequently transcribed genes 
. These genes do not completely overlap with those that receive the highest levels of H3K36me3, nor do they all produce cryptic transcripts, although all genes that produce cryptic transcripts fall within Cluster 2 (data not shown) 
. Since we were specifically interested in cryptic internal initiation of transcription, we focused on Cluster 2 and filtered the dataset for genes with an ORF that was represented by two or more probes. As previously demonstrated, rco1
Δ mutants showed an increase in acetylated H4 across a gene ORF when compared to the wild type strain (, 
). When the RSC complex mutants were compared to wild type, rsc2
Δ showed no change, while the rsc1
Δ strain showed an increase in AcH4 at the promoter region. Therefore, the RSC1 complex suppresses histone acetylation at yeast promoters. Neither the rsc1
Δ nor the rsc2
Δ strain had cryptic transcripts at STE11
(), which was consistent with the lack of ORF hyperacetylation in either of these mutants.
We wanted to know if the occupancy profiles of either Rsc1 or Rsc2 changed in rco1
Δ mutants. Specifically, ChIP was performed using myc-tagged Rsc1 and Rsc2 in wild type 
Δ strains, followed by PCR with primers spanning the STE11
ORF (). There was no significant change in Rsc2 occupancy; however, Rsc1 occupancy was decreased by approximately forty percent in the rco1
Δ mutant compared to the wild type strain. We do not see a direct association of the Rpd3S and RSC1 complexes by mass spectrometry (data not shown). Therefore, given that Rsc1 played a role in repression of acetylation at promoter regions in a subset of genes (), it is possible that retention of the RSC1 complex at certain ORFs is related to the histone deacetylation activity of Rpd3S. We attempted to examine this possibility through determination of genome-wide occupancy of Rsc1 and Rsc2 in rco1
Δ mutants, but the data was inconclusive due to inconsistent results between biological replicates (data not shown).
If the RSC complex is involved in nucleosome remodeling at cryptic promoters, then disruption of RSC subunits in an rco1
Δ background should suppress cryptic transcription. Due to difficulties with making either rsc1
Δ, or rsc2
Δ double mutants, we created an Rco1-degron strain (Rco1-deg), using the system described in Kanemaki et al
. The C-terminus of the Rco1 protein was tagged with a FLAG tag for detection by western blot (see materials and methods
section). RNA and protein samples were extracted at 0, 40, 80, and 160 minutes following induction of Rco1 protein degradation (). Cryptic transcription was visualized by northern blot at the STE11
gene beginning at 40 minutes post-induction (, Lanes 1–4). Rco1 protein levels did not change in a control with FLAG-tagged Rco1 protein that lacked the degron tag in the degron strain background (, upper panel, lanes 1–4). The Rco1-deg strain had no visible Rco1 protein after 80 minutes (, upper panel, lanes 5–8), which was consistent with the appearance of cryptic transcripts (, lanes 1–4).
Deletion of RSC1 in an Rco1-degron background results in a partial suppression of the small STE11 cryptic transcript.
We examined the effects of RSC on cryptic transcript formation by deleting either Rsc1 or Rsc2 in the Rco1-degron background. In both of these deletion strains, Rco1 protein was no longer visible after 80 minutes (, bottom panel, lanes 1–8). When STE11 transcript levels were evaluated by northern blot, however, formation of the small cryptic transcript was delayed in the rsc1Δ Rco1-deg strain compared to either Rco1-deg, or rsc2Δ Rco1-deg strains (, compare lanes 5–8 to lanes 1–4 and 9–12). Densitometry of northern blots from three biological repeats of this experiment showed that formation of the small cryptic transcript was suppressed by approximately 50% in the rsc1Δ Rco1-deg strain, compared to Rco1-deg alone (). Formation of the large cryptic transcript in the rsc1Δ Rco1-deg strain was comparable to Rco1-degron alone (). Therefore the RSC1 complex, and not the RSC2 complex suppresses formation of the small cryptic transcript at the STE11 gene in Rpd3S mutants. This finding also indicates that there is differential regulation of each cryptic promoter as the large cryptic promoter was not sensitive to RSC1 deletion.
We next used ChIP to assess the status of acetylated H4 occupancy at the STE11 locus at 40, 80, and 160 minutes compared to 0 minutes post-degron induction (). Maximum acetylated H4 occupancy occurred in the ORF, rather than the promoter region in all three strains at all three time points examined (), despite the fact that there was a difference in the intensity of the small cryptic transcript in the rsc1Δ Rco1-deg strain at 40 minutes (). Therefore differential regulation of cryptic promoters at STE11 by the RSC1 complex is determined by events downstream of ORF acetylation.
Deletion of the bromodomain-containing protein, Bdf1, completely suppresses the small STE11 cryptic transcript
Bdf1, like Rsc1, is a tandem bromodomain-containing protein that is important for recruitment and retention of TFIID at TATA-less promoters 
. In yeast, Bdf1 serves as the bromodomain-containing portion of Taf1 
, and is important for recruitment of TFIID to TATA-less promoters 
. Since Bdf1-dependent recruitment of TFIID plays an early role in transcription activation, we examined the role of this bromodomain-containing protein in cryptic transcript formation.
We deleted Bdf1 from the Rco1-degron strain and determined the formation of cryptic transcripts over time at the STE11 ORF (, top panel). Western blots showed that Rco1 degradation in the bdf1Δ Rco1-deg strain was comparable to that in Rco1-deg alone (, compare lanes 1–4 and 5–8). When we examined transcript formation by northern blot, however, we were surprised to find that the small cryptic transcript was completely suppressed in the bdf1Δ Rco1-deg strain (, top panel, compare lanes 4 and 8). Also interesting, was the fact that both the large cryptic transcript and the full-length transcript increased in intensity. These results indicate that dependence on co-activators for transcription activation varies from one cryptic promoter to the next. We also looked at cryptic transcript formation at the FLO8 locus, which has a single cryptic transcript. Compared to the Rco1-deg strain alone, the bdf1Δ Rco1-deg strain showed a dramatic increase in both the full-length and cryptic transcript at the FLO8 gene (, middle panel). When AcH4 occupancy at the STE11 locus was determined by ChIP, both Rco1-deg and the bdf1Δ Rco1-deg strain had maximum occupancy of this modification in the gene ORF (). Therefore, like RSC, Bdf1 affects cryptic promoter activity downstream of histone acetylation.
Deletion of BDF1 in an Rco1-degron strain suppresses the small STE11 cryptic transcript.
Bdf1 also interacts with the SWR1 complex, which is responsible for deposition of the H2A.Z histone variant 
. We wanted to determine if the suppression of the small cryptic transcript in the bdf1
Δ Rco1-deg strain was related to Bdf1 recruitment of SWR1. Deletion of the catalytic subunit, Swr1, from the Rco1-degron strain had no effect on the formation of cryptic transcripts at the STE11
ORF (, Lanes 2–4). Thus, the function of Bdf1 at cryptic promoters is probably independent of its role in the recruitment of the SWR1 complex. An interesting future experiment would be to compare the genome-wide occupancy of Bdf1 and other components of the TFIID complex to the locations of cryptic transcription in rco1
Genes that rely on Bdf1 for TFIID recruitment are generally not associated with SAGA 
. In fact, Bdf1 has been linked to repression of SAGA-dependent genes 
. Since there was a loss of the small cryptic transcript at STE11
, but an increase in the intensity of the large cryptic and full-length transcripts (), we compared SAGA occupancy at this gene between the Rco1-degron and bdf1
Δ Rco1-degron strains. ChIP was performed with an antibody directed against the Ada2 subunit of SAGA, followed by qPCR with primers directed against the STE11
locus (). In the Rco1-degron strain (black bars), maximal Ada2 occupancy occurs at the full-length STE11
promoter region. Occupancy at the large (+790) and small (+1790) cryptic promoter regions was comparable to that of a probe located in a region that does not contain a cryptic promoter (+390). This high baseline of Ada2 occupancy is likely to contribute to the increased levels of acetylation across the ORF as shown in . In the absence of Bdf1, however (grey bars), maximal occupancy at the large promoter (+790) increased to a level comparable to the full-length promoter (−11). There was no significant change at the small cryptic promoter in the presence or absence of Bdf1 (+1790). These results, along with the northern blot data (), suggest that both the full-length and large cryptic promoters are SAGA-dependent, while the small cryptic promoter is SAGA-independent for expression.
Overall, cryptic promoters are independently regulated by a variety of co-activators. In this sense, they resemble canonical gene promoters, which may explain why the location of these cryptic transcription start sites does not vary like the levels of expression.