Sebastián Chávez

A protein complex containing Tho2, Hpr1, Mft1 and a novel protein, Thp2, connects transcription elongation with mitotic recombination in Saccharomyces cerevisiae

Transcription‐induced recombination has been reported in all organisms from bacteria to mammals. We have shown previously that the yeast genes HPR1 and THO2 may be keys to the understanding of transcription‐associated recombination, as they both affect transcription elongation and hyper‐recombination in a concerted manner. Using a yeast strain that has the wild‐type THO2 gene replaced by one encoding a His6‐HA‐tagged version, we have isolated an oligomeric complex containing four proteins: Tho2, Hpr1, Mft1 and a novel protein that we have named Thp2. We have reciprocally identified a complex containing Hpr1, Tho2 and Mft1 using anti‐Mft1 antibodies in immunoprecipitation experiments. The protein complex is mainly nuclear; therefore, Tho2 and Hpr1 are physically associated. Like hpr1Δ and tho2Δ cells, mft1Δ and thp2Δ cells show mitotic hyper‐ recombination and impaired transcription elongation, in particular, through the bacterial lacZ sequence. Hyper‐recombination conferred by mft1Δ and thp2Δ is only observed in DNA regions under transcription conditions. We propose that this protein complex acts as a functional unit connecting transcription elongation with the incidence of mitotic recombination.

Cytoplasmic 5 -3 exonuclease Xrn1p is also a genome-wide transcription factor in yeast

The 5′ to 3′ exoribonuclease Xrn1 is a large protein involved in cytoplasmatic mRNA degradation as a critical component of the major decaysome. Its deletion in the yeast Saccharomyces cerevisiae is not lethal, but it has multiple physiological effects. In a previous study, our group showed that deletion of all tested components of the yeast major decaysome, including XRN1, results in a decrease in the synthetic rate and an increase in half-life of most mRNAs in a compensatory manner. Furthermore, the same study showed that the all tested decaysome components are also nuclear proteins that bind to the 5′ region of a number of genes. In the present work, we show that disruption of Xrn1 activity preferentially affects both the synthesis and decay of a distinct subpopulation of mRNAs. The most affected mRNAs are the transcripts of the highly transcribed genes, mainly those encoding ribosome biogenesis and translation factors. Previously, we proposed that synthegradases play a key role in regulating both mRNA synthesis and degradation. Evidently, Xrn1 functions as a synthegradase, whose selectivity might help coordinating the expression of the protein synthetic machinery. We propose to name the most affected genes “Xrn1 synthegradon.”

Transcriptional regulation by steroid hormones.

Steroid hormones influence the transcription of a large number of genes by virtue of their interaction with intracellular receptors, which are modular proteins composed of a ligand binding domain, a DNA binding domain, and several transactivation functions distributed along the molecule. The DNA binding domain is organized around two zinc ions and allows the receptors to bind as homodimers to palindromic DNA sequences, the hormones responsive elements (HRE), is such a way that each homodimer contacts one half of the palindrome. Since the two halves are separated by three base pairs, the two homodimers contact the same face of the double helix. Before hormone binding, the receptors are part of a complex with multiple chaperones which maintain the receptor in its steroid binding conformation. Following hormone binding, the complex dissociates and the receptors bind to HREs in chromatin. Regulation of gene expression by hormones involves an interaction of the DNA-bound receptors with other sequence-specific transcription factors and with the general transcription factors, which is partly mediated by co-activators and co-repressors. The specific array of cis regulatory elements in a particular promoter/enhancer region, as well as the organization of the DNA sequences in nucleosomes, specifies the network of receptor interactions. Depending on the nature of these interactions, the final outcome can be induction or repression of transcription. The various levels at which these interactions are modulated are discussed using as an example the promoter of the Mouse Mammary Tumor Virus and its organization in chromatin.

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.

Hpr1 is preferentially required for transcription of either long or G+C-rich DNA sequences in Saccharomyces cerevisiae.

ABSTRACT Hpr1 forms, together with Tho2, Mft1, and Thp2, the THO complex, which controls transcription elongation and genome stability inSaccharomyces cerevisiae. Mutations in genes encoding the THO complex confer strong transcription-impairment and hyperrecombination phenotypes in the bacterial lacZgene. In this work we demonstrate that Hpr1 is a factor required for transcription of long as well as G+C-rich DNA sequences. Using different lacZ segments fused to the GAL1promoter, we show that the negative effect of lacZsequences on transcription depends on their distance from the promoter. In parallel, we show that transcription of either a longLYS2 fragment or the S. cerevisiae YAT1G+C-rich open reading frame fused to the GAL1 promoter is severely impaired in hpr1 mutants, whereas transcription of LAC4, the Kluyveromyces lactis ortholog of lacZ but with a lower G+C content, is only slightly affected. The hyperrecombination behavior of the DNA sequences studied is consistent with the transcriptional defects observed in hpr1 cells. These results indicate that both length and G+C content are important elements influencing transcription in vivo. We discuss their relevance for the understanding of the functional role of Hpr1 and, by extension, the THO complex.

Recruitment of a chromatin remodelling complex by the Hog1 MAP kinase to stress genes

For efficient transcription, RNA PolII must overcome the presence of nucleosomes. The p38‐related MAPK Hog1 is an important regulator of transcription upon osmostress in yeast and thereby it is involved in initiation and elongation. However, the role of this protein kinase in elongation has remained unclear. Here, we show that during stress there is a dramatic change in the nucleosome organization of stress‐responsive loci that depends on Hog1 and the RSC chromatin remodelling complex. Upon stress, the MAPK Hog1 physically interacts with RSC to direct its association with the ORF of osmo‐responsive genes. In RSC mutants, PolII accumulates on stress promoters but not in coding regions. RSC mutants also display reduced stress gene expression and enhanced sensitivity to osmostress. Cell survival under acute osmostress might thus depend on a burst of transcription that in turn could occur only with efficient nucleosome eviction. Our results suggest that the selective targeting of the RSC complex by Hog1 provides the necessary mechanistic basis for this event.

Sus1 is recruited to coding regions and functions during transcription elongation in association with SAGA and TREX2.

Gene transcription, RNA biogenesis, and mRNA transport constitute a complicated process essential for all eukaryotic cells. The transcription/export factor Sus1 plays a key role in coupling transcription activation with mRNA export, and it resides in both the SAGA and TREX2 complexes. Moreover, Sus1 is responsible for GAL1 gene gating at the nuclear periphery, which is important for its transcriptional status. Here, we show that Sus1 is required during transcription elongation and is associated with the elongating form of RNA Polymerase II (RNAP II) phosphorylated on Ser5 and Ser2 of the C-terminal domain (CTD). In addition, Sus1 copurifies with the essential mRNA export factors Yra1 and Mex67, which bind to the mRNA cotranscriptionally. Consistently, ChIP analysis reveals that Sus1 is present at coding regions dependent on transcription in a manner stimulated by Kin28-dependent CTD phosphorylation. Strikingly, eliminating the TREX2 component Sac3 or the SAGA subunit Ubp8 partially impairs Sus1 targeting to coding sequences and upstream activating sequences (UAS). We found, unexpectedly, that Sgf73 is necessary for association of Sus1 with both SAGA and TREX2, and that its absence dramatically reduces Sus1 occupancy of UAS and ORF sequences. Our results reveal that Sus1 plays a key role in coordinating gene transcription and mRNA export by working at the interface between the SAGA and TREX2 complexes during transcription elongation.

A Complete Set of Nascent Transcription Rates for Yeast Genes

The amount of mRNA in a cell is the result of two opposite reactions: transcription and mRNA degradation. These reactions are governed by kinetics laws, and the most regulated step for many genes is the transcription rate. The transcription rate, which is assumed to be exercised mainly at the RNA polymerase recruitment level, can be calculated using the RNA polymerase densities determined either by run-on or immunoprecipitation using specific antibodies. The yeast Saccharomyces cerevisiae is the ideal model organism to generate a complete set of nascent transcription rates that will prove useful for many gene regulation studies. By combining genomic data from both the GRO (Genomic Run-on) and the RNA pol ChIP-on-chip methods we generated a new, more accurate nascent transcription rate dataset. By comparing this dataset with the indirect ones obtained from the mRNA stabilities and mRNA amount datasets, we are able to obtain biological information about posttranscriptional regulation processes and a genomic snapshot of the location of the active transcriptional machinery. We have obtained nascent transcription rates for 4,670 yeast genes. The median RNA polymerase II density in the genes is 0.078 molecules/kb, which corresponds to an average of 0.096 molecules/gene. Most genes have transcription rates of between 2 and 30 mRNAs/hour and less than 1% of yeast genes have >1 RNA polymerase molecule/gene. Histone and ribosomal protein genes are the highest transcribed groups of genes and other than these exceptions the transcription of genes is an infrequent phenomenon in a yeast cell.

Mitotic recombination in yeast: elements controlling its incidence.

Mitotic recombination is an important mechanism of DNA repair in eukaryotic cells. Given the redundancy of the eukaryotic genomes and the presence of repeated DNA sequences, recombination may also be an important source of genomic instability. Here we review the data, mainly from the budding yeast S. cerevisiae, that may help to understand the spontaneous origin of mitotic recombination and the different elements that may control its occurrence. We cover those observations suggesting a putative role of replication defects and DNA damage, including double‐strand breaks, as sources of mitotic homologous recombination. An important part of the review is devoted to the experimental evidence suggesting that transcription and chromatin structure are important factors modulating the incidence of mitotic recombination. This is of great relevance in order to identify the causes and risk factors of genomic instability in eukaryotes. Copyright

Eukaryotic mRNA Decay: Methodologies, Pathways, and Links to Other Stages of Gene Expression

mRNA concentration depends on the balance between transcription and degradation rates. On both sides of the equilibrium, synthesis and degradation show, however, interesting differences that have conditioned the evolution of gene regulatory mechanisms. Here, we discuss recent genome-wide methods for determining mRNA half-lives in eukaryotes. We also review pre- and posttranscriptional regulons that coordinate the fate of functionally related mRNAs by using protein- or RNA-based trans factors. Some of these factors can regulate both transcription and decay rates, thereby maintaining proper mRNA homeostasis during eukaryotic cell life.