Chromatin- and transcription-related factors repress transcription from within coding regions throughout the Saccharomyces cerevisiae genome

Abstract

Previous studies in Saccharomyces cerevisiae have demonstrated that cryptic promoters within coding regions activate transcription in particular mutants. We have performed a comprehensive analysis of cryptic transcription in order to identify factors that normally repress cryptic promoters, to determine the amount of cryptic transcription genome-wide, and to study the potential for expression of genetic information by cryptic transcription. Our results show that a large number of factors that control chromatin structure and transcription are required to repress cryptic transcription from at least 1,000 locations across the S. cerevisiae genome. Two results suggest that some cryptic transcripts are translated. First, as expected, many cryptic transcripts contain an ATG and an open reading frame of at least 100 codons. Second, several cryptic transcripts are translated into proteins. Furthermore, a subset of cryptic transcripts tested is transiently induced in wild-type cells following a nutritional shift, suggesting a possible physiological role in response to a change in growth conditions. Taken together, our results demonstrate that, during normal growth, the global integrity of gene expression is maintained by a wide range of factors and suggest that, under altered genetic or physiological conditions, the expression of alternative genetic information may occur.

Figure 1. Detection of Cryptic Initiation with the FLO8-HIS3 Reporter

(A) Diagram of FLO8-HIS3 reporter. The FLO8 promoter is replaced by the GAL1 promoter, and the 3′ end of the FLO8 ORF is replaced with the HIS3 ORF. The approximate position of the FLO8 internal cryptic TATA site (base pair position +1,626) is shown. The expected FLO8-HIS3 full-length and HIS3 short transcripts are indicated beneath the diagram. (B) His⁺ phenotypes of wild-type and spt6-1004 strains carrying the FLO8-HIS3 reporter. Cells were replica-plated onto the indicated medium (SC or SC-His), and plates were grown at 30 °C for 5 d. (C) Northern analysis of wild-type and spt6-1004 strains carrying the FLO8-HIS3 reporter. RNA was isolated from cells either grown at 30 °C or shifted to 37 °C for 80 min. The probe for the northern analysis was generated against HIS3, and SNR190 was used as a loading control. The arrow indicates full-length FLO8-HIS3 RNA transcripts, and the asterisk indicates HIS3 short transcripts resulting from cryptic initiation.

Figure 2. Analysis of Cryptic Initiation Mutants

(A) His⁺ phenotypes of cryptic initiation mutants carrying the FLO8-HIS3 reporter. Cells were spotted in a 10-fold dilution series from 1 × 10⁸ to 1 × 10³ cells/ml on the indicated medium. Growth on media containing galactose (Gal) induces expression of the full-length FLO8-HIS3 construct, which can affect activation of the FLO8 cryptic promoter in several mutants. Growth on media containing 3-aminotriazole (3AT), a competitive inhibitor of histidine, is indicative of higher expression levels of the HIS3 transcript. Plates were grown at 30 °C for 6 d. The ctk1 and ctk2 mutants are unable to use galactose as a carbon source; therefore, they only grow on the plates with glucose as the carbon source regardless of the presence or absence of histidine in the growth medium. (B) Northern analysis of FLO8, SPB4, and STE11 in cryptic initiation mutants. RNA was isolated from cells grown at 30 °C, except for the spt6-1004 and spt16-197 mutants, which were shifted to 37 °C for 80 min as indicated. SNR190 was used as a loading control. Arrows indicate full-length RNA transcripts, and asterisks indicate short transcripts resulting from cryptic initiation.

Figure 3. Western Analysis of Histone H3 K36 Methylation Levels in Cryptic Initiation Mutants

Whole-cell extracts were prepared from cells grown at 30 °C, except for strains FY2425 and FY347, which were shifted to 37 °C for 80 min as indicated. Probes for the western analyses were generated with antibodies specific for total histone H3, dimethylated H3 K36, and trimethylated H3 K36. WT, wild type.

Figure 4. Microarray Analysis of Genes with Cryptic Promoters in spt6 and spt16 Mutants

(A) Northern analysis of cryptic promoter genes identified by microarrays in spt6-1004 and spt16-197 mutants. RNA was isolated from cells after an 80-min temperature shift from 30 °C to 37 °C. Probes for northern analyses were generated with APM2, DDC1, OMS1, PUS4, and SYF1. SNR190 was used as a loading control. Arrows indicate full-length RNA transcripts, and asterisks indicate short transcripts. (B) Venn diagram comparing the number of genes with cryptic promoters in spt6-1004 and spt16-197 mutants. Microarray results predict 960 genes to express short transcripts in an spt6-1004 mutant and 1,130 genes to express short transcripts in an spt16-197 mutant, based on a 3′/5′ ratio threshold of 2.5 for each gene. Among these, the overlap is 709 genes. (C) spt16-197 versus spt6-1004 plot of cryptic promoter microarray results. The log₂ value of the 3′/5′ ratio for probe 6 of each gene in the spt16-197 microarray was plotted against the log₂ (3′/5′ ratio) value for probe 6 of the corresponding gene in the spt6-1004 microarray. The correlation coefficient, r, between the two datasets is 0.8345.

Figure 5. Analysis of Short Protein Expression in an spt6 Mutant

All Western analyses used whole-cell extracts prepared from cells after an 80-min temperature shift from 30 °C to 37 °C. Probes for the western analyses were generated with an antibody recognizing the TAP epitope tag. Pgk1 was used as a loading control. Arrows indicate full-length proteins, and asterisks indicate short proteins. WT. wild type. (A) Western analysis of short protein expression in an spt6-1004 mutant. (B) Western analysis of short-protein expression in wild-type and spt6-1004 strains containing APM2 and PUS4 ATG mutations. The lower molecular weight bands in lane 6 are likely to be degradation products.

Figure 6. Northern Analysis of Cryptic Initiation during a Nutritional Shift from YPD Rich Medium to SD Minimal Medium for the Indicated Times

Lanes 1 and 2 contain RNA isolated from cells shifted from 30 °C to 37 °C for 80 min as indicated. All other RNA was isolated from cells grown at 30 °C. SNR190 was used as a loading control. Asterisks indicate short transcripts. SNR190 served as a loading control.