Cells perceive their microenvironment not only through soluble signals but also through physical and mechanical cues, such as extracellular matrix (ECM) stiffness or confined adhesiveness. By mechanotransduction systems, cells translate these stimuli into biochemical signals controlling multiple aspects of cell behaviour, including growth, differentiation and cancer malignant progression, but how rigidity mechanosensing is ultimately linked to activity of nuclear transcription factors remains poorly understood. Here we report the identification of the Yorkie-homologues YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif, also known as WWTR1) as nuclear relays of mechanical signals exerted by ECM rigidity and cell shape. This regulation requires Rho GTPase activity and tension of the actomyosin cytoskeleton, but is independent of the Hippo/LATS cascade. Crucially, YAP/TAZ are functionally required for differentiation of mesenchymal stem cells induced by ECM stiffness and for survival of endothelial cells regulated by cell geometry; conversely, expression of activated YAP overrules physical constraints in dictating cell behaviour. These findings identify YAP/TAZ as sensors and mediators of mechanical cues instructed by the cellular microenvironment.

Cancer stem cells (CSCs) are proposed to drive tumor initiation and progression. Yet, our understanding of the cellular and molecular mechanisms that underlie CSC properties is limited. Here we show that the activity of TAZ, a transducer of the Hippo pathway, is required to sustain self-renewal and tumor-initiation capacities in breast CSCs. TAZ protein levels and activity are elevated in prospective CSCs and in poorly differentiated human tumors and have prognostic value. Gain of TAZ endows self-renewal capacity to non-CSCs. In epithelial cells, TAZ forms a complex with the cell-polarity determinant Scribble, and loss of Scribble--or induction of the epithelial-mesenchymal transition (EMT)--disrupts the inhibitory association of TAZ with the core Hippo kinases MST and LATS. This study links the CSC concept to the Hippo pathway in breast cancer and reveals a mechanistic basis of the control of Hippo kinases by cell polarity.

YAP/TAZ are nuclear effectors of the Hippo pathway regulating organ growth and tumorigenesis. Yet, their function as transcriptional regulators remains underinvestigated. By ChIP-seq analyses in breast cancer cells, we discovered that the YAP/TAZ transcriptional response is pervasively mediated by a dual element: TEAD factors, through which YAP/TAZ bind to DNA, co-occupying chromatin with activator protein-1 (AP-1, dimer of JUN and FOS proteins) at composite cis-regulatory elements harbouring both TEAD and AP-1 motifs. YAP/TAZ/TEAD and AP-1 form a complex that synergistically activates target genes directly involved in the control of S-phase entry and mitosis. This control occurs almost exclusively from distal enhancers that contact target promoters through chromatin looping. YAP/TAZ-induced oncogenic growth is strongly enhanced by gain of AP-1 and severely blunted by its loss. Conversely, AP-1-promoted skin tumorigenesis is prevented in YAP/TAZ conditional knockout mice. This work highlights a new layer of signalling integration, feeding on YAP/TAZ function at the chromatin level.

Key Points• Identification of the biologic requirements for memory stem T cell (T SCM ) generation and expansion from naive precursors ex vivo.• Differentiation, expansion, and genetic manipulation of human T SCM for cancer adoptive cellular therapy. Long-living memory stem T cells (T SCM) IntroductionAdaptive immunity is a potent and flexible system able to combat microbes and cancer cells. 1,2 In the presence of infections or cancer, antigen-specific lymphocytes expand and differentiate into effectors devoted to rapidly clearing the pathogen and memory cells able to persist long-term to patrol the entire organism for recurrence and minimal residual disease. 3,4 However, the mechanism and hierarchical differentiation path underlying the generation of memory precursors and terminal effector cells remain to be fully elucidated. 5 This process has been proposed to involve a self-renewing, stem cell-like memory T-cell subset capable of differentiating into effectors on antigen reencounter. 6,7 This T-cell subset, referred to as memory stem T cells (T SCM ), and initially described in mice, 8,9 begins to be unveiled in humans. 10 T SCM potential biodistribution and long-term persistence represent appealing features to overcome the current limitations of cancer adoptive immune-gene therapy. [11][12][13] At present, clinical-grade protocols able to obtain or preserve T SCM functional and phenotypic characteristics remain to be defined. We previously showed that costimulation of unselected T cells and culture with ␥-chain cytokines allow the preferential generation of gene-modified T cells with a functional central memory (T CM ) phenotype, superior to effector/effector memory (T EM ) counterparts for expansion potential and antitumor activity. 14,15 Compared with T CM and T EM lymphocytes, naive T cells (T N ) are endowed with the highest developmental plasticity and are unique in the ability to generate daughter cells with potential to enter the entire spectrum of immunologic memory, including T SCM . We thus hypothesized that, starting from naive precursors, we could differentiate and genetically engineer human T SCM . We report that IL-7 and IL-15 support the generation of postmitotic costimulated CD8 ϩ T cells with molecular and functional features of T SCM cells. These cells-defined by the expression of CD45RA, CD45R0, CD62L, CCR7, IL-7R␣, and CD95-can be identified among healthy subjects, are selectively enriched in hematopoietic stem cell transplant (HSCT) recipients, and reveal a phenotypic and functional profile distinct from that of T CM and T EM cells for extensive expansion capacity and ability Submitted May 22, 2012; accepted October 25, 2012. Prepublished online as Blood First Edition paper, November 15, 2012; DOI 10.1182 DOI 10. /blood-2012 There is an Inside Blood commentary on this article in this issue.The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this arti...

Wnt growth factors are fundamental regulators of cell fate, but how the Wnt signal is translated into biological responses is incompletely understood. Here, we report that TAZ, a biologically potent transcriptional coactivator, serves as a downstream element of the Wnt/β-catenin cascade. This function of TAZ is independent from its well-established role as mediator of Hippo signaling. In the absence of Wnt activity, the components of the β-catenin destruction complex--APC, Axin, and GSK3--are also required to keep TAZ at low levels. TAZ degradation depends on phosphorylated β-catenin that bridges TAZ to its ubiquitin ligase β-TrCP. Upon Wnt signaling, escape of β-catenin from the destruction complex impairs TAZ degradation and leads to concomitant accumulation of β-catenin and TAZ. At the genome-wide level, a substantial portion of Wnt transcriptional responses is mediated by TAZ. TAZ activation is a general feature of Wnt signaling and is functionally relevant to mediate Wnt biological effects.

Circadian rhythms control metabolism and energy homeostasis, but the role of the skeletal muscle clock has never been explored. We generated conditional and inducible mouse lines with muscle-specific ablation of the core clock gene Bmal1. Skeletal muscles from these mice showed impaired insulin-stimulated glucose uptake with reduced protein levels of GLUT4, the insulin-dependent glucose transporter, and TBC1D1, a Rab-GTPase involved in GLUT4 translocation. Pyruvate dehydrogenase (PDH) activity was also reduced due to altered expression of circadian genes Pdk4 and Pdp1, coding for PDH kinase and phosphatase, respectively. PDH inhibition leads to reduced glucose oxidation and diversion of glycolytic intermediates to alternative metabolic pathways, as revealed by metabolome analysis. The impaired glucose metabolism induced by muscle-specific Bmal1 knockout suggests that a major physiological role of the muscle clock is to prepare for the transition from the rest/fasting phase to the active/feeding phase, when glucose becomes the predominant fuel for skeletal muscle.

Hi-C is a genome-wide sequencing technique to investigate the 3D chromatin conformation inside the nucleus. The most studied structures that can be identified from Hi-C - chromatin interactions and topologically associating domains (TADs) - require computational methods to analyze genome-wide contact probability maps. We quantitatively compared the performances of 13 algorithms for the analysis of Hi-C data from 6 landmark studies and simulations. The comparison revealed clear differences in the performances of methods to identify chromatin interactions and more comparable results of algorithms for TAD detection.

Cancer cells rely on dysregulated gene expression. This establishes specific transcriptional addictions that may be therapeutically exploited. Yet, the mechanisms that are ultimately responsible for these addictions are poorly understood. Here, we investigated the transcriptional dependencies of transformed cells to the transcription factors YAP and TAZ. YAP/TAZ physically engage the general coactivator bromodomain-containing protein 4 (BRD4), dictating the genome-wide association of BRD4 to chromatin. YAP/TAZ flag a large set of enhancers with super-enhancer-like functional properties. YAP/TAZ-bound enhancers mediate the recruitment of BRD4 and RNA polymerase II at YAP/TAZ-regulated promoters, boosting the expression of a host of growth-regulating genes. Treatment with small-molecule inhibitors of BRD4 blunts YAP/TAZ pro-tumorigenic activity in several cell or tissue contexts, causes the regression of pre-established, YAP/TAZ-addicted neoplastic lesions and reverts drug resistance. This work sheds light on essential mediators, mechanisms and genome-wide regulatory elements that are responsible for transcriptional addiction in cancer and lays the groundwork for a rational use of BET inhibitors according to YAP/TAZ biology.