The zinc-finger protein Z4 cooperates with condensin II to regulate somatic chromosome pairing and 3D chromatin organization.

Chromosome pairing constitutes an important level of genome organization, yet the mechanisms that regulate pairing in somatic cells and the impact on 3D chromatin organization are still poorly understood. Here, we address these questions in Drosophila, an organism with robust somatic pairing. In Drosophila, pairing preferentially occurs at loci consisting of numerous architectural protein binding sites (APBSs), suggesting a role of architectural proteins (APs) in pairing regulation. Amongst these, the anti-pairing function of the condensin II subunit CAP-H2 is well established. However, the factors that regulate CAP-H2 localization and action at APBSs remain largely unknown. Here, we identify two factors that control CAP-H2 occupancy at APBSs and, therefore, regulate pairing. We show that Z4, interacts with CAP-H2 and is required for its localization at APBSs. We also show that hyperosmotic cellular stress induces fast and reversible unpairing in a Z4/CAP-H2 dependent manner. Moreover, by combining the opposite effects of Z4 depletion and osmostress, we show that pairing correlates with the strength of intrachromosomal 3D interactions, such as active (A) compartment interactions, intragenic gene-loops, and polycomb (Pc)-mediated chromatin loops. Altogether, our results reveal new players in CAP-H2-mediated pairing regulation and the intimate interplay between inter-chromosomal and intra-chromosomal 3D interactions.

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.

Somatic chromosome pairing has a determinant impact on 3D chromatin organization.

In the nucleus, chromatin is intricately structured into multiple layers of 3D organization important for genome activity. How distinct layers influence each other is not well understood. In particular, the contribution of chromosome pairing to 3D chromatin organization has been largely neglected. Here, we address this question in Drosophila, an organism that shows robust chromosome pairing in interphasic somatic cells. The extent of chromosome pairing depends on the balance between pairing and anti-pairing factors, with the anti-pairing activity of the CAP-H2 condensin II subunit being the best documented. Here, we identify the zinc-finger protein Z4 as a strong anti-pairer that interacts with and mediates the chromatin binding of CAP-H2. We also report that hyperosmotic cellular stress induces fast and reversible chromosome unpairing that depends on Z4/CAP-H2. And, most important, by combining Z4 depletion and osmostress, we show that chromosome pairing reinforces intrachromosomal 3D interactions. On the one hand, pairing facilitates RNAPII occupancy that correlates with enhanced intragenic gene-loop interactions. In addition, acting at a distance, pairing reinforces chromatin-loop interactions mediated by Polycomb (Pc). In contrast, chromosome pairing does not affect which genomic intervals segregate to active (A) and inactive (B) compartments, with only minimal effects on the strength of A-A compartmental interactions. Altogether, our results unveil the intimate interplay between inter-chromosomal and intra-chromosomal 3D interactions, unraveling the interwoven relationship between different layers of chromatin organization and the essential contribution of chromosome pairing.

Positive feedback induces switch between distributive and processive phosphorylation of Hog1.

Cellular decision making often builds on ultrasensitive MAPK pathways. The phosphorylation mechanism of MAP kinase has so far been described as either distributive or processive, with distributive mechanisms generating ultrasensitivity in theoretical analyses. However, the in vivo mechanism of MAP kinase phosphorylation and its activation dynamics remain unclear. Here, we characterize the regulation of the MAP kinase Hog1 in Saccharomyces cerevisiae via topologically different ODE models, parameterized on multimodal activation data. Interestingly, our best fitting model switches between distributive and processive phosphorylation behavior regulated via a positive feedback loop composed of an affinity and a catalytic component targeting the MAP kinase-kinase Pbs2. Indeed, we show that Hog1 directly phosphorylates Pbs2 on serine 248 (S248), that cells expressing a non-phosphorylatable (S248A) or phosphomimetic (S248E) mutant show behavior that is consistent with simulations of disrupted or constitutively active affinity feedback and that Pbs2-S248E shows significantly increased affinity to Hog1 in vitro. Simulations further suggest that this mixed Hog1 activation mechanism is required for full sensitivity to stimuli and to ensure robustness to different perturbations.

A Genome-Wide Functional Screen Identifies Enhancer and Protective Genes for Amyloid Beta-Peptide Toxicity.

Alzheimer's disease (AD) is known to be caused by amyloid ß-peptide (Aß) misfolded into ß-sheets, but this knowledge has not yet led to treatments to prevent AD. To identify novel molecular players in Aß toxicity, we carried out a genome-wide screen in Saccharomyces cerevisiae, using a library of 5154 gene knock-out strains expressing Aß1-42. We identified 81 mammalian orthologue genes that enhance Aß1-42 toxicity, while 157 were protective. Next, we performed interactome and text-mining studies to increase the number of genes and to identify the main cellular functions affected by Aß oligomers (oAß). We found that the most affected cellular functions were calcium regulation, protein translation and mitochondrial activity. We focused on SURF4, a protein that regulates the store-operated calcium channel (SOCE). An in vitro analysis using human neuroblastoma cells showed that SURF4 silencing induced higher intracellular calcium levels, while its overexpression decreased calcium entry. Furthermore, SURF4 silencing produced a significant reduction in cell death when cells were challenged with oAß1-42, whereas SURF4 overexpression induced Aß1-42 cytotoxicity. In summary, we identified new enhancer and protective activities for Aß toxicity and showed that SURF4 contributes to oAß1-42 neurotoxicity by decreasing SOCE activity.

Implementing re-configurable biological computation with distributed multicellular consortia.

The use of synthetic biological circuits to deal with numerous biological challenges has been proposed in several studies, but its implementation is still remote. A major problem encountered is the complexity of the cellular engineering needed to achieve complex biological circuits and the lack of general-purpose biological systems. The generation of re-programmable circuits can increase circuit flexibility and the scalability of complex cell-based computing devices. Here we present a new architecture to produce reprogrammable biological circuits that allow the development of a variety of different functions with minimal cell engineering. We demonstrate the feasibility of creating several circuits using only a small set of engineered cells, which can be externally reprogrammed to implement simple logics in response to specific inputs. In this regard, depending on the computation needs, a device composed of a number of defined cells can generate a variety of circuits without the need of further cell engineering or rearrangements. In addition, the inclusion of a memory module in the circuits strongly improved the digital response of the devices. The reprogrammability of biological circuits is an intrinsic capacity that is not provided in electronics and it may be used as a tool to solve complex biological problems.

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.

Regulation of Claspin by the p38 stress-activated protein kinase protects cells from DNA damage.

Stress-activated protein kinases (SAPKs) enhance survival in response to environmental changes. In yeast, the Hog1 SAPK and Mrc1, a protein required for DNA replication, define a safeguard mechanism that allows eukaryotic cells to prevent genomic instability upon stress during S-phase. Here we show that, in mammals, the p38 SAPK and Claspin-the functional homolog of Mrc1-protect cells from DNA damage upon osmostress during S-phase. We demonstrate that p38 phosphorylates Claspin and either the mutation of the p38-phosphorylation sites in Claspin or p38 inhibition suppresses the protective role of Claspin on DNA damage. In addition, wild-type Claspin but not the p38-unphosphorylatable mutant has a protective effect on cell survival in response to cisplatin treatment. These findings reveal a role of Claspin in response to chemotherapeutic drugs. Thus, this pathway protects S-phase integrity from different insults and it is conserved from yeast to mammals.

The HOG pathway and the regulation of osmoadaptive responses in yeast.

Cells coordinate intracellular activities in response to changes in the extracellular environment to maximize their probability of survival and proliferation. Eukaryotic cells need to adapt to constant changes in the osmolarity of their environment. In yeast, the high-osmolarity glycerol (HOG) pathway is responsible for the response to high osmolarity. Activation of the Hog1 stress-activated protein kinase (SAPK) induces a complex program required for cellular adaptation that includes temporary arrest of cell cycle progression, adjustment of transcription and translation patterns, and the regulation of metabolism, including the synthesis and retention of the compatible osmolyte glycerol. Hog1 is a member of the family of p38 SAPKs, which are present across eukaryotes. Many of the properties of the HOG pathway and downstream-regulated proteins are conserved from yeast to mammals. This review addresses the global view of this signaling pathway in yeast, as well as the contribution of Dr Hohmann's group to its understanding.

Understanding Retinoblastoma Post-Translational Regulation for the Design of Targeted Cancer Therapies.

The retinoblastoma protein (Rb1) is a prototypical tumor suppressor protein whose role was described more than 40 years ago. Together with p107 (also known as RBL1) and p130 (also known as RBL2), the Rb1 belongs to a family of structurally and functionally similar proteins that inhibits cell cycle progression. Given the central role of Rb1 in regulating proliferation, its expression or function is altered in most types of cancer. One of the mechanisms underlying Rb-mediated cell cycle inhibition is the binding and repression of E2F transcription factors, and these processes are dependent on Rb1 phosphorylation status. However, recent work shows that Rb1 is a convergent point of many pathways and thus the regulation of its function through post-translational modifications is more complex than initially expected. Moreover, depending on the context, downstream signaling can be both E2F-dependent and -independent. This review seeks to summarize the most recent research on Rb1 function and regulation and discuss potential avenues for the design of novel cancer therapies.

Data-driven identification of inherent features of eukaryotic stress-responsive genes.

Living organisms are continuously challenged by changes in their environment that can propagate to stresses at the cellular level, such as rapid changes in osmolarity or oxygen tension. To survive these sudden changes, cells have developed stress-responsive mechanisms that tune cellular processes. The response of Saccharomyces cerevisiae to osmostress includes a massive reprogramming of gene expression. Identifying the inherent features of stress-responsive genes is of significant interest for understanding the basic principles underlying the rewiring of gene expression upon stress. Here, we generated a comprehensive catalog of osmostress-responsive genes from 5 independent RNA-seq experiments. We explored 30 features of yeast genes and found that 25 (83%) were distinct in osmostress-responsive genes. We then identified 13 non-redundant minimal osmostress gene traits and used statistical modeling to rank the most stress-predictive features. Intriguingly, the most relevant features of osmostress-responsive genes are the number of transcription factors targeting them and gene conservation. Using data on HeLa samples, we showed that the same features that define yeast osmostress-responsive genes can predict osmostress-responsive genes in humans, but with changes in the rank-ordering of feature-importance. Our study provides a holistic understanding of the basic principles of the regulation of stress-responsive gene expression across eukaryotes.

LRRC8A-containing chloride channel is crucial for cell volume recovery and survival under hypertonic conditions.

Regulation of cell volume is essential for tissue homeostasis and cell viability. In response to hypertonic stress, cells need rapid electrolyte influx to compensate water loss and to prevent cell death in a process known as regulatory volume increase (RVI). However, the molecular component able to trigger such a process was unknown to date. Using a genome-wide CRISPR/Cas9 screen, we identified LRRC8A, which encodes a chloride channel subunit, as the gene most associated with cell survival under hypertonic conditions. Hypertonicity activates the p38 stress-activated protein kinase pathway and its downstream MSK1 kinase, which phosphorylates and activates LRRC8A. LRRC8A-mediated Cl- efflux facilitates activation of the with-no-lysine (WNK) kinase pathway, which in turn, promotes electrolyte influx via Na+/K+/2Cl- cotransporter (NKCC) and RVI under hypertonic stress. LRRC8A-S217A mutation impairs channel activation by MSK1, resulting in reduced RVI and cell survival. In summary, LRRC8A is key to bidirectional osmotic stress responses and cell survival under hypertonic conditions.

The regulation of Net1/Cdc14 by the Hog1 MAPK upon osmostress unravels a new mechanism regulating mitosis.

During evolution, cells have developed a plethora of mechanisms to optimize survival in a changing and unpredictable environment. In this regard, they have evolved networks that include environmental sensors, signaling transduction molecules and response mechanisms. Hog1 (yeast) and p38 (mammals) stress-activated protein kinases (SAPKs) are activated upon stress and they drive a full collection of cell adaptive responses aimed to maximize survival. SAPKs are extensively used to learn about the mechanisms through which cells adapt to changing environments. In addition to regulating gene expression and metabolism, SAPKs control cell cycle progression. In this review, we will discuss the latest findings related to the SAPK-driven regulation of mitosis upon osmostress in yeast.

Hog1 activation delays mitotic exit via phosphorylation of Net1.

Adaptation to environmental changes is crucial for cell fitness. In Saccharomyces cerevisiae, variations in external osmolarity trigger the activation of the stress-activated protein kinase Hog1 (high-osmolarity glycerol 1), which regulates gene expression, metabolism, and cell-cycle progression. The activation of this kinase leads to the regulation of G1, S, and G2 phases of the cell cycle to prevent genome instability and promote cell survival. Here we show that Hog1 delays mitotic exit when cells are stressed during metaphase. Hog1 phosphorylates the nucleolar protein Net1, altering its affinity for the phosphatase Cdc14, whose activity is essential for mitotic exit and completion of the cell cycle. The untimely release of Cdc14 from the nucleolus upon activation of Hog1 is linked to a defect in ribosomal DNA (rDNA) and telomere segregation, and it ultimately delays cell division. A mutant of Net1 that cannot be phosphorylated by Hog1 displays reduced viability upon osmostress. Thus, Hog1 contributes to maximizing cell survival upon stress by regulating mitotic exit.

The p38 Pathway: From Biology to Cancer Therapy.

The p38 MAPK pathway is well known for its role in transducing stress signals from the environment. Many key players and regulatory mechanisms of this signaling cascade have been described to some extent. Nevertheless, p38 participates in a broad range of cellular activities, for many of which detailed molecular pictures are still lacking. Originally described as a tumor-suppressor kinase for its inhibitory role in RAS-dependent transformation, p38 can also function as a tumor promoter, as demonstrated by extensive experimental data. This finding has prompted the development of specific inhibitors that have been used in clinical trials to treat several human malignancies, although without much success to date. However, elucidating critical aspects of p38 biology, such as isoform-specific functions or its apparent dual nature during tumorigenesis, might open up new possibilities for therapy with unexpected potential. In this review, we provide an extensive description of the main biological functions of p38 and focus on recent studies that have addressed its role in cancer. Furthermore, we provide an updated overview of therapeutic strategies targeting p38 in cancer and promising alternatives currently being explored.

A genetic analysis reveals novel histone residues required for transcriptional reprogramming upon stress.

Cells have the ability to sense, respond and adapt to environmental fluctuations. Stress causes a massive reorganization of the transcriptional program. Many examples of histone post-translational modifications (PTMs) have been associated with transcriptional activation or repression under steady-state growth conditions. Comparatively less is known about the role of histone PTMs in the cellular adaptive response to stress. Here, we performed high-throughput genetic screenings that provide a novel global map of the histone residues required for transcriptional reprogramming in response to heat and osmotic stress. Of note, we observed that the histone residues needed depend on the type of gene and/or stress, thereby suggesting a 'personalized', rather than general, subset of histone requirements for each chromatin context. In addition, we identified a number of new residues that unexpectedly serve to regulate transcription. As a proof of concept, we characterized the function of the histone residues H4-S47 and H4-T30 in response to osmotic and heat stress, respectively. Our results uncover novel roles for the kinases Cla4 and Ste20, yeast homologs of the mammalian PAK2 family, and the Ste11 MAPK as regulators of H4-S47 and H4-T30, respectively. This study provides new insights into the role of histone residues in transcriptional regulation under stress conditions.

Functional Network Analysis Reveals the Relevance of SKIIP in the Regulation of Alternative Splicing by p38 SAPK.

Alternative splicing is a prevalent mechanism of gene regulation that is modulated in response to a wide range of extracellular stimuli. Stress-activated protein kinases (SAPKs) play a key role in controlling several steps of mRNA biogenesis. Here, we show that osmostress has an impact on the regulation of alternative splicing (AS), which is partly mediated through the action of p38 SAPK. Splicing network analysis revealed a functional connection between p38 and the spliceosome component SKIIP, whose depletion abolished a significant fraction of p38-mediated AS changes. Importantly, p38 interacted with and directly phosphorylated SKIIP, thereby altering its activity. SKIIP phosphorylation regulated AS of GADD45α, the upstream activator of the p38 pathway, uncovering a negative feedback loop involving AS regulation. Our data reveal mechanisms and targets of SAPK function in stress adaptation through the regulation of AS.

Sensitive high-throughput single-cell RNA-seq reveals within-clonal transcript correlations in yeast populations.

Single-cell RNA sequencing has revealed extensive cellular heterogeneity within many organisms, but few methods have been developed for microbial clonal populations. The yeast genome displays unusually dense transcript spacing, with interleaved and overlapping transcription from both strands, resulting in a minuscule but complex pool of RNA that is protected by a resilient cell wall. Here, we have developed a sensitive, scalable and inexpensive yeast single-cell RNA-seq (yscRNA-seq) method that digitally counts transcript start sites in a strand- and isoform-specific manner. YscRNA-seq detects the expression of low-abundance, noncoding RNAs and at least half of the protein-coding genome in each cell. In clonal cells, we observed a negative correlation for the expression of sense-antisense pairs, whereas paralogs and divergent transcripts co-expressed. By combining yscRNA-seq with index sorting, we uncovered a linear relationship between cell size and RNA content. Although we detected an average of ~3.5 molecules per gene, the number of expressed isoforms is restricted at the single-cell level. Remarkably, the expression of metabolic genes is highly variable, whereas their stochastic expression primes cells for increased fitness towards the corresponding environmental challenge. These findings suggest that functional transcript diversity acts as a mechanism that provides a selective advantage to individual cells within otherwise transcriptionally heterogeneous populations.

Yeast Single-cell RNA-seq, Cell by Cell and Step by Step.

Single-cell RNA-seq (scRNA-seq) has become an established method for uncovering the intrinsic complexity within populations. Even within seemingly homogenous populations of isogenic yeast cells, there is a high degree of heterogeneity that originates from a compact and pervasively transcribed genome. Research with microorganisms such as yeast represents a major challenge for single-cell transcriptomics, due to their small size, rigid cell wall, and low RNA content per cell. Because of these technical challenges, yeast-specific scRNA-seq methodologies have recently started to appear, each one of them relying on different cell-isolation and library-preparation methods. Consequently, each approach harbors unique strengths and weaknesses that need to be considered. We have recently developed a yeast single-cell RNA-seq protocol (yscRNA-seq), which is inexpensive, high-throughput and easy-to-implement, tailored to the unique needs of yeast. yscRNA-seq provides a unique platform that combines single-cell phenotyping via index sorting with the incorporation of unique molecule identifiers on transcripts that allows to digitally count the number of molecules in a strand- and isoform-specific manner. Here, we provide a detailed, step-by-step description of the experimental and computational steps of yscRNA-seq protocol. This protocol will ease the implementation of yscRNA-seq in other laboratories and provide guidelines for the development of novel technologies.

Rapid reversible changes in compartments and local chromatin organization revealed by hyperosmotic shock.

Nuclear architecture is decisive for the assembly of transcriptional responses. However, how chromosome organization is dynamically modulated to permit rapid and transient transcriptional changes in response to environmental challenges remains unclear. Here we show that hyperosmotic stress disrupts different levels of chromosome organization, ranging from A/B compartment changes to reduction in the number and insulation of topologically associating domains (TADs). Concomitantly, transcription is greatly affected, TAD borders weaken, and RNA Polymerase II runs off from hundreds of transcription end sites. Stress alters the binding profiles of architectural proteins, which explains the disappearance of local chromatin organization. These processes are dynamic, and cells rapidly reconstitute their default chromatin conformation after stress removal, uncovering an intrinsic organization. Transcription is not required for local chromatin reorganization, while compartment recovery is partially transcription-dependent. Thus, nuclear organization in mammalian cells can be rapidly modulated by environmental changes in a reversible manner.