Shaping the Transcriptional Landscape through MAPK Signaling

A change in the transcriptional landscape is an equilibrium-breaking event important for many biological processes. Mitogen-activated protein kinase (MAPK) signaling pathways are dedicated to sensing extracellular cues and are highly conserved across eukaryotes. Modulation of gene expression in response to the extracellular environment is one of the main mechanisms by which MAPK regulates proteome homeostasis to orchestrate adaptive responses that determine cell fate. A massive body of knowledge generated from population and single-cell analyses has led to an understanding of how MAPK pathways operate. MAPKs have thus emerged as fundamental transcriptome regulators that function through a multi-layered control of gene expression, a process often deregulated in disease, which therefore provides an attractive target for therapeutic strategies. Here, we summarize the current understanding of the mechanisms underlying MAPK-mediated gene expression in organisms ranging from yeast to mammals

Structural disruption of BAF chromatin remodeller impairs neuroblastoma metastasis by reverting an invasiveness epigenomic program

Background Epigenetic programming during development is essential for determining cell lineages, and alterations in this programming contribute to the initiation of embryonal tumour development. In neuroblastoma, neural crest progenitors block their course of natural differentiation into sympathoadrenergic cells, leading to the development of aggressive and metastatic paediatric cancer. Research of the epigenetic regulators responsible for oncogenic epigenomic networks is crucial for developing new epigenetic-based therapies against these tumours. Mammalian switch/sucrose non-fermenting (mSWI/SNF) ATP-dependent chromatin remodelling complexes act genome-wide translating epigenetic signals into open chromatin states. The present study aimed to understand the contribution of mSWI/SNF to the oncogenic epigenomes of neuroblastoma and its potential as a therapeutic target. Methods Functional characterisation of the mSWI/SNF complexes was performed in neuroblastoma cells using proteomic approaches, loss-of-function experiments, transcriptome and chromatin accessibility analyses, and in vitro and in vivo assays. Results Neuroblastoma cells contain three main mSWI/SNF subtypes, but only BRG1-associated factor (BAF) complex disruption through silencing of its key structural subunits, ARID1A and ARID1B, impairs cell proliferation by promoting cell cycle blockade. Genome-wide chromatin remodelling and transcriptomic analyses revealed that BAF disruption results in the epigenetic repression of an extensive invasiveness-related expression program involving integrins, cadherins, and key mesenchymal regulators, thereby reducing adhesion to the extracellular matrix and the subsequent invasion in vitro and drastically inhibiting the initiation and growth of neuroblastoma metastasis in vivo. Conclusions We report a novel ATPase-independent role for the BAF complex in maintaining an epigenomic program that allows neuroblastoma invasiveness and metastasis, urging for the development of new BAF pharmacological structural disruptors for therapeutic exploitation in metastatic neuroblastoma

Control of Ubp3 ubiquitin protease activity by the Hog1 SAPK modulates transcription upon osmostress

Here, the Hog1 kinase interacts with and activates the ubiquitin protease Ubp3 in a stress-dependent manner. The phosphorylation of Ubp3 enhances RNA polymerase II occupancy on osmotic stress-responsive genes

Control of transcription by the stress activated Hog1 kinase

A fundamental property of living cells is the ability to sense and robustly respond to fluctuations in their environment. In budding yeast (Saccharomyces cerevisiae) changes in extracellular osmolarity are sensed by the HOG pathway, which evokes the program for cell adaptation required for cell survival. The aim of this thesis was to further characterize the molecular mechanisms by which Hog1 regulates gene expression upon osmostress. A genome-wide genetic screen lead to the identification of several activities required for regulation of gene expression. Here we describe the characterization of a novel substrate for the SAPK whose activity is required for proper transcription initiation and elongation in response to stress. This thesis also aimed to globally characterize the role of Hog1 in reprogramming the transcriptome of S. cerevisiae under osmostress conditions. By the combination of molecular approaches coupled to genome-wide techniques (ChIP-seq, MNase-seq and Tiling arrays) we have been able to fully characterize the localization of the key components that drive osmoresponsive transcription, providing for the first time a complete picture of the transcription process. The high resolution of the genome-wide approaches, has allowed us to identify new transcriptional roles for the SAPK such as the targeting of RNA Pol III machinery, and the regulation of a novel class of functional long noncoding RNAs (lncRNA). In summary, results presented in this thesis provide novel insights into the mechanisms by which the Hog1 SAPK modulates gene expression.Una propietat cel·lular fonamental és l’habilitat de detectar i respondre de forma robusta a les fluctuacions en el seu entorn. En cèl·lules de llevat (Saccharomyces cerevisiae), els canvis en l’ osmolaritat extracel·lular són detectats per la via de senyalització de HOG, que coordina el procés d’adaptació cel·lular imprescindible per sobreviure a un estrès osmòtic. L’objectiu d’aquest estudi és identificar i caracteritzar els mecanismes moleculars utilitzats per Hog1 per regular l’expressió gènica en resposta a estrès osmòtic. Fent servir un crivatge genètic a gran escala dissenyat per identificar activitats necessàries per la regulació de l’expressió gènica en resposta a estrès osmòtic, hem identificat un nou substrat de Hog1, l’activitat del qual és requereix tan per la iniciació com l’ elongació de la transcripció. En aquest treball també ens hem centrat en caracteritzar el paper global de Hog1 en la reorganització del transcriptoma de S. cerevisae en condicions d’ estrès osmòtic. Mitjançant la combinació de tècniques moleculars amb tècniques de seqüenciació (ChIP-seq, MNase-seq, Tiling arrays) hem definit el posicionament en el genoma dels components claus que regulen la transcripció, oferint per primera vegada una visió general del procés de transcripció en resposta a estrès osmòtic L’alta resolució d’aquestes tècniques ens ha permès identificar noves dianes transcripcionals de Hog1, com és la regulació d’una altra maquinària transcripcional (RNA Pol III) i la regulació de la transcripció de una nova classe de RNAs no codificants (lncRNAs). En conjunt, els resultats presentats en aquesta tesi proporcionen una nova visió dels mecanismes per els quals Hog1 modula l’expressió gènic

The rise of single-cell transcriptomics in yeast

The field of single-cell omics has transformed our understanding of biological processes and is constantly advancing both experimentally and computationally. One of the most significant developments is the ability to measure the transcriptome of individual cells by single-cell RNA-seq (scRNA-seq), which was pioneered in higher eukaryotes. While yeast has served as a powerful model organism in which to test and develop transcriptomic technologies, the implementation of scRNA-seq has been significantly delayed in this organism, mainly because of technical constraints associated with its intrinsic characteristics, namely the presence of a cell wall, a small cell size and little amounts of RNA. In this review, we examine the current technologies for scRNA-seq in yeast and highlight their strengths and weaknesses. Additionally, we explore opportunities for developing novel technologies and the potential outcomes of implementing single-cell transcriptomics and extension to other modalities. Undoubtedly, scRNA-seq will be invaluable for both basic and applied yeast research, providing unique insights into fundamental biological processes.The laboratories of FP and EdeN are supported by a coordinated grant from the Ministry of Science, Innovation, and Universities (PID2021-124723NB-C21/C22 and FEDER). We also gratefully acknowledge institutional funding from the Ministry of Science, Innovation and Universities through the Centres of Excellence Severo Ochoa Award, and from the CERCA Programme of the Government of Catalonia and the Unidad de Excelencia María de Maeztu, funded by the AEI (CEX2018-000792-M)

A novel role for lncRNAs in cell cycle control during stress adaptation

Eukaryotic cells have developed sophisticated systems to constantly monitor changes in the extracellular environment and to orchestrate a proper cellular response. To maximize survival, cells delay cell-cycle progression in response to environmental changes. In response to extracellular insults, stress-activated protein kinases (SAPKs) modulate cell-cycle progression and gene expression. In yeast, osmostress induces activation of the p38-related SAPK Hog1, which plays a key role in reprogramming gene expression upon osmostress. Genomic analysis has revealed the existence of a large number of long non-coding RNAs (lncRNAs) with different functions in a variety of organisms, including yeast. Upon osmostress, hundreds of lncRNAs are induced by the SAPK p38/Hog1. One gene that expresses Hog1-dependent lncRNA in an antisense orientation is the CDC28 gene, which encodes CDK1 kinase that controls the cell cycle in yeast. Cdc28 lncRNA mediates the induction of CDC28 expression and this increase in the level of Cdc28 results in more efficient re-entry of the cells into the cell cycle after stress. Thus, the control of lncRNA expression as a new mechanism for the regulation of cell-cycle progression opens new avenues to understand how stress adaptation can be accomplished in response to changing environments.The laboratory of FP and EN is supported by grants from the Spanish Government (BFU2012-33503 and FEDER to FP, BFU2011-26722 to EN), an ERC Advanced Grant Number 294294 from the EU seventh framework program (SYNCOM) and the Fundación Marcelino Botín (FMB) to FP. FP and EN are recipients of an ICREA Acadèmia (Generalitat de Catalunya). The authors declare no competing financial interes

Control of Cdc28 CDK1 by a stress-induced lncRNA

Genomic analysis has revealed the existence of a large number of long noncoding RNAs (lncRNAs) with different functions in a variety of organisms, including yeast. Cells display dramatic changes of gene expression upon environmental changes. Upon osmostress, hundreds of stress-responsive genes are induced by the stress-activated protein kinase (SAPK) p38/Hog1. Using whole-genome tiling arrays, we found that Hog1 induces a set of lncRNAs upon stress. One of the genes expressing a Hog1-dependent lncRNA in antisense orientation is CDC28, the cyclin-dependent kinase 1 (CDK1) that controls the cell cycle in yeast. Cdc28 lncRNA mediates the establishment of gene looping and the relocalization of Hog1 and RSC from the 3′ UTR to the +1 nucleosome to induce CDC28 expression. The increase in the levels of Cdc28 results in cells able to reenter the cell cycle more efficiently after stress. This may represent a general mechanism to prime expression of genes needed after stresses are alleviated.This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (BFU2012-33503 and FEDER to F.P., BFU2011-26722 to E.d.N.), the Fundación Marcelino Botín (FMB), and the Consolider Ingenio 2010 programme CSD2007-0015 (to F.P.). This work was supported by the National Institutes of Health and Deutsche Forschungsgemeinschaft (L.M.S.). F.P. and E.d.N. are recipients of an ICREA Acadèmia award (Generalitat de Catalunya)

Control of Ubp3 ubiquitin protease activity by the Hog1 SAPK modulates transcription upon osmostress

Protein ubiquitylation is a key process in the regulation of many cellular processes. The balance between the activity of ubiquitin ligases and that of proteases controls the level of ubiquitylation. In response to extracellular stimuli, stress-activated protein kinases (SAPK) modulate gene expression to maximize cell survival. In yeast, the Hog1 SAPK has a key role in reprogramming the gene expression pattern required for cell survival upon osmostress. Here, we show that the Ubp3 ubiquitin protease is a target for the Hog1 SAPK to modulate gene expression. ubp3 mutant cells are defective in expression of osmoresponsive genes. Hog1 interacts with and phosphorylates Ubp3 at serine 695, which is essential to determine the extent of transcriptional activation in response to osmostress. Furthermore, Ubp3 is recruited to osmoresponsive genes to modulate transcriptional initiation as well as elongation. Therefore, Ubp3 activity responds to external stimuli and is required for transcriptional activation upon osmostress.MN is the recipient of an FIS (Spanish Government) fellowship and FP is the recipient of an ICREA Acadèmia (Generalitat de Catalunya). This work was supported by Fundación Marcelino Botín (FMB) and grants from the Spanish Ministry of Science and Innovation (BFU2008-00530 to EN and BIO2009-07762 to FP) and the FP7 (UNICELLSYS) framework programme

Hog1 bypasses stress-mediated down-regulation of transcription by RNA polymerase II redistribution and chromatin remodeling

Cells are subjected to dramatic changes of gene expression upon environmental changes. Stress/ncauses a general down-regulation of gene expression together with the induction of a set of stress-responsive/ngenes. The p38-related stress-activated protein kinase Hog1 is an important regulator of transcription upon/nosmostress in yeast. Genome-wide localization studies of RNA polymerase II (RNA Pol II) and Hog1 showed that stress induced major changes in RNA Pol II localization, with a shift toward stress-responsive genes relative to housekeeping genes. RNA Pol II relocalization required Hog1, which was also localized to stress-responsive loci. In addition to RNA Pol II-bound genes, Hog1 also localized to RNA polymerase III-bound genes, pointing to a wider role for Hog1 in transcriptional control than initially expected. Interestingly, an increasing association of Hog1 with stressresponsive genes was strongly correlated with chromatin remodeling and increased gene expression. Remarkably, MNase-Seq analysis showed that although chromatin structure was not significantly altered at a genome-wide level in response to stress, there was pronounced chromatin remodeling for those genes that displayed Hog1 association. Hog1 serves to bypass the general down-regulation of gene expression that occurs in response to osmostress, and does so both by targeting RNA Pol II machinery and by inducing chromatin remodeling at stressresponsive loci.MN is a recipient of an FIS fellowship. This work was supported by grants from the Spanish Government (BIO2011-23920 to EE, BIO2009-07762 and BFU2012-33503 to FP, BFU2011-26722 to EN), the Consolider Ingenio 2010/nprogramme CSD2007-0015 to FP and FP7 UNICELLSYS grant 201142, the Fundación Marcelino Botín (FMB) to FP. EN and FP are recipients of an ICREA Acadèmia (Generalitat de Catalunya)