Here, we report dynamic whole-genome mapping of PolII occupancy during several different stress time courses. Our major findings are: 1, transcriptional changes in response to stress are only partly reflected in mRNA abundance; 2, widespread cryptic transcription likely contributes to gene regulation during stress response; and 3, some PolII maintains contact with the genome, and is even recruited, well after mRNA synthesis is thought to have stopped in the rpb1-1 mutant.
Most interestingly, we find widespread mismatches between changes in PolII and changes in mRNA abundance during two stress response time courses. While PolII recruitment to a given gene is correlated with an increase in expression of that gene, the quantitative level of mRNA change for a given relative change in PolII recruitment is highly variable. A number of factors could explain variability in mRNA production per PolII, such as regulated mRNA stability [2
]. Indeed, we find that genes with excess mRNA production tended to exhibit longer half-lives (Additional file 14
). However, genome-wide mRNA half-lives have typically been measured in the rpb1-1
strain, which appears to upregulate stress genes for at least some time during the shift to the restrictive temperature [1
], indicating that the long mRNA half-lives for stress-related transcripts likely include effects of increased mRNA production during these time courses.
Here, we additionally find that genes exhibiting unusually high levels of mRNA produced per change in PolII generally exhibit greater overlap with cryptic and stable noncoding/unannotated transcripts (Figure ; Additional file 12c
). Furthermore, we note that many poorly expressed mRNAs are associated with overlapping transcripts before stress (Figure ). Together, these observations support a model where some of the PolII associated with such a gene in mid-log growth is engaged in nonproductive (in some cases regulatory) transcription [22
] (Additional file 10
). Upon stress, upregulation of the ORF promoter, downregulation of the CUT/SUT promoter, or both, would result in a higher proportion of PolII molecules associated with a gene being engaged in productive transcription. We suspect that each of these three possibilities occurs at different genes.
We further speculate, then, that mismatches in which less mRNA is produced per PolII change represent genes where nonproductive transcription is induced in stress (see, for example, altered SUT expression in stress in [36
]). As genome-wide datasets that identified CUTs and SUTs in budding yeast were not derived under stress conditions, these putative stress-specific transcripts would not have been identified in prior studies.
Interestingly, we found that genes involved in carbohydrate metabolism as a class are more subject to excess mRNA production than other gene sets (Additional file 8
). Previous studies of cryptic unstable transcripts found that genes involved in glucose metabolism were significantly enriched for sense CUTs [37
], consistent with our finding that genes exhibiting excess mRNA production were associated with overlapping CUTs or SUTs (Figure ). What is the biological rationale for regulation of carbohydrate-related genes by overlapping transcription? In the cases of nucleotide metabolism and termination factors [6
], regulation by CUTs appears to provide a mechanism for feedback regulation of the relevant genes. In the case of carbohydrate metabolism the basis for direct feedback is less clear, although given the widespread mechanisms by which a cell's metabolic state can influence chromatin regulators' activities [38
], we speculate that control of CUT transcription or termination could globally respond to NAD/NADH ratios or some other aspects of global cellular metabolism.
Finally, we extensively characterize the widely used temperature-sensitive rpb1-1
mutant in PolII. Many or most published studies use 15 minutes of inactivation of this allele to address the role of transcription in a given process (nuclear pore association, nucleosome positioning, and so on). However, here we find that PolII remains associated with the genome for approximately an hour before dissociating. Furthermore, we find that after 10 minutes in restrictive temperature PolII can still be recruited to newly activated genes. Prior studies with this mutant have shown a decrease in mRNA production [15
] and in permanganate sensitivity [39
] after 15 minutes of heat inactivation of this mutant, while our results show continued genomic association of PolII with the genome for at least another 15 minutes after this time. These different assays suggest that inactivating this mutant results first in loss of productive transcription without concomitant dissociation from the genome, followed after some time by dissociation from DNA. Thus, these experiments indicate that care must be used when interpreting the results of experiments with this mutant, and that longer incubation at restrictive temperature is required before PolII disengages from the genome.
Together, our results provide a broad perspective on the relationship between PolII and gene expression. These results have particular importance for studies attempting to use genomic sequence to understand transcriptional regulation - while the role of promoter sequence in the regulation of transcription is of course a major factor in the transcriptome, a great deal of variability in mRNA abundance may result from upstream or downstream regulatory promoters. Future computational studies will no doubt need to take local genomic structure-mediated effects such as these into account [40
] in order to achieve a quantitative predictive understanding of how gene regulation derives from genomic sequence.