Set1 is the catalytic subunit and the central component of the evolutionarily conserved Set1 complex (Set1C) that methylates histone 3 lysine 4 (H3K4). Here we have determined protein/protein interactions within the complex and related the substructure to function. The loss of individual Set1C subunits differentially affects Set1 stability, complex integrity, global H3K4 methylation, and distribution of H3K4 methylation along active genes. The complex requires Set1, Swd1, and Swd3 for integrity, and Set1 amount is greatly reduced in the absence of the Swd1-Swd3 heterodimer. Bre2 and Sdc1 also form a heteromeric subunit, which requires the SET domain for interaction with the complex, and Sdc1 strongly interacts with itself. Inactivation of either Bre2 or Sdc1 has very similar effects. Neither is required for complex integrity, and their removal results in an increase of H3K4 mono- and dimethylation and a severe decrease of trimethylation at the 5' end of active coding regions but a decrease of H3K4 dimethylation at the 3' end of coding regions. Cells lacking Spp1 have a reduced amount of Set1 and retain a fraction of trimethylated H3K4, whereas cells lacking Shg1 show slightly elevated levels of both di- and trimethylation. Set1C associates with both serine 5- and serine 2-phosphorylated forms of polymerase II, indicating that the association persists to the 3' end of transcribed genes. Taken together, our results suggest that Set1C subunits stimulate Set1 catalytic activity all along active genes.

RNA polymerases frequently deal with a number of obstacles during transcription elongation that need to be removed for transcription resumption. One important type of hindrance consists of DNA lesions, which are removed by transcription-coupled repair (TC-NER), a specific sub-pathway of nucleotide excision repair. To improve our knowledge of transcription elongation and its coupling to TC-NER, we used the yeast library of non-essential knock-out mutations to screen for genes conferring resistance to the transcription-elongation inhibitor mycophenolic acid and the DNA-damaging agent 4-nitroquinoline-N-oxide. Our data provide evidence that subunits of the SAGA and Ccr4-Not complexes, Mediator, Bre1, Bur2, and Fun12 affect transcription elongation to different extents. Given the dependency of TC-NER on RNA Polymerase II transcription and the fact that the few proteins known to be involved in TC-NER are related to transcription, we performed an in-depth TC-NER analysis of a selection of mutants. We found that mutants of the PAF and Ccr4-Not complexes are impaired in TC-NER. This study provides evidence that PAF and Ccr4-Not are required for efficient TC-NER in yeast, unraveling a novel function for these transcription complexes and opening new perspectives for the understanding of TC-NER and its functional interconnection with transcription elongation. Dealing with DNA lesions is one of the most important tasks of both prokaryotic and eukaryotic cells. This is particularly relevant for damage occurring inside genes, in the DNA strands that are actively transcribed, because transcription cannot proceed through a damaged site and the persisting lesion can cause either genome instability or cell death. Cells have evolved specific mechanisms to repair these DNA lesions, the malfunction of which leads to severe genetic syndromes in humans. Despite many years of intensive research, the mechanisms underlying transcription-coupled repair is still poorly understood. To gain insight into this phenomenon, we undertook a genome-wide screening in the model eukaryotic organism Saccharomyces cerevisiae for genes that affect this type of repair that is coupled to transcription. Our study has permitted us to identify and demonstrate new roles in DNA repair for factors with a previously known function in transcription, opening new perspectives for the understanding of DNA repair and its functional interconnection with transcription.

Transcription elongation by RNA polymerase II was often considered an invariant non-regulated process. However, genome-wide studies have shown that transcriptional pausing during elongation is a frequent phenomenon in tightly-regulated metazoan genes. Using a combination of ChIP-on-chip and genomic run-on approaches, we found that the proportion of transcriptionally active RNA polymerase II (active versus total) present throughout the yeast genome is characteristic of some functional gene classes, like those related to ribosomes and mitochondria. This proportion also responds to regulatory stimuli mediated by protein kinase A and, in relation to cytosolic ribosomal-protein genes, it is mediated by the silencing domain of Rap1. We found that this inactive form of RNA polymerase II, which accumulates along the full length of ribosomal protein genes, is phosphorylated in the Ser5 residue of the CTD, but is hypophosphorylated in Ser2. Using the same experimental approach, we show that the in vivo–depletion of FACT, a chromatin-related elongation factor, also produces a regulon-specific effect on the expression of the yeast genome. This work demonstrates that the regulation of transcription elongation is a widespread, gene class–dependent phenomenon that also affects housekeeping genes. Transcription of DNA–encoded information into RNA is the first step in gene regulation. RNA polymerases initiate transcription at the promoter region and elongate the transcripts traveling throughout the gene until reaching the termination sequences. Classical models of transcriptional regulation were focused on the initiation step, but there is increasing evidence for gene regulation after initiation. We have investigated the importance of elongation in gene regulation using the yeast Saccharomyces cerevisiae, one of the main experimental systems in modern biology. By comparing the genomic distribution of RNA polymerase molecules with the actual transcriptional signal across the genome, we have detected that many genes are regulated at the elongation level. We show that yeast cells use this step to modulate the expression of several groups of genes, which have to be simultaneously regulated in a very coordinated manner. Genes encoding essential functions, like those related to protein synthesis and respiration, change their transcriptional activities in response to environmental stimuli, without changing in the same extension the amount of RNA polymerase that is physically associated to them. We also show that this kind of regulation, in spite of taking place during the elongation step, can be mediated by promoter-binding transcription factors.