Transcriptomes of six mutants in the Sen1 pathway reveal combinatorial control of transcription termination across the Saccharomyces cerevisiae genome.

Transcriptome studies on eukaryotic cells have revealed an unexpected abundance and diversity of noncoding RNAs synthesized by RNA polymerase II (Pol II), some of which influence the expression of protein-coding genes. Yet, much less is known about biogenesis of Pol II non-coding RNA than mRNAs. In the budding yeast Saccharomyces cerevisiae, initiation of non-coding transcripts by Pol II appears to be similar to that of mRNAs, but a distinct pathway is utilized for termination of most non-coding RNAs: the Sen1-dependent or "NNS" pathway. Here, we examine the effect on the S. cerevisiae transcriptome of conditional mutations in the genes encoding six different essential proteins that influence Sen1-dependent termination: Sen1, Nrd1, Nab3, Ssu72, Rpb11, and Hrp1. We observe surprisingly diverse effects on transcript abundance for the different proteins that cannot be explained simply by differing severity of the mutations. Rather, we infer from our results that termination of Pol II transcription of non-coding RNA genes is subject to complex combinatorial control that likely involves proteins beyond those studied here. Furthermore, we identify new targets and functions of Sen1-dependent termination, including a role in repression of meiotic genes in vegetative cells. In combination with other recent whole-genome studies on termination of non-coding RNAs, our results provide promising directions for further investigation.

The Modifier of Transcription 1 (Mot1) ATPase and Spt16 Histone Chaperone Co-regulate Transcription through Preinitiation Complex Assembly and Nucleosome Organization.

Modifier of transcription 1 (Mot1) is a conserved and essential Swi2/Snf2 ATPase that can remove TATA-binding protein (TBP) from DNA using ATP hydrolysis and in so doing exerts global effects on transcription. Spt16 is also essential and functions globally in transcriptional regulation as a component of the facilitates chromatin transcription (FACT) histone chaperone complex. Here we demonstrate that Mot1 and Spt16 regulate a largely overlapping set of genes in Saccharomyces cerevisiae. As expected, Mot1 was found to control TBP levels at co-regulated promoters. In contrast, Spt16 did not affect TBP recruitment. On a global scale, Spt16 was required for Mot1 promoter localization, and Mot1 also affected Spt16 localization to genes. Interestingly, we found that Mot1 has an unanticipated role in establishing or maintaining the occupancy and positioning of nucleosomes at the 5' ends of genes. Spt16 has a broad role in regulating chromatin organization in gene bodies, including those nucleosomes affected by Mot1. These results suggest that the large scale overlap in Mot1 and Spt16 function arises from a combination of both their unique and shared functions in transcription complex assembly and chromatin structure regulation.

Measuring chromatin interaction dynamics on the second time scale at single-copy genes.

The chromatin immunoprecipitation (ChIP) assay is widely used to capture interactions between chromatin and regulatory proteins, but it is unknown how stable most native interactions are. Although live-cell imaging suggests short-lived interactions at tandem gene arrays, current methods cannot measure rapid binding dynamics at single-copy genes. We show, by using a modified ChIP assay with subsecond temporal resolution, that the time dependence of formaldehyde cross-linking can be used to extract in vivo on and off rates for site-specific chromatin interactions varying over a ~100-fold dynamic range. By using the method, we show that a regulatory process can shift weakly bound TATA-binding protein to stable promoter interactions, thereby facilitating transcription complex formation. This assay provides an approach for systematic, quantitative analyses of chromatin binding dynamics in vivo.