RNase H activities counteract a toxic effect of Polymerase η in cells replicating with depleted dNTP pools.

RNA:DNA hybrids are transient physiological intermediates that arise during several cellular processes such as DNA replication. In pathological situations, they may stably accumulate and pose a threat to genome integrity. Cellular RNase H activities process these structures to restore the correct DNA:DNA sequence. Yeast cells lacking RNase H are negatively affected by depletion of deoxyribonucleotide pools necessary for DNA replication. Here we show that the translesion synthesis DNA polymerase η (Pol η) plays a role in DNA replication under low deoxyribonucleotides condition triggered by hydroxyurea. In particular, the catalytic reaction performed by Pol η is detrimental for RNase H deficient cells, causing DNA damage checkpoint activation and G2/M arrest. Moreover, a Pol η mutant allele with enhanced ribonucleotide incorporation further exacerbates the sensitivity to hydroxyurea of cells lacking RNase H activities. Our data are compatible with a model in which Pol η activity facilitates the formation or stabilization of RNA:DNA hybrids at stalled replication forks. However, in a scenario where RNase H activity fails to restore DNA, these hybrids become highly toxic for cells.

Abf1 and other general regulatory factors control ribosome biogenesis gene expression in budding yeast.

Ribosome biogenesis in Saccharomyces cerevisiae involves a regulon of >200 genes (Ribi genes) coordinately regulated in response to nutrient availability and cellular growth rate. Two cis-acting elements called PAC and RRPE are known to mediate Ribi gene repression in response to nutritional downshift. Here, we show that most Ribi gene promoters also contain binding sites for one or more General Regulatory Factors (GRFs), most frequently Abf1 and Reb1, and that these factors are enriched in vivo at Ribi promoters. Abf1/Reb1/Tbf1 promoter association was required for full Ribi gene expression in rich medium and for its modulation in response to glucose starvation, characterized by a rapid drop followed by slow recovery. Such a response did not entail changes in Abf1 occupancy, but it was paralleled by a quick increase, followed by slow decrease, in Rpd3L histone deacetylase occupancy. Remarkably, Abf1 site disruption also abolished Rpd3L complex recruitment in response to starvation. Extensive mutational analysis of the DBP7 promoter revealed a complex interplay of Tbf1 sites, PAC and RRPE in the transcriptional regulation of this Ribi gene. Our observations point to GRFs as new multifaceted players in Ribi gene regulation both during exponential growth and under repressive conditions.

The telomere-binding protein Tbf1 demarcates snoRNA gene promoters in Saccharomyces cerevisiae.

Small nucleolar RNAs (snoRNAs) play a key role in ribosomal RNA biogenesis, yet factors controlling their expression are unknown. We found that the majority of Saccharomyces snoRNA promoters display an aRCCCTaa sequence motif at the upstream border of a TATA-containing nucleosome-free region. Genome-wide ChIP-seq analysis showed that these motifs are bound by Tbf1, a telomere-binding protein known to recognize mammalian-like T(2)AG(3) repeats at subtelomeric regions. Tbf1 has over 100 additional promoter targets, including several other genes involved in ribosome biogenesis and the TBF1 gene itself. Tbf1 is required for full snoRNA expression, yet it does not influence nucleosome positioning at snoRNA promoters. In contrast, Tbf1 contributes to nucleosome exclusion at non-snoRNA promoters, where it selectively colocalizes with the Tbf1-interacting zinc-finger proteins Vid22 and Ygr071c. Our data show that, besides the ribosomal protein gene regulator Rap1, a second telomere-binding protein also functions as a transcriptional regulator linked to yeast ribosome biogenesis.

Promoter architecture and transcriptional regulation of Abf1-dependent ribosomal protein genes in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, ribosomal protein gene (RPG) promoters display binding sites for either Rap1 or Abf1 transcription factors. Unlike Rap1-associated promoters, the small cohort of Abf1-dependent RPGs (Abf1-RPGs) has not been extensively investigated. We show that RPL3, RPL4B, RPP1A, RPS22B and RPS28A/B share a common promoter architecture, with an Abf1 site upstream of a conserved element matching the sequence recognized by Fhl1, a transcription factor which together with Ifh1 orchestrates Rap1-associated RPG regulation. Abf1 and Fhl1 promoter association was confirmed by ChIP and/or gel retardation assays. Mutational analysis revealed a more severe requirement of Abf1 than Fhl1 binding sites for RPG transcription. In the case of RPS22B an unusual Tbf1 binding site promoted both RPS22B and intron-hosted SNR44 expression. Abf1-RPG down-regulation upon TOR pathway inhibition was much attenuated at defective mutant promoters unable to bind Abf1. TORC1 inactivation caused the expected reduction of Ifh1 occupancy at RPS22B and RPL3 promoters, but unexpectedly it entailed largely increased Abf1 association with Abf1-RPG promoters. We present evidence that Abf1 recruitment upon nutritional stress, also observed for representative ribosome biogenesis genes, favours RPG transcriptional rescue upon nutrient replenishment, thus pointing to nutrient-regulated Abf1 dynamics at promoters as a novel mechanism in ribosome biogenesis control.

Multiple roles of the general regulatory factor Abf1 in yeast ribosome biogenesis.

In Saccharomyces cerevisiae, the large majority of the genes coding for cytoplasmic ribosomal proteins (RPs) depend on the general regulatory factor Rap1 for their transcription, but a small cohort of them relies on Abf1 regulatory activity. A recent study showed that unlike Rap1, whose association with RP gene promoters is not affected by environmental changes causing RP gene repression/reactivation, Abf1 association with both RP gene and ribosome biogenesis (Ribi) gene promoters dynamically responds to changes in growth conditions. This observation changes the paradigm of general regulatory factors as relatively static DNA-binding proteins constitutively bound to highly active promoters, and point to Abf1, which binds hundreds of non-RPG promoters within the yeast genome, as a possible key regulatory switch in nutrient- and stress-dependent transcriptional modulation. Moreover, the frequent presence of Abf1 binding sites in the promoters of mitochondrial RP genes evokes the possibility that Abf1 might orchestrate still unexplored levels of co-regulation involving growth-related gene networks in yeast cells.

Transcription reinitiation by RNA polymerase III.

The retention of transcription proteins at an actively transcribed gene contributes to maintenance of the active transcriptional state and increases the rate of subsequent transcription cycles relative to the initial cycle. This process, called transcription reinitiation, generates the abundant RNAs in living cells. The persistence of stable preinitiation intermediates on activated genes representing at least a subset of basal transcription components has long been recognized as a shared feature of RNA polymerase (Pol) I, II and III-dependent transcription in eukaryotes. Studies of the Pol III transcription machinery and its target genes in eukaryotic genomes over the last fifteen years, has uncovered multiple details on transcription reinitiation. In addition to the basal transcription factors that recruit the polymerase, Pol III itself can be retained on the same gene through multiple transcription cycles by a facilitated recycling pathway. The molecular bases for facilitated recycling are progressively being revealed with advances in structural and functional studies. At the same time, progress in our understanding of Pol III transcriptional regulation in response to different environmental cues points to the specific mechanism of Pol III reinitiation as a key target of signaling pathway regulation of cell growth. This article is part of a Special Issue entitled: Transcription by Odd Pols.

Transcriptional control of yeast ribosome biogenesis: A multifaceted role for general regulatory factors.

In Saccharomyces cerevisiae, a group of more than 200 co-regulated genes (Ribi genes) is involved in ribosome biogenesis. This regulon has recently been shown to rely on a small set of transcriptional regulators (mainly Abf1, but also Reb1, Tbf1 and Rap1) previously referred to as general regulatory factors (GRFs) because of their widespread binding and action at many promoters and other specialized genomic regions. Intriguingly, Abf1 binding to Ribi genes is differentially modulated in response to distinct nutrition signaling pathways. Such a dynamic promoter association has the potential to orchestrate both activation and repression of Ribi genes in synergy with neighboring regulatory sites and through the functional interplay of histone acetyltransferases and deacetylases.

Investigating transcription reinitiation through in vitro approaches.

By influencing the number of RNA molecules repeatedly synthesized from the same gene, the control of transcription reinitiation has the potential to shape the transcriptome. Transcription reinitiation mechanisms have been mainly addressed in vitro, through approaches based on both crude and reconstituted systems. These studies support the notion that transcription reinitiation and its regulation rely on dedicated networks of molecular interactions within transcription machineries. At the same time, comparison with in vivo transcription rates suggests that additional mechanisms, factors and conditions must exist in the nucleus, whose biochemical elucidation is a fascinating challenge for future in vitro transcription studies.

Promoter architectures in the yeast ribosomal expression program.

Ribosome biogenesis begins with the orchestrated expression of hundreds of genes, including the three large classes of ribosomal protein, ribosome biogenesis and snoRNA genes. Current knowledge about the corresponding promoters suggests the existence of novel class-specific transcriptional strategies and crosstalk between telomere length and cell growth control.