Spt6 is a highly conserved histone chaperone that interacts directly with both RNA polymerase II and histones to regulate gene expression. To gain a comprehensive understanding of the roles of Spt6, we performed genome-wide analyses of transcription, chromatin structure, and histone modifications in a Schizosaccharomyces pombe spt6 mutant. Our results demonstrate dramatic changes to transcription and chromatin structure in the mutant, including elevated antisense transcripts at >70% of all genes and general loss of the ؉1 nucleosome. Furthermore, Spt6 is required for marks associated with active transcription, including trimethylation of histone H3 on lysine 4, previously observed in humans but not Saccharomyces cerevisiae, and lysine 36. Taken together, our results indicate that Spt6 is critical for the accuracy of transcription and the integrity of chromatin, likely via its direct interactions with RNA polymerase II and histones. Studies over the last few years have revealed that transcription across eukaryotic genomes is much more widespread and complex than previously believed (1). Although it was once thought that transcription occurs primarily across protein-coding regions, genome-wide studies have now shown that transcription is also prevalent in intergenic regions and on antisense strands, in organisms ranging from yeast to humans (2, 3). Although roles for a small amount of this transcription have been established, for most, we have little understanding of its biological functions. Furthermore, while some factors have been shown to control the level of noncoding and antisense transcripts, many questions remain regarding the regulation of their synthesis and stability.One factor that plays a prominent role in the genome-wide control of transcription is Spt6. Originally identified in Saccharomyces cerevisiae (4, 5), Spt6 is conserved throughout eukaryotes and also has homology to the prokaryotic activator Tex (6). Spt6 interacts directly with several important factors, including RNA polymerase II (RNAPII) (7-11), histones (12, 13), and the transcription factor Iws1/Spn1 (7,14,15), suggesting that it is multifunctional. Recent studies in mammalian cells show that Spt6 also interacts directly with other chromatin related factors, including H3K27 demethylases (16, 17). Several gene-specific studies have demonstrated roles for Spt6 in transcription initiation (18)(19)(20), elongation (21, 22), and termination (23, 24). In addition, Spt6 is required for H3K36 methylation (25-28) and regulates nucleosome positioning and occupancy, particularly over highly expressed genes (12,19,29). Finally, Spt6 can assemble nucleosomes in vitro in an ATP-independent fashion (12). These results suggest that Spt6 acts as a histone chaperone by restoring nucleosomes in the wake of RNAPII transcription (30,31).In vivo, Spt6 is critical for normal growth and transcription. It is either essential or nearly essential for viability in all organisms tested, and viable spt6 mutations cause severe defects. In S. cerevisiae spt6 mutants,...

Yeast (Saccharomyces cerevisiae) has served as a key model system in biology and as a benchmark for "omics" technology. Although near-complete proteomes of log phase yeast have been measured, protein abundance in yeast is dynamic, particularly during the transition from log to stationary phase. Defining the dynamics of proteomic changes during this transition, termed the diauxic shift, is important to understand the basic biology of proliferative versus quiescent cells. Here, we perform temporal quantitative proteomics to fully capture protein induction and repression during the diauxic shift. Accurate and sensitive quantitation at a high temporal resolution and depth of proteome coverage was achieved using TMT10 reagents and LC-MS3 analysis on an Orbitrap Fusion tribrid mass spectrometer deploying synchronous precursor selection. Triplicate experiments were analyzed using the time-course R package and a simple template matching strategy was used to reveal groups of proteins with similar temporal patterns of protein induction and repression. Within these groups are functionally distinct types of proteins such as those of glyoxylate metabolism and many proteins of unknown function not previously associated with the diauxic shift (e.g. YNR034W-A and FMP16). We also perform a dual time-course experiment to determine Hap2-dependent proteins during the diauxic shift. These data serve as an important basic model for fermentative versus respiratory growth of yeast and other eukaryotes and are a benchmark for temporal quantitative proteomics. Molecular & Cellular

Transcription elongation factor GreA induces nucleolytic activity of bacterial RNA polymerase (RNAP). In vitro, transcript cleavage by GreA contributes to transcription efficiency by (i) suppressing pauses and arrests, (ii) stimulating RNAP promoter escape, and (iii) enhancing transcription fidelity. However, it is unclear which of these functions is (are) most relevant in vivo. By comparing global gene expression profiles of Escherichia coli strains lacking Gre factors and strains expressing either the wild type (wt) or a functionally inactive GreA mutant, we identified genes that are potential targets of GreA action. Data analysis revealed that in the presence of chromosomally expressed GreA, 19 genes are upregulated; an additional 105 genes are activated upon overexpression of the wt but not the mutant GreA. Primer extension reactions with selected transcription units confirmed the gene array data. The most prominent stimulatory effect (threefold to about sixfold) of GreA was observed for genes of ribosomal protein operons and the tna operon, suggesting that transcript cleavage by GreA contributes to optimal expression levels of these genes in vivo. In vitro transcription assays indicated that the stimulatory effect of GreA upon the transcription of these genes is mostly due to increased RNAP recycling due to facilitated promoter escape. We propose that transcript cleavage during early stages of initiation is thus the main in vivo function of GreA. Surprisingly, the presence of the wt GreA also led to the decreased transcription of many genes. The mechanism of this effect is unknown and may be indirect.In bacteria, the transcription process is initiated when the RNA polymerase (RNAP) holoenzyme binds to a promoter DNA sequence and forms an open promoter complex (RPo). In the presence of nucleoside triphosphates (NTPs), RNAP in the RPo synthesizes short (typically 2 to 9 nucleotides [nt] long) transcripts that rapidly dissociate from the complex. However, some of these "abortive" transcripts are extended beyond a threshold of 9 to 12 nt, which allows RNAP to start transcript elongation (36,30). RNAP in the elongation complex (EC) can transcribe over long distances until it reaches a terminator, where the EC dissociates into individual components (the DNA template, the RNA product, and RNAP, which can reinitiate transcription). Every stage of the transcription cycle can be limiting to the overall process and subject to regulation.Transcription elongation can be slowed or even blocked at certain points of the template, with the resultant formation of paused or arrested complexes, respectively. In these complexes, RNAP shifts along the DNA template in the direction opposite to that of transcription. As a result of such backtracking (18, 24) the 3Ј end of RNA disengages from the RNAP catalytic center, making further elongation impossible. An arrested complex can resume transcript elongation only following endonucleolytic cleavage of the nascent RNA that generates a new 3Ј end of the transcript in the RNAP catalyti...

Elevated production of collagen-rich extracellular matrix is a hallmark of cancer-associated fibroblasts (CAFs) and a central driver of cancer aggressiveness. Here we find that proline, a highly abundant amino acid in collagen proteins, is newly synthesized from glutamine in CAFs to make tumour collagen in breast cancer xenografts. PYCR1 is a key enzyme for proline synthesis and highly expressed in the stroma of breast cancer patients and in CAFs. Reducing PYCR1 levels in CAFs is sufficient to reduce tumour collagen production, tumour growth and metastatic spread in vivo and cancer cell proliferation in vitro. Both collagen and glutamine-derived proline synthesis in CAFs are epigenetically upregulated by increased pyruvate dehydrogenase-derived acetyl-CoA levels. PYCR1 is a cancer cell vulnerability and potential target for therapy; therefore, our work provides evidence that targeting PYCR1 may have the additional benefit of halting the production of a pro-tumorigenic extracellular matrix. Our work unveils new roles for CAF metabolism to support pro-tumorigenic collagen production.