Interacciones entre activadores y represores en el inicio de la transcripción en Saccharomyces cerevisiae

Autosomic dominant lateral temporal epilepsy (ADLTE) is a form of partial epilepsy with secondary generalized seizures, caused by mutations in LGI1. This gene was initially identified in 1998, as a tumour suppressor in multiform glioblastoma, affecting to the malignancy of these tumours. In silico analysis of the primary sequence of LGI1, revealed four paralogues of the gene, which shared a very similar domain structure and were named as LGI1, LGI2, LGI3 and LGI4. The existence of similar types of epilepsy with common features for that seen for ADLTE, suggests that some other members of this gene family could be involved in these pathologies. Moreover, the presence of four paralogues, makes the idea of a certain degree of functional redundancy, possible. This fact, could be explained by the complementary distribution of cells expressing the genes of this family in the brain. In this work, we show a detailed expression analysis of the mRNA of the members of the LGI family in adult mouse brain. The distribution of these genes is regionally heterogeneous, suggesting that since their origin as a common ancestral gene, a subfunctionalization might have occurred. In this work, we try to shed light on these possible functions.Negative cofactor 2 (NC2) has been described as an essential and evolutionarily conserved transcriptional repressor, although in vitro and in vivo experiments suggest that it can function as both a positive and a negative effector of transcription. NC2 operates by interacting with the core promoter and components of the basal transcription machinery, like the TBP. In this work, we have isolated mutants that suppress the growth defect caused by depletion of NC2. We have identified mutations affecting components of three different complexes involved in the control of basal transcription: the mediator, TFIIH, and RNA pol II itself. Mutations in RNA pol II include both overexpression of truncated forms of the two largest subunits (Rpb1p and Rpb2p) and reduced levels of these proteins. Suppression of NC2 depletion was also observed by reducing the amounts of the mediator essential components Nut2p and Med7p, as well as by deleting any of the nonessential mediator components, except Med2p, Med3p, and Gal11p subunits. Interestingly, the Med2p/Med3p/Gal11p triad forms a submodule within the mediator tail. Our results support the existence of different components within the basic transcription complexes that antagonistically interact with NC2 repressor and suggest that the correct balance between the activities of specific positive and negative components is essential for cell growth. In the second part of this work, we study a novel and highly conserved factor that physically interacts with most of the RNA pol II subunits, Iwr1p. Here we show that Iwr1p genetically interacts with components of the basal transcription machinery and plays a role in both basal and regulated transcription. We report that mutation of IWR1 gene is able to bypass the otherwise essential requirement for the transcriptional regulator NC2, which occurs with different components of the basal transcription machinery, including TFIIA and subunits of the mediator complex. Deletion of the IWR1 gene leads to an altered expression of specific genes, including phosphate-responsive genes and SUC2. Our results show that Iwr1p is a nucleocytoplasmic shuttling protein and suggest that Iwr1p acts early in the formation of the pre-initiation complex by mediating the interaction of certain activators with the basal transcription apparatus

Insights into mRNP Biogenesis Provided by New Genetic Interactions Among Export and Transcription Factors

The various steps of mRNP biogenesis (transcription, processing and export) are interconnected. It has been shown that the transcription machinery plays a pivotal role in mRNP assembly, since several mRNA export factors are recruited during transcription and physically interact with components of the transcription machinery. Although the shuttling DEAD-box protein Dbp5p is concentrated on the cytoplasmic fibrils of the NPC, previous studies demonstrated that it interacts physically and genetically with factors involved in transcription initiation. We investigated the effect of mutations affecting various components of the transcription initiation apparatus on the phenotypes of mRNA export mutant strains. Our results show that growth and mRNA export defects of dbp5 and mex67 mutant strains can be suppressed by mutation of specific transcription initiation components, but suppression was not observed for mutants acting in the very first steps of the pre-initiation complex (PIC) formation

Specific Defects in Different Transcription Complexes Compensate for the Requirement of the Negative Cofactor 2 Repressor in Saccharomyces cerevisiae

Negative cofactor 2 (NC2) has been described as an essential and evolutionarily conserved transcriptional repressor, although in vitro and in vivo experiments suggest that it can function as both a positive and a negative effector of transcription. NC2 operates by interacting with the core promoter and components of the basal transcription machinery, like the TATA-binding protein (TBP). In this work, we have isolated mutants that suppress the growth defect caused by the depletion of NC2. We have identified mutations affecting components of three different complexes involved in the control of basal transcription: the mediator, TFIIH, and RNA pol II itself. Mutations in RNA pol II include both overexpression of truncated forms of the two largest subunits (Rpb1 and Rpb2) and reduced levels of these proteins. Suppression of NC2 depletion was also observed by reducing the amounts of the mediator essential components Nut2 and Med7, as well as by deleting any of the nonessential mediator components, except Med2, Med3, and Gal11 subunits. Interestingly, the Med2/Med3/Gal11 triad forms a submodule within the mediator tail. Our results support the existence of different components within the basic transcription complexes that antagonistically interact with the NC2 repressor and suggest that the correct balance between the activities of specific positive and negative components is essential for cell growth

Insights into mRNP biogenesis provided by new genetic interactions among export and transcription factors

<p>Abstract</p> <p>Background</p> <p>The various steps of mRNP biogenesis (transcription, processing and export) are interconnected. It has been shown that the transcription machinery plays a pivotal role in mRNP assembly, since several mRNA export factors are recruited during transcription and physically interact with components of the transcription machinery. Although the shuttling DEAD-box protein Dbp5p is concentrated on the cytoplasmic fibrils of the NPC, previous studies demonstrated that it interacts physically and genetically with factors involved in transcription initiation.</p> <p>Results</p> <p>We investigated the effect of mutations affecting various components of the transcription initiation apparatus on the phenotypes of mRNA export mutant strains. Our results show that growth and mRNA export defects of <it>dbp5</it> and <it>mex67</it> mutant strains can be suppressed by mutation of specific transcription initiation components, but suppression was not observed for mutants acting in the very first steps of the pre-initiation complex (PIC) formation.</p> <p>Conclusions</p> <p>Our results indicate that mere reduction in the amount of mRNP produced is not sufficient to suppress the defects caused by a defective mRNA export factor. Suppression occurs only with mutants affecting events within a narrow window of the mRNP biogenesis process. We propose that reducing the speed with which transcription converts from initiation and promoter clearance to elongation may have a positive effect on mRNP formation by permitting more effective recruitment of partially-functional mRNP proteins to the nascent mRNP.</p

Role of glutathione in the regulation of epigenetic mechanisms in disease

Epigenetics is a rapidly growing field that studies gene expression modifications not involving changes in the DNA sequence. Histone H3, one of the basic proteins in the nucleosomes that make up chromatin, is S-glutathionylated in mammalian cells and tissues, making Gamma-L-glutamyl-L-cysteinylglycine, glutathione (GSH), a physiological antioxidant and second messenger in cells, a new post-translational modifier of the histone code that alters the structure of the nucleosome. However, the role of GSH in the epigenetic mechanisms likely goes beyond a mere structural function. Evidence supports the hypothesis that there is a link between GSH metabolism and the control of epigenetic mechanisms at different levels (i.e., substrate availability, enzymatic activity for DNA methylation, changes in the expression of microRNAs, and participation in the histone code). However, little is known about the molecular pathways by which GSH can control epigenetic events. Studying mutations in enzymes involved in GSH metabolism and the alterations of the levels of cofactors affecting epigenetic mechanisms appears challenging. However, the number of diseases induced by aberrant epigenetic regulation is growing, so elucidating the intricate network between GSH metabolism, oxidative stress and epigenetics could shed light on how their deregulation contributes to the development of neurodegeneration, cancer, metabolic pathologies and many other types of diseases.Sin financiación6.020 JCR (2017) Q1, 39/292 Biochemistry and Molecular Biology, 17/143 Endocrinology and MetabolismUE

In search of an evidence-based strategy for quality assessment of human tissue samples: report of the tissue biospecimen research working group of the spanish biobank network

The purpose of the present work is to underline the importance of obtaining a standardized procedure to ensure and evaluate both clinical and research usability of human tissue samples. The study, which was carried out by the Biospecimen Science Working Group of the Spanish Biobank Network, is based on a general overview of the cur‑ rent situation about quality assurance in human tissue biospecimens. It was conducted an exhaustive review of the analytical techniques used to evaluate the quality of human tissue samples over the past 30 years, as well as their reference values if they were published, and classifed them according to the biomolecules evaluated: (i) DNA, (ii) RNA, and (iii) soluble or/and fxed proteins for immunochemistry. More than 130 publications released between 1989 and 2019 were analysed, most of them reporting results focused on the analysis of tumour and biopsy samples. A qual‑ ity assessment proposal with an algorithm has been developed for both frozen tissue samples and formalin-fxed parafn-embedded (FFPE) samples, according to the expected quality of sample based on the available pre-analytical information and the experience of the participants in the Working Group. The high heterogeneity of human tissue samples and the wide number of pre-analytic factors associated to quality of samples makes it very difcult to harmo‑ nize the quality criteria. However, the proposed method to assess human tissue sample integrity and antigenicity will not only help to evaluate whether stored human tissue samples ft for the purpose of biomarker development, but will also allow to perform further studies, such as assessing the impact of diferent pre-analytical factors on very well characterized samples or evaluating the readjustment of tissue sample collection, processing and storing procedur