Alfonso Rodríguez-Gil

Protein interactions within the SET1 complex and their roles in the regulation of histone 3 lysine 4 methylation

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.

Genome-wide analysis of factors affecting transcription elongation and DNA repair: a new role for PAF and Ccr4-not in transcription-coupled repair

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.

Regulon-specific control of transcription elongation across the yeast genome.

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.

The distribution of active RNA polymerase II along the transcribed region is gene-specific and controlled by elongation factors.

In order to study the intragenic profiles of active transcription, we determined the relative levels of active RNA polymerase II present at the 3′- and 5′-ends of 261 yeast genes by run-on. The results obtained indicate that the 3′/5′ run-on ratio varies among the genes studied by over 12 log2 units. This ratio seems to be an intrinsic characteristic of each transcriptional unit and does not significantly correlate with gene length, G + C content or level of expression. The correlation between the 3′/5′ RNA polymerase II ratios measured by run-on and those obtained by chromatin immunoprecipitation is poor, although the genes encoding ribosomal proteins present exceptionally low ratios in both cases. We detected a subset of elongation-related factors that are important for maintaining the wild-type profiles of active transcription, including DSIF, Mediator, factors related to the methylation of histone H3-lysine 4, the Bur CDK and the RNA polymerase II subunit Rpb9. We conducted a more detailed investigation of the alterations caused by rpb9Δ to find that Rpb9 contributes to the intragenic profiles of active transcription by influencing the probability of arrest of RNA polymerase II.

Yeast Genetic Analysis Reveals the Involvement of Chromatin Reassembly Factors in Repressing HIV-1 Basal Transcription

Rebound of HIV viremia after interruption of anti-retroviral therapy is due to the small population of CD4+ T cells that remain latently infected. HIV-1 transcription is the main process controlling post-integration latency. Regulation of HIV-1 transcription takes place at both initiation and elongation levels. Pausing of RNA polymerase II at the 5′ end of HIV-1 transcribed region (5′HIV-TR), which is immediately downstream of the transcription start site, plays an important role in the regulation of viral expression. The activation of HIV-1 transcription correlates with the rearrangement of a positioned nucleosome located at this region. These two facts suggest that the 5′HIV-TR contributes to inhibit basal transcription of those HIV-1 proviruses that remain latently inactive. However, little is known about the cell elements mediating the repressive role of the 5′HIV-TR. We performed a genetic analysis of this phenomenon in Saccharomyces cerevisiae after reconstructing a minimal HIV-1 transcriptional system in this yeast. Unexpectedly, we found that the critical role played by the 5′HIV-TR in maintaining low levels of basal transcription in yeast is mediated by FACT, Spt6, and Chd1, proteins so far associated with chromatin assembly and disassembly during ongoing transcription. We confirmed that this group of factors plays a role in HIV-1 postintegration latency in human cells by depleting the corresponding human orthologs with shRNAs, both in HIV latently infected cell populations and in particular single-integration clones, including a latent clone with a provirus integrated in a highly transcribed gene. Our results indicate that chromatin reassembly factors participate in the establishment of the equilibrium between activation and repression of HIV-1 when it integrates into the human genome, and they open the possibility of considering these factors as therapeutic targets of HIV-1 latency.

The Prefoldin Complex Regulates Chromatin Dynamics during Transcription Elongation

Transcriptional elongation requires the concerted action of several factors that allow RNA polymerase II to advance through chromatin in a highly processive manner. In order to identify novel elongation factors, we performed systematic yeast genetic screening based on the GLAM (Gene Length-dependent Accumulation of mRNA) assay, which is used to detect defects in the expression of long transcription units. Apart from well-known transcription elongation factors, we identified mutants in the prefoldin complex subunits, which were among those that caused the most dramatic phenotype. We found that prefoldin, so far involved in the cytoplasmic co-translational assembly of protein complexes, is also present in the nucleus and that a subset of its subunits are recruited to chromatin in a transcription-dependent manner. Prefoldin influences RNA polymerase II the elongation rate in vivo and plays an especially important role in the transcription elongation of long genes and those whose promoter regions contain a canonical TATA box. Finally, we found a specific functional link between prefoldin and histone dynamics after nucleosome remodeling, which is consistent with the extensive network of genetic interactions between this factor and the machinery regulating chromatin function. This study establishes the involvement of prefoldin in transcription elongation, and supports a role for this complex in cotranscriptional histone eviction.

Application of flow focusing to the break-up of a magnetite suspension jet for the production of paramagnetic microparticles

Paramagnetic particles offer an extensive improvement in the magnetic separation or purification of a wide variety of protein molecules. Most commercial paramagnetic particles are synthesized by laborious and costly procedures. A straightforward production of paramagnetic microparticles with homogeneous and selectable sizes using flow focusing (FF) technology is described in this work. The development of an initial formulation of a stable iron oxide suspension compatible with the FF requirements is also reported. The obtained particles, below 10 microns in diameter and presenting smooth and reactive surface, were codified with an organic fluorophore and showed excellent properties for covalent attachment of biomolecules such as proteins and its subsequent recognition by flow cytometry. Furthermore, particles with suitable magnetite content resulted as well-suited for commercial magnet separators for these purposes.