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.

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.

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.

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.

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 novel mechanism for the prevention of transcription replication conflicts.

Transcription and replication complexes can coincide in space and time. Such coincidences may result in collisions that trigger genomic instability. The phosphorylation of Mrc1 by different signaling kinases is part of a general mechanism that serves to delay replication in response to different stresses that trigger a massive transcriptional response in S phase. This mechanism prevents Transcription-Replication Conflicts and maintains genomic integrity in response to unscheduled massive transcription during S phase.

Phosphorylation and Proteasome Recognition of the mRNA-Binding Protein Cth2 Facilitates Yeast Adaptation to Iron Deficiency.

Iron is an indispensable micronutrient for all eukaryotic organisms due to its participation as a redox cofactor in many metabolic pathways. Iron imbalance leads to the most frequent human nutritional deficiency in the world. Adaptation to iron limitation requires a global reorganization of the cellular metabolism directed to prioritize iron utilization for essential processes. In response to iron scarcity, the conserved Saccharomyces cerevisiae mRNA-binding protein Cth2, which belongs to the tristetraprolin family of tandem zinc finger proteins, coordinates a global remodeling of the cellular metabolism by promoting the degradation of multiple mRNAs encoding highly iron-consuming proteins. In this work, we identify a critical mechanism for the degradation of Cth2 protein during the adaptation to iron deficiency. Phosphorylation of a patch of Cth2 serine residues within its amino-terminal region facilitates recognition by the SCFGrr1 ubiquitin ligase complex, accelerating Cth2 turnover by the proteasome. When Cth2 degradation is impaired by either mutagenesis of the Cth2 serine residues or deletion of GRR1, the levels of Cth2 rise and abrogate growth in iron-depleted conditions. Finally, we uncover that the casein kinase Hrr25 phosphorylates and promotes Cth2 destabilization. These results reveal a sophisticated posttranslational regulatory pathway necessary for the adaptation to iron depletion.IMPORTANCE Iron is a vital element for many metabolic pathways, including the synthesis of DNA and proteins, and the generation of energy via oxidative phosphorylation. Therefore, living organisms have developed tightly controlled mechanisms to properly distribute iron, since imbalances lead to nutritional deficiencies, multiple diseases, and vulnerability against pathogens. Saccharomyces cerevisiae Cth2 is a conserved mRNA-binding protein that coordinates a global reprogramming of iron metabolism in response to iron deficiency in order to optimize its utilization. Here we report that the phosphorylation of Cth2 at specific serine residues is essential to regulate the stability of the protein and adaptation to iron depletion. We identify the kinase and ubiquitination machinery implicated in this process to establish a posttranscriptional regulatory model. These results and recent findings for both mammals and plants reinforce the privileged position of E3 ubiquitin ligases and phosphorylation events in the regulation of eukaryotic iron homeostasis.

Osteoblast-Secreted Factors Mediate Dormancy of Metastatic Prostate Cancer in the Bone via Activation of the TGFßRIII-p38MAPK-pS249/T252RB Pathway.

Bone metastasis from prostate cancer can occur years after prostatectomy, due to reactivation of dormant disseminated tumor cells (DTC) in the bone, yet the mechanism by which DTCs are initially induced into a dormant state in the bone remains to be elucidated. We show here that the bone microenvironment confers dormancy to C4-2B4 prostate cancer cells, as they become dormant when injected into mouse femurs but not under the skin. Live-cell imaging of dormant cells at the single-cell level revealed that conditioned medium from differentiated, but not undifferentiated, osteoblasts induced C4-2B4 cellular quiescence, suggesting that differentiated osteoblasts present locally around the tumor cells in the bone conferred dormancy to prostate cancer cells. Gene array analyses identified GDF10 and TGFß2 among osteoblast-secreted proteins that induced quiescence of C4-2B4, C4-2b, and PC3-mm2, but not 22RV1 or BPH-1 cells, indicating prostate cancer tumor cells differ in their dormancy response. TGFß2 and GDF10 induced dormancy through TGFßRIII to activate phospho-p38MAPK, which phosphorylates retinoblastoma (RB) at the novel N-terminal S249/T252 sites to block prostate cancer cell proliferation. Consistently, expression of dominant-negative p38MAPK in C4-2b and C4-2B4 prostate cancer cell lines abolished tumor cell dormancy both in vitro and in vivo Lower TGFßRIII expression in patients with prostate cancer correlated with increased metastatic potential and decreased survival rates. Together, our results identify a dormancy mechanism by which DTCs are induced into a dormant state through TGFßRIII-p38MAPK-pS249/pT252-RB signaling and offer a rationale for developing strategies to prevent prostate cancer recurrence in the bone.Significance: These findings provide mechanistic insights into the dormancy of metastatic prostate cancer in the bone and offer a rationale for developing strategies to prevent prostate cancer recurrence in the bone. Cancer Res; 78(11); 2911-24. ©2018 AACR.

The Hog1p kinase regulates Aft1p transcription factor to control iron accumulation.

Iron acquisition systems have to be tightly regulated to assure a continuous supply of iron, since it is essential for survival, but simultaneously to prevent iron overload that is toxic to the cells. In budding yeast, the low­iron sensing transcription factor Aft1p is a master regulator of the iron regulon. Our previous work revealed that bioactive sphingolipids modulate iron homeostasis as yeast cells lacking the sphingomyelinase Isc1p exhibit an upregulation of the iron regulon. In this study, we show that Isc1p impacts on iron accumulation and localization. Notably, Aft1p is activated in isc1Δ cells due to a decrease in its phosphorylation and an increase in its nuclear levels. Consistently, the expression of a phosphomimetic version of Aft1p-S210/S224 that favours its nuclear export abolished iron accumulation in isc1Δ cells. Notably, the Hog1p kinase, homologue of mammalian p38, interacts with and directly phosphorylates Aft1p at residues S210 and S224. However, Hog1p-Aft1p interaction decreases in isc1Δ cells, which likely contributes to Aft1p dephosphorylation and consequently to Aft1p activation and iron overload in isc1Δ cells. These results suggest that alterations in sphingolipid composition in isc1Δ cells may impact on iron homeostasis by disturbing the regulation of Aft1p by Hog1p. To our knowledge, Hog1p is the first kinase reported to directly regulate Aft1p, impacting on iron homeostasis.

An RB insensitive to CDK regulation.

The N-term phosphorylation of Retinoblastoma (RB) by the p38 stress-activated protein kinase (SAPK) makes RB insensitive to cyclin-dependent kinase (CDK)-Cyclin inhibition, which enhances the transcriptional repression of E2F-driven promoters and delays tumor cell growth. This novel mechanism of RB regulation opens up a window for developing new cancer drug treatments for tumors harboring high CDK-Cyclin activity and a wild-type RB gene.

The N-Terminal Phosphorylation of RB by p38 Bypasses Its Inactivation by CDKs and Prevents Proliferation in Cancer Cells.

Control of the G1/S phase transition by the Retinoblastoma (RB) tumor suppressor is critical for the proliferation of normal cells in tissues, and its inactivation is one of the most fundamental events leading to cancer. Cyclin-dependent kinase (CDK) phosphorylation inactivates RB to promote cell cycle-regulated gene expression. Here we show that, upon stress, the p38 stress-activated protein kinase (SAPK) maximizes cell survival by downregulating E2F gene expression through the targeting of RB. RB undergoes selective phosphorylation by p38 in its N terminus; these phosphorylations render RB insensitive to the inactivation by CDKs. p38 phosphorylation of RB increases its affinity toward the E2F transcription factor, represses gene expression, and delays cell-cycle progression. Remarkably, introduction of a RB phosphomimetic mutant in cancer cells reduces colony formation and decreases their proliferative and tumorigenic potential in mice.

H3K4 monomethylation dictates nucleosome dynamics and chromatin remodeling at stress-responsive genes.

Chromatin remodeling is essential for proper adaptation to extracellular stimuli. The p38-related Hog1 SAPK is an important regulator of transcription that mediates chromatin remodeling upon stress. Hog1 targets the RSC chromatin remodeling complex to stress-responsive genes and rsc deficient cells display reduced induction of gene expression. Here we show that the absence of H3K4 methylation, either achieved by deletion of the SET1 methyltransferase or by amino acid substitution of H3K4, bypasses the requirement of RSC for stress-responsive gene expression. Monomethylation of H3K4 is specifically inhibiting RSC-independent chromatin remodeling and thus, it prevents osmostress-induced gene expression. The absence of H3K4 monomethylation permits that the association of alternative remodelers with stress-responsive genes and the Swr1 complex (SWR-C) is instrumental in the induction of gene expression upon stress. Accordingly, the absence of SWR-C or histone H2A.Z results in compromised chromatin remodeling and impaired gene expression in the absence of RSC and H3K4 methylation. These results indicate that expression of stress-responsive genes is controlled by two remodeling mechanisms: RSC in the presence of monomethylated H3K4, and SWR-C in the absence of H3K4 monomethylation. Our findings point to a novel role for H3K4 monomethylation in dictating the specificity of chromatin remodeling, adding an extra layer of regulation to the transcriptional stress response.

Cell cycle control and HIV-1 susceptibility are linked by CDK6-dependent CDK2 phosphorylation of SAMHD1 in myeloid and lymphoid cells.

Proliferating cells are preferentially susceptible to infection by retroviruses. Sterile α motif and HD domain-containing protein-1 (SAMHD1) is a recently described deoxynucleotide phosphohydrolase controlling the size of the intracellular deoxynucleotide triphosphate (dNTP) pool, a limiting factor for retroviral reverse transcription in noncycling cells. Proliferating (Ki67(+)) primary CD4(+) T cells or macrophages express a phosphorylated form of SAMHD1 that corresponds with susceptibility to infection in cell culture. We identified cyclin-dependent kinase (CDK) 6 as an upstream regulator of CDK2 controlling SAMHD1 phosphorylation in primary T cells and macrophages susceptible to infection by HIV-1. In turn, CDK2 was strongly linked to cell cycle progression and coordinated SAMHD1 phosphorylation and inactivation. CDK inhibitors specifically blocked HIV-1 infection at the reverse transcription step in a SAMHD1-dependent manner, reducing the intracellular dNTP pool. Our findings identify a direct relationship between control of the cell cycle by CDK6 and SAMHD1 activity, which is important for replication of lentiviruses, as well as other viruses whose replication may be regulated by intracellular dNTP availability.

Gene expression profiling of yeasts overexpressing wild type or misfolded Pma1 variants reveals activation of the Hog1 MAPK pathway.

Dominant negative PMA1 mutants render misfolded proteins that are retained in the endoplasmic reticulum (ER) and slowly degraded by ER-associated degradation. Accumulation of misfolded proteins in the ER activates an ER-to-nucleus signalling pathway termed the unfolded protein response (UPR). We have used a PMA1-D378T dominant negative mutant to analyse its impact on UPR induction. Our results show that overexpression of the misfolded mutant Pma1 does not lead to activation of the UPR. In addition, in mutants with a constitutively activated UPR the turnover rate of the mutant ATPase is not altered. To determine if the expression of the misfolded mutant protein induces some other kind of response we performed global gene expression analysis experiments in yeasts overexpressing either wild type or dominant lethal PMA1 alleles. The results suggest that the high osmolarity glycerol (Hog1) mitogen-activated protein kinase (MAPK) pathway is activated by both wild type and mutant ATPases. We show that expression of the PMA1 alleles induces phosphorylation of Hog1 and activation of the Hog1 MAPK cascade. This activation is mediated by the Sln1 branch of the stress-dependent Hog1 MAPK network. Finally, we show that at least two other plasma membrane proteins are also able to activate the Hog1 MAPK system.

Distributed biological computation with multicellular engineered networks.

Ongoing efforts within synthetic and systems biology have been directed towards the building of artificial computational devices using engineered biological units as basic building blocks. Such efforts, inspired in the standard design of electronic circuits, are limited by the difficulties arising from wiring the basic computational units (logic gates) through the appropriate connections, each one to be implemented by a different molecule. Here, we show that there is a logically different form of implementing complex Boolean logic computations that reduces wiring constraints thanks to a redundant distribution of the desired output among engineered cells. A practical implementation is presented using a library of engineered yeast cells, which can be combined in multiple ways. Each construct defines a logic function and combining cells and their connections allow building more complex synthetic devices. As a proof of principle, we have implemented many logic functions by using just a few engineered cells. Of note, small modifications and combination of those cells allowed for implementing more complex circuits such as a multiplexer or a 1-bit adder with carry, showing the great potential for re-utilization of small parts of the circuit. Our results support the approach of using cellular consortia as an efficient way of engineering complex tasks not easily solvable using single-cell implementations.

The p38 SAPK is recruited to chromatin via its interaction with transcription factors.

In mammals, the stress-activated protein kinase (SAPK) p38 coordinates a rapid and complex transcriptional program to adapt to sudden changes in the extracellular environment. Although a number of genes have been reported to be under the control of p38, the basic mechanisms of transcriptional regulation by this SAPK remain uncharacterized. Here we show that in response to osmotic shock, anisomycin- or TNFα-activated p38 SAPK is recruited to stress-induced genes. The MAPKK MKK6 is also found at stress-responsive promoters. The recruitment of RNA polymerase II complex to the target promoters requires p38 activity. Moreover, when tethered to DNA as a LexA fusion protein, p38 activates transcription in a stress-regulated manner. Thus, p38 activity allows for recruitment of RNA polymerase and transcription initiation. p38 directly phosphorylates and interacts with the transcription factor Elk1. p38 activity is necessary for the recruitment of Elk1 to the c-Fos promoter, and knocking down Elk1 by siRNAs compromises both p38 recruitment to the c-Fos promoter and c-Fos transcriptional up-regulation upon osmostress. In addition, p38 recruitment to the osmoinducible gene Cox2 and the TNFα target gene IL8 is mediated by the transcription factors AP1 and NFκB, respectively. Therefore, anchoring of active SAPK to target genes is mediated by transcription factors. The presence of active p38 at open reading frames also suggests the involvement of the SAPK in elongation. Taken together, SAPK recruitment to target genes appears to be a broad mechanism to regulate transcription that has been preserved from yeast to mammals.

Whole genome analysis of p38 SAPK-mediated gene expression upon stress.

BACKGROUND: Cells have the ability to respond and adapt to environmental changes through activation of stress-activated protein kinases (SAPKs). Although p38 SAPK signalling is known to participate in the regulation of gene expression little is known on the molecular mechanisms used by this SAPK to regulate stress-responsive genes and the overall set of genes regulated by p38 in response to different stimuli. RESULTS: Here, we report a whole genome expression analyses on mouse embryonic fibroblasts (MEFs) treated with three different p38 SAPK activating-stimuli, namely osmostress, the cytokine TNFalpha and the protein synthesis inhibitor anisomycin. We have found that the activation kinetics of p38alpha SAPK in response to these insults is different and also leads to a complex gene pattern response specific for a given stress with a restricted set of overlapping genes. In addition, we have analysed the contribution of p38alpha the major p38 family member present in MEFs, to the overall stress-induced transcriptional response by using both a chemical inhibitor (SB203580) and p38alpha deficient (p38alpha-/-) MEFs. We show here that p38 SAPK dependency ranged between 60% and 88% depending on the treatments and that there is a very good overlap between the inhibitor treatment and the ko cells. Furthermore, we have found that the dependency of SAPK varies depending on the time the cells are subjected to osmostress. CONCLUSIONS: Our genome-wide transcriptional analyses shows a selective response to specific stimuli and a restricted common response of up to 20% of the stress up-regulated early genes that involves an important set of transcription factors, which might be critical for either cell adaptation or preparation for continuous extra-cellular changes. Interestingly, up to 85% of the up-regulated genes are under the transcriptional control of p38 SAPK. Thus, activation of p38 SAPK is critical to elicit the early gene expression program required for cell adaptation to stress.