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Nothapodytes nimmoniana is a medicinally important plant producing anticancer monoterpene indole alkaloid (MIA), camptothecin (CPT). The CPT is synthesised through the strictosidine intermediate following the MIA pathway; however, transcriptional regulation of CPT pathway is still elusive in N. nimmoniana. Biosynthesis of MIA is regulated by various transcription factors (TFs) belonging to AP2/ERF, bHLH, MYB, and WRKY families. The present study identified transcriptionally active full-length 105 AP2/ERF and 68 bHLH family TFs from the N. nimmoniana. AP2/ERF TFs were divided into three subfamilies along with a soloist, while bHLH TFs were divided into 10 subfamilies according to their phylogenetic similarities. Three group IXa ERFs, Nn-ERF22, Nn-ERF29, and Nn-ERF41, one subfamily IVa TF Nn-bHLH7, and three subfamilies IIIe Nn-bHLH33, Nn-bHLH51, and Nn-bHLH52 clustered with the TFs regulating alkaloid biosynthesis in Catharanthus roseus, tomato, tobacco, and Artemisia annua. Expression of these TFs in N. nimmoniana was higher in roots, which is a primary CPT accumulating tissue. Moreover, genome skimming approach was used to reconstruct the promoter regions of candidate ERF genes to identify the cis-regulatory elements. The presence of G-boxes and other jasmonic acid-responsive elements in the promoter suggests the regulation of ERFs by bHLHs. The present study effectively generated and used genomics resource for characterisation of regulatory TFs from non-model medicinal plant. Key words: transcription factors, alkaloid biosynthesis, AP2/ERF, bHLH, N. nimmoniana, 1. Introduction Transcriptional regulation is a major component of plant's response to internal and external stimuli. Transcription factors (TFs) are important elements of the transcriptional regulation. TFs are the DNA [...]

Author(s): Omar Khan [sup.1] [sup.2] [sup.3] [sup.14] , Josephine R. Giles [sup.1] [sup.2] [sup.3] , Sierra McDonald [sup.4] [sup.5] [sup.6] , Sasikanth Manne [sup.1] [sup.2] , Shin Foong Ngiow [sup.1] [...], Exhausted CD8.sup.+ T (T.sub.ex) cells in chronic infections and cancer have limited effector function, high co-expression of inhibitory receptors and extensive transcriptional changes compared with effector (T.sub.eff) or memory (T.sub.mem) CD8.sup.+ T cells. T.sub.ex cells are important clinical targets of checkpoint blockade and other immunotherapies. Epigenetically, T.sub.ex cells are a distinct immune subset, with a unique chromatin landscape compared with T.sub.eff and T.sub.mem cells. However, the mechanisms that govern the transcriptional and epigenetic development of T.sub.ex cells remain unknown. Here we identify the HMG-box transcription factor TOX as a central regulator of T.sub.ex cells in mice. TOX is largely dispensable for the formation of T.sub.eff and T.sub.mem cells, but it is critical for exhaustion: in the absence of TOX, T.sub.ex cells do not form. TOX is induced by calcineurin and NFAT2, and operates in a feed-forward loop in which it becomes calcineurin-independent and sustained in T.sub.ex cells. Robust expression of TOX therefore results in commitment to T.sub.ex cells by translating persistent stimulation into a distinct T.sub.ex cell transcriptional and epigenetic developmental program. The transcription factor TOX is a central regulator of the transcriptional and epigenetic development of exhausted T cells.

Introduction Acute myeloid leukemia (AML) is the most common hematological malignancy in adults and the second most common one in children (1). Aberrant epigenetic events associated with DNA/chromatin modification that [...], The antileukemic effect of inhibiting bromodomain and extra-terminal domain-containing (BET-containing) proteins (BETPs) such as BRD4 has largely been largely attributed to transcriptional downregulation of cellular anabolic and antiapoptotic processes, but its effect on the bone marrow microenvironment, a sanctuary favoring the persistence of leukemic stem/progenitor cells, is unexplored. Sustained degradation of BETP with the small-molecule BET proteolysistargeting chimera (PROTAC) ARV-825 resulted in a marked downregulation of surface CXCR4 and CD44, key proteins in leukemia-microenvironment interactions, in acute myeloid leukemia (AML) cells. Abrogation of surface CXCR4 expression impaired SDF-1[alpha]-directed migration and was mediated through transcriptional downregulation of PIM1 kinase, which in turn phosphorylates CXCR4 and facilitates its surface localization. Downregulation of CD44, including isoforms CD44v8- 10 impaired cystine uptake, lowered intracellular reduced glutathione, and increased oxidative stress. More important, BETP degradation markedly decreased the [CD34.sup.+][CD38.sup.-][CD90.sup.-][CD45RA.sup.+] leukemic stem cell population and, alone or in combination with cytarabine, prolonged survival in a mouse model of human leukemia that included AML patient-derived xenografts (AML-PDX). Gene expression profiling and single-cell proteomics confirmed a downregulation of the gene signatures associated with "sternness" in AML and Wnt/[beta]-catenin and Myc pathways. Hence, BETP degradation by ARV-825 simultaneously targets cell-intrinsic signaling, stromal interactions, and metabolism in AML.

Author(s): Lorna A. Farrelly [sup.1] , Robert E. Thompson [sup.2] , Shuai Zhao [sup.3] [sup.4] , Ashley E. Lepack [sup.1] , Yang Lyu [sup.1] , Natarajan V. Bhanu [sup.5] , [...], Chemical modifications of histones can mediate diverse DNA-templated processes, including gene transcription.sup.1-3. Here we provide evidence for a class of histone post-translational modification, serotonylation of glutamine, which occurs at position 5 (Q5ser) on histone H3 in organisms that produce serotonin (also known as 5-hydroxytryptamine (5-HT)). We demonstrate that tissue transglutaminase 2 can serotonylate histone H3 tri-methylated lysine 4 (H3K4me3)-marked nucleosomes, resulting in the presence of combinatorial H3K4me3Q5ser in vivo. H3K4me3Q5ser displays a ubiquitous pattern of tissue expression in mammals, with enrichment observed in brain and gut, two organ systems responsible for the bulk of 5-HT production. Genome-wide analyses of human serotonergic neurons, developing mouse brain and cultured serotonergic cells indicate that H3K4me3Q5ser nucleosomes are enriched in euchromatin, are sensitive to cellular differentiation and correlate with permissive gene expression, phenomena that are linked to the potentiation of TFIID.sup.4-6 interactions with H3K4me3. Cells that ectopically express a H3 mutant that cannot be serotonylated display significantly altered expression of H3K4me3Q5ser-target loci, which leads to deficits in differentiation. Taken together, these data identify a direct role for 5-HT, independent from its contributions to neurotransmission and cellular signalling, in the mediation of permissive gene expression. In serotonin-rich tissues, tissue transglutaminase 2 is able to attach serotonin to a glutamine residue in histone H3; this modification mediates permissive gene expression in these tissues.

Author(s): Germán Camargo Ortega [sup.1] [sup.2] [sup.3] , Sven Falk [sup.1] [sup.2] , Pia A. Johansson [sup.1] [sup.2] [sup.20] , Elise Peyre [sup.4] , Loïc Broix [sup.4] , Sanjeeb Kumar [...], The expansion of brain size is accompanied by a relative enlargement of the subventricular zone during development. Epithelial-like neural stem cells divide in the ventricular zone at the ventricles of the embryonic brain, self-renew and generate basal progenitors.sup.1 that delaminate and settle in the subventricular zone in enlarged brain regions.sup.2. The length of time that cells stay in the subventricular zone is essential for controlling further amplification and fate determination. Here we show that the interphase centrosome protein AKNA has a key role in this process. AKNA localizes at the subdistal appendages of the mother centriole in specific subtypes of neural stem cells, and in almost all basal progenitors. This protein is necessary and sufficient to organize centrosomal microtubules, and promote their nucleation and growth. These features of AKNA are important for mediating the delamination process in the formation of the subventricular zone. Moreover, AKNA regulates the exit from the subventricular zone, which reveals the pivotal role of centrosomal microtubule organization in enabling cells to both enter and remain in the subventricular zone. The epithelial-to-mesenchymal transition is also regulated by AKNA in other epithelial cells, demonstrating its general importance for the control of cell delamination. The interphase centrosome protein AKNA is necessary and sufficient for the organization of centrosomal microtubules, mediates delamination in the formation of the subventricular zone and regulates exit from this zone.

Introduction Atherosclerosis has become one of the major causes of death worldwide because of its severe complications, such as acute myocardial infarction and stroke (1). Vascular endothelial cell (EC) activation [...], Local flow patterns determine the uneven distribution of atherosclerotic lesions. This research aims to elucidate the mechanism of regulation of nuclear translocation of Yes-associated protein (YAP) under oscillatory shear stress (OSS) in the atheroprone phenotype of endothelial cells (ECs). We report here that OSS led to tyrosine phosphorylation and strong, continuous nuclear translocation of YAP in ECs that is dependent on integrin [alpha]5[beta]1 activation. YAP overexpression in ECs blunted the anti-atheroprone effect of an integrin [alpha]5[beta]1-blocking peptide (ATN161) in Apoe~h mice. Activation of integrin [alpha]5[beta]1 induced tyrosine, but not serine, phosphorylation of YAP in ECs. Blockage of integrin [alpha]5[beta]1 with ATN161 abolished the phosphorylation of YAP at [Y.sup.357] induced by OSS. Mechanistic studies showed that c-Abl inhibitor attenuated the integrin [alpha]5[beta]1-induced YAP tyrosine phosphorylation. Furthermore, the phosphorylation of c-Abl and [YAP.sup.Y357] was significantly increased in ECs in atherosclerotic vessels of mice and in human plaques versus normal vessels. Finally, bosutinib, a tyrosine kinase inhibitor, markedly reduced the level of [YAP.sup.Y357] and the development of atherosclerosis in [Apoe.sup.-/-] mice. The c-Abl/[YAP.sup.Y357] pathway serves as a mechanism for the activation of integrin [alpha]5[beta]1 and the atherogenic phenotype of ECs in response to OSS, and provides a potential therapeutic strategy for atherogenesis.

Introduction High-throughput sequencing has revolutionized molecular medicine. In hematology, genomic technologies have unveiled the complex genomic landscape of blood pathologies including myelodys-plastic syndrome (MDS) and acute myeloid leukemia (AML) (1-3). [...], The initiation and evolution of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are driven by genomic events that disrupt multiple genes controlling hematopoiesis. Human genetic studies have discovered germline mutations in single genes that instigate familial MDS/AML. The best understood of these genes encode transcription factors, such as GATA-2, RUNX1, ETV6, and C/EBP[alpha], which establish and maintain genetic networks governing the genesis and function of blood stem and progenitor cells. Many questions remain unanswered regarding how genes and circuits within these networks function in physiology and disease and whether network integrity is exquisitely sensitive to or efficiently buffered from perturbations. In familial MDS/AML, mutations change the coding sequence of a gene to generate a mutant protein with altered activity or introduce frameshifts or stop codons or disrupt regulatory elements to alter protein expression. Each mutation has the potential to exert quantitatively and qualitatively distinct influences on networks. Consistent with this mechanistic diversity, disease onset is unpredictable and phenotypic variability can be considerable. Efforts to elucidate mechanisms and forge prognostic and therapeutic strategies must therefore contend with a spectrum of patient-specific leukemogenic scenarios. Here we illustrate mechanistic advances in our understanding of familial MDS/AML syndromes caused by germline mutations of hematopoietic transcription factors.

Author(s): Shunsuke Miyashima [sup.1] [sup.2] , Pawel Roszak [sup.1] [sup.3] , Iris Sevilem [sup.1] , Koichi Toyokura [sup.3] [sup.4] , Bernhard Blob [sup.3] , Jung-ok Heo [sup.1] [sup.3] , Nathan [...], Apical growth in plants initiates upon seed germination, whereas radial growth is primed only during early ontogenesis in procambium cells and activated later by the vascular cambium.sup.1. Although it is not known how radial growth is organized and regulated in plants, this system resembles the developmental competence observed in some animal systems, in which pre-existing patterns of developmental potential are established early on.sup.2,3. Here we show that in Arabidopsis the initiation of radial growth occurs around early protophloem-sieve-element cell files of the root procambial tissue. In this domain, cytokinin signalling promotes the expression of a pair of mobile transcription factors--PHLOEM EARLY DOF 1 (PEAR1) and PHLOEM EARLY DOF 2 (PEAR2)--and their four homologues (DOF6, TMO6, OBP2 and HCA2), which we collectively name PEAR proteins. The PEAR proteins form a short-range concentration gradient that peaks at protophloem sieve elements, and activates gene expression that promotes radial growth. The expression and function of PEAR proteins are antagonized by the HD-ZIP III proteins, well-known polarity transcription factors.sup.4--the expression of which is concentrated in the more-internal domain of radially non-dividing procambial cells by the function of auxin, and mobile miR165 and miR166 microRNAs. The PEAR proteins locally promote transcription of their inhibitory HD-ZIP III genes, and thereby establish a negative-feedback loop that forms a robust boundary that demarks the zone of cell division. Taken together, our data establish that during root procambial development there exists a network in which a module that links PEAR and HD-ZIP III transcription factors integrates spatial information of the hormonal domains and miRNA gradients to provide adjacent zones of dividing and more-quiescent cells, which forms a foundation for further radial growth.Radial growth in the roots of Arabidopsis, which is mediated by gene expression activated by the mobile PEAR1 and PEAR2 transcription factors, is initiated around protophloem-sieve-element cell files of procambial tissue.

Author(s): Anant Mamidi [sup.1] , Christy Prawiro [sup.1] , Philip A. Seymour [sup.1] , Kristian Honnens de Lichtenberg [sup.1] , Abigail Jackson [sup.1] , Palle Serup [sup.1] , Henrik Semb [...], The pancreas originates from two epithelial evaginations of the foregut, which consist of multipotent epithelial progenitors that organize into a complex tubular epithelial network. The trunk domain of each epithelial branch consists of bipotent pancreatic progenitors (bi-PPs) that give rise to both duct and endocrine lineages, whereas the tips give rise to acinar cells.sup.1. Here we identify the extrinsic and intrinsic signalling mechanisms that coordinate the fate-determining transcriptional events underlying these lineage decisions.sup.1,2. Single-cell analysis of pancreatic bipotent pancreatic progenitors derived from human embryonic stem cells reveal that cell confinement is a prerequisite for endocrine specification, whereas spreading drives the progenitors towards a ductal fate. Mechanistic studies identify the interaction of extracellular matrix (ECM) with integrin [alpha]5 as the extracellular cue that cell-autonomously, via the F-actin-YAP1-Notch mechanosignalling axis, controls the fate of bipotent pancreatic progenitors. Whereas ECM-integrin [alpha]5 signalling promotes differentiation towards the duct lineage, endocrinogenesis is stimulated when this signalling cascade is disrupted. This cascade can be disrupted pharmacologically or genetically to convert bipotent pancreatic progenitors derived from human embryonic stem cells to hormone-producing islet cells. Our findings identify the cell-extrinsic and intrinsic mechanotransduction pathway that acts as gatekeeper in the fate decisions of bipotent pancreatic progenitors in the developing pancreas. Single-cell analysis reveals that interactions with the extracellular matrix via integrin [alpha]5 and mechanotransducer YAP1 determine whether pancreatic progenitors develop along the duct or endocrine lineages.

Triple-negative breast cancer (TNBC) is particularly aggressive, with enhanced incidence of tumor relapse, resistance to chemotherapy, and metastases. As the mechanistic basis for this aggressive phenotype is unclear, treatment options are limited. Here, we showed an increased population of myeloid-derived immunosuppressor cells (MDSCs) in TNBC patients compared with non-TNBC patients. We found that high levels of the transcription factor [DELTA]Np63 correlate with an increased number of MDSCs in basal TNBC patients, and that [DELTA]Np63 promotes tumor growth, progression, and metastasis in human and mouse TNBC cells. Furthermore, we showed that MDSC recruitment to the primary tumor and metastatic sites occurs via direct [DELTA]Np63-dependent activation of the chemokines CXCL2 and CCL22. CXCR2/CCR4 inhibitors reduced MDSC recruitment, angiogenesis, and metastasis, highlighting a novel treatment option for this subset of TNBC patients. Finally, we found that MDSCs secrete prometastatic factors such as MMP9 and chitinase 3-like 1 to promote TNBC cancer stem cell function, thereby identifying a nonimmunologic role for MDSCs in promoting TNBC progression. These findings identify a unique crosstalk between [DELTA][Np63.sup.+] TNBC cells and MDSCs that promotes tumor progression and metastasis, which could be exploited in future combined immunotherapy/chemotherapy strategies for TNBC patients., Introduction Breast cancer is a devastating disease that, despite significantly advanced treatment options, claims around 50 million lives per year worldwide. Breast cancer is classified into subtypes based on the [...]

Author(s): Lei Chang [sup.1] , Luca Azzolin [sup.1] , Daniele Di Biagio [sup.1] , Francesca Zanconato [sup.1] , Giusy Battilana [sup.1] , Romy Lucon Xiccato [sup.1] , Mariaceleste Aragona [sup.1] [...], Inactivation of ARID1A and other components of the nuclear SWI/SNF protein complex occurs at very high frequencies in a variety of human malignancies, suggesting a widespread role for the SWI/SNF complex in tumour suppression.sup.1. However, the underlying mechanisms remain poorly understood. Here we show that ARID1A-containing SWI/SNF complex (ARID1A-SWI/SNF) operates as an inhibitor of the pro-oncogenic transcriptional coactivators YAP and TAZ.sup.2. Using a combination of gain- and loss-of-function approaches in several cellular contexts, we show that YAP/TAZ are necessary to induce the effects of the inactivation of the SWI/SNF complex, such as cell proliferation, acquisition of stem cell-like traits and liver tumorigenesis. We found that YAP/TAZ form a complex with SWI/SNF; this interaction is mediated by ARID1A and is alternative to the association of YAP/TAZ with the DNA-binding platform TEAD. Cellular mechanotransduction regulates the association between ARID1A-SWI/SNF and YAP/TAZ. The inhibitory interaction of ARID1A-SWI/SNF and YAP/TAZ is predominant in cells that experience low mechanical signalling, in which loss of ARID1A rescues the association between YAP/TAZ and TEAD. At high mechanical stress, nuclear F-actin binds to ARID1A-SWI/SNF, thereby preventing the formation of the ARID1A-SWI/SNF-YAP/TAZ complex, in favour of an association between TEAD and YAP/TAZ. We propose that a dual requirement must be met to fully enable the YAP/TAZ responses: promotion of nuclear accumulation of YAP/TAZ, for example, by loss of Hippo signalling, and inhibition of ARID1A-SWI/SNF, which can occur either through genetic inactivation or because of increased cell mechanics. This study offers a molecular framework in which mechanical signals that emerge at the tissue level together with genetic lesions activate YAP/TAZ to induce cell plasticity and tumorigenesis.The ARID1A-containing SWI/SNF complex operates as an inhibitor of the pro-oncogenic transcriptional coactivators YAP and TAZ; this interaction is regulated by cellular mechanotransduction.

Author(s): Leonardo Beccari [sup.1] , Naomi Moris [sup.2] , Mehmet Girgin [sup.3] , David A. Turner [sup.2] , Peter Baillie-Johnson [sup.2] [sup.5] , Anne-Catherine Cossy [sup.4] , Matthias P. Lutolf [...], The emergence of multiple axes is an essential element in the establishment of the mammalian body plan. This process takes place shortly after implantation of the embryo within the uterus and relies on the activity of gene regulatory networks that coordinate transcription in space and time. Whereas genetic approaches have revealed important aspects of these processes.sup.1, a mechanistic understanding is hampered by the poor experimental accessibility of early post-implantation stages. Here we show that small aggregates of mouse embryonic stem cells (ESCs), when stimulated to undergo gastrulation-like events and elongation in vitro, can organize a post-occipital pattern of neural, mesodermal and endodermal derivatives that mimic embryonic spatial and temporal gene expression. The establishment of the three major body axes in these 'gastruloids'.sup.2,3 suggests that the mechanisms involved are interdependent. Specifically, gastruloids display the hallmarks of axial gene regulatory systems as exemplified by the implementation of collinear Hox transcriptional patterns along an extending antero-posterior axis. These results reveal an unanticipated self-organizing capacity of aggregated ESCs and suggest that gastruloids could be used as a complementary system to study early developmental events in the mammalian embryo.Cultures grown from small aggregates of mouse embryonic stem cells can be induced to organize spatial and temporal patterns of gene expression that parallel those of the early embryo, offering a potentially useful system for studying early development.

Introduction Progression of chronic kidney disease (CKD) toward end-stage renal disease (ESRD) is an unmet biomedical challenge, just like chronic progressive failure of any other parenchymal organ, because effective therapies [...], Progression of chronic kidney disease associated with progressive fibrosis and impaired tubular epithelial regeneration is still an unmet biomedical challenge because, once chronic lesions have manifested, no effective therapies are available as of yet for clinical use. Prompted by various studies across multiple organs demonstrating that preconditioning regimens to induce endogenous regenerative mechanisms protect various organs from later incurring acute injuries, we here aimed to gain insights into the molecular mechanisms underlying successful protection and to explore whether such pathways could be utilized to inhibit progression of chronic organ injury. We identified a protective mechanism controlled by the transcription factor ARNT that effectively inhibits progression of chronic kidney injury by transcriptional induction of ALK3, the principal mediator of antifibrotic and proregenerative bone morphogenetic protein-signaling (BMP-signaling) responses. We further report that ARNT expression itself is controlled by the FKBP12/YY1 transcriptional repressor complex and that disruption of such FKBP12/YY1 complexes by picomolar FK506 at subimmunosuppressive doses increases ARNT expression, subsequently leading to homodimeric ARNT-induced ALK3 transcription. Direct targeting of FKBP12/YY1 with in vivo morpholino approaches or small molecule inhibitors, including GPI-1046, was equally effective for inducing ARNT expression, with subsequent activation of ALK3-dependent canonical BMP-signaling responses and attenuated chronic organ failure in models of chronic kidney disease, and also cardiac and liver injuries. In summary, we report an organ-protective mechanism that can be pharmacologically modulated by immunophilin ligands FK506 and GPI-1046 or therapeutically targeted by in vivo morpholino approaches.

Introduction Squamous cell carcinomas (SCCs) are the most common forms of human cancer (1) and are a major cause of death. These heterogeneous tumors originate from surface epithelia, such as [...], Notch 1/2 genes play tumor-suppressing functions in squamous cell carcinoma (SCC), a very common malignancy in skin and internal organs. In contrast with Notch, we show that the transcription factor CSL (also known as RBP-J[kappa]), a key effector of canonical Notch signaling endowed with intrinsic transcription-repressive functions, plays a tumor-promoting function in SCC development. Expression of this gene decreased in upper epidermal layers and human keratinocytes (HKCs) undergoing differentiation, while it increased in premalignant and malignant SCC lesions from skin, head/neck, and lung. Increased CSL levels enhanced the proliferative potential of HKCs and SCC cells, while silencing of CSL induced growth arrest and apoptosis. In vivo, SCC cells with increased CSL levels gave rise to rapidly expanding tumors, while cells with silenced CSL formed smaller and more differentiated tumors with enhanced inflammatory infiltrate. Global transcriptomic analysis of HKCs and SCC cells with silenced CSL revealed major modulation of apoptotic, cell-cycle, and proinflammatory genes. We also show that the histone demethylase KDM6B is a direct CSL-negative target, with inverse roles of CSL in HKC and SCC proliferative capacity, tumorigenesis, and tumor- associated inflammatory reaction. CSL/KDM6B protein expression could be used as a biomarker of SCC development and indicator of cancer treatment.

Introduction The coordinated proliferation and differentiation of adult stem cells provide sustainable cell sources for tissue repair and regeneration. In support of long-term tissue homeostasis, many adult stem cell populations [...], The remarkable regeneration capability of skeletal muscle depends on the coordinated proliferation and differentiation of satellite cells (SCs). The self-renewal of SCs is critical for long-term maintenance of muscle regeneration potential. Hypoxia profoundly affects the proliferation, differentiation, and self-renewal of cultured myoblasts. However, the physiological relevance of hypoxia and hypoxia signaling in SCs in vivo remains largely unknown. Here, we demonstrate that SCs are in an intrinsic hypoxic state in vivo and express hypoxia-inducible factor 2A (HIF2A). HIF2A promotes the stemness and long-term homeostatic maintenance of SCs by maintaining their quiescence, increasing their self-renewal, and blocking their myogenic differentiation. HIF2A stabilization in SCs cultured under normoxia augments their engraftment potential in regenerative muscle. Conversely, HIF2A ablation leads to the depletion of SCs and their consequent regenerative failure in the long-term. In contrast, transient pharmacological inhibition of HIF2A accelerates muscle regeneration by increasing SC proliferation and differentiation. Mechanistically, HIF2A induces the quiescence and self-renewal of SCs by binding the promoter of the Spryl gene and activating Spryl expression. These findings suggest that HIF2A is a pivotal mediator of hypoxia signaling in SCs and may be therapeutically targeted to improve muscle regeneration.

Author(s): Monika Bambouskova [sup.1] , Laurent Gorvel [sup.1] , Vicky Lampropoulou [sup.1] , Alexey Sergushichev [sup.2] , Ekaterina Loginicheva [sup.1] , Kendall Johnson [sup.3] , Daniel Korenfeld [sup.1] , Mary [...], Metabolic regulation has been recognized as a powerful principle guiding immune responses. Inflammatory macrophages undergo extensive metabolic rewiring.sup.1 marked by the production of substantial amounts of itaconate, which has recently been described as an immunoregulatory metabolite.sup.2. Itaconate and its membrane-permeable derivative dimethyl itaconate (DI) selectively inhibit a subset of cytokines.sup.2, including IL-6 and IL-12 but not TNF. The major effects of itaconate on cellular metabolism during macrophage activation have been attributed to the inhibition of succinate dehydrogenase.sup.2,3, yet this inhibition alone is not sufficient to account for the pronounced immunoregulatory effects observed in the case of DI. Furthermore, the regulatory pathway responsible for such selective effects of itaconate and DI on the inflammatory program has not been defined. Here we show that itaconate and DI induce electrophilic stress, react with glutathione and subsequently induce both Nrf2 (also known as NFE2L2)-dependent and -independent responses. We find that electrophilic stress can selectively regulate secondary, but not primary, transcriptional responses to toll-like receptor stimulation via inhibition of I[kappa]B[zeta] protein induction. The regulation of I[kappa]B[zeta] is independent of Nrf2, and we identify ATF3 as its key mediator. The inhibitory effect is conserved across species and cell types, and the in vivo administration of DI can ameliorate IL-17-I[kappa]B[zeta]-driven skin pathology in a mouse model of psoriasis, highlighting the therapeutic potential of this regulatory pathway. Our results demonstrate that targeting the DI-I[kappa]B[zeta] regulatory axis could be an important new strategy for the treatment of IL-17-I[kappa]B[zeta]-mediated autoimmune diseases. The immunoregulatory metabolite itaconate and its dimethyl derivative induce electrophilic stress and react with glutathione to induce both Nrf2-dependent and Nrf2-independent responses, resulting in AF3-mediated inhibition of the inflammation-related protein I[kappa]B[zeta].

Introduction Autophagy is an intracellular self-digesting process that promotes degradation of damaged cellular organelles through trafficking to the lysosome (1). Three different forms of autophagy have been identified: macroautophagy, microautophagy, [...], Autophagy modulates lipid turnover, cell survival, inflammation, and atherogenesis. Scavenger receptor class B type I (SR-BI) plays a crucial role in lysosome function. Here, we demonstrate that SR-BI regulates autophagy in atherosclerosis. SR-BI deletion attenuated lipid-induced expression of autophagy mediators in macrophages and atherosclerotic aortas. Consequently, SR-BI deletion resulted in 1.8- and 2.5-fold increases in foam cell formation and apoptosis, respectively, and increased oxidized LDL-induced inflammatory cytokine expression. Pharmacological activation of autophagy failed to reduce lipid content or apoptosis in Sr-[b1.sup.-/-] macrophages. SR-BI deletion reduced both basal and inducible levels of transcription factor EB (TFEB), a master regulator of autophagy, causing decreased expression of autophagy genes encoding VPS34 and Beclin-1. Notably, SR-BI regulated Tfeb expression by enhancing PPAR[alpha] activation. Moreover, intracellular macrophage SR-BI localized to autophagosomes, where it formed cholesterol domains resulting in enhanced association of Barkor and recruitment of the VPS34-Beclin-1 complex. Thus, SR-BI deficiency led to lower VPS34 activity in macrophages and in atherosclerotic aortic tissues. Overexpression of Tfeb or Vps34 rescued the defective autophagy in Sr-[b1.sup.-/-] macrophages. Taken together, our results show that macrophage SR-BI regulates autophagy via Tfeb expression and recruitment of the VPS34-Beclin-1 complex, thus identifying previously unrecognized roles for SR-BI and potentially novel targets for the treatment of atherosclerosis.

Introduction The vast majority of human colorectal cancer (CRC) is triggered by inactivation of the tumor suppressor adenomatous polyposis coli (APC) in one of several intestinal stem cells (ISCs) present [...], p120-Catenin (p120) functions as a tumor suppressor in intestinal cancer, but the mechanism is unclear. Here, using conditional p120 knockout in Ape-sensitized mouse models of intestinal cancer, we have identified p120 as an "obligatory" haploinsufficient tumor suppressor. Whereas monoallelic loss of p120 was associated with a significant increase in tumor multiplicity, loss of both alleles was never observed in tumors from these mice. Moreover, forced ablation of the second allele did not further enhance tumorigenesis, but instead induced synthetic lethality in combination with Ape loss of heterozygosity. In tumor-derived organoid cultures, elimination of both p120 alleles resulted in caspase-3-dependent apoptosis that was blocked by inhibition of Rho kinase (ROCK). With ROCK inhibition, however, p120-ablated organoids exhibited a branching phenotype and a substantial increase in cell proliferation. Access to data from Sleeping Beauty mutagenesis screens afforded an opportunity to directly assess the tumorigenic impact of p120 haploinsufficiency relative to other candidate drivers. Remarkably, p120 ranked third among the 919 drivers identified. Cofactors [alpha]-catenin and epithelial cadherin (E-cadherin) were also among the highest scoring candidates, indicating a mechanism at the level of the intact complex that may play an important role at very early stages of of intestinal tumorigenesis while simultaneously restricting outright loss via synthetic lethality.

Author(s): Sabine Harlander [1, 2]; Désirée Schönenberger [1]; Nora C Toussaint [3, 4]; Michael Prummer [3, 4]; Antonella Catalano [1, 5, 6]; Laura Brandt [1]; Holger Moch [7]; Peter J [...], Clear cell renal cell carcinomas (ccRCCs) frequently exhibit inactivation of the von Hippel-Lindau tumor-suppressor gene, VHL, and often harbor multiple copy-number alterations in genes that regulate cell cycle progression. We show here that modeling these genetic alterations by combined deletion of Vhl, Trp53 and Rb1 specifically in renal epithelial cells in mice caused ccRCC. These tumors arose from proximal tubule epithelial cells and shared molecular markers and mRNA expression profiles with human ccRCC. Exome sequencing revealed that mouse and human ccRCCs exhibit recurrent mutations in genes associated with the primary cilium, uncovering a mutational convergence on this organelle and implicating a subset of ccRCCs as genetic ciliopathies. Different mouse tumors responded differently to standard therapies for advanced human ccRCC, mimicking the range of clinical behaviors in the human disease. Inhibition of hypoxia-inducible factor (HIF)-[alpha] transcription factors with acriflavine as third-line therapy had therapeutic effects in some tumors, providing preclinical evidence for further investigation of HIF-[alpha] inhibition as a ccRCC treatment. This autochthonous mouse ccRCC model represents a tool to investigate the biology of ccRCC and to identify new treatment strategies.

Introduction The E2F family of transcription factors consists of 8 members that can be organized into 3 subcategories based on function and expression patterns during the cell cycle: activators (E2F1-3), [...], Disruption of the retinoblastoma (RB) tumor suppressor pathway, either through genetic mutation of upstream regulatory components or mutation of RB1 itself, is believed to be a required event in cancer. However, genetic alterations in the RB-regulated E2F family of transcription factors are infrequent, casting doubt on a direct role for E2Fs in driving cancer. In this work, a mutation analysis of human cancer revealed subtle but impactful copy number gains in E2F1 and E2F3 in hepatocellular carcinoma (HCC). Using a series of loss- and gain-of-function alleles to dial E2F transcriptional output, we have shown that copy number gains in E2f1 or E2f3b resulted in dosage-dependent spontaneous HCC in mice without the involvement of additional organs. Conversely, germ-line loss of E2f1 or E2f3b, but not E2f3a, protected mice against HCC. Combinatorial mapping of chromatin occupancy and transcriptome profiling identified an E2F1- and E2F3B-driven transcriptional program that was associated with development and progression of HCC. These findings demonstrate a direct and cell-autonomous role for E2F activators in human cancer.

HepcidinHepcidin is produced primarily in the liver by hepatocytes, which sense plasma iron through a complex network of membrane-bound proteins that include the HLA class I--like protein HFE, transferrin receptor [...], Hepcidin is the master regulator of iron metabolism. It plays a key role in the regulation of iron transport across the duodenal epithelium, which in turn is dependent on the oxygen-regulated transcription factor hypoxia-inducible factor 2[alpha] (HIF-2[alpha]). In this issue of the JCI, Schwartz and colleagues show that duodenal HIF-2[alpha] is itself regulated by hepcidin, thereby indicating that this transcription factor is not only regulated by oxygen, but also by iron. This work indicates that the crosstalk between liver hepcidin and intestinal HIF-2[alpha] plays an important role during iron overload, systemic iron deficiency, and anemia.

DNA-binding with one finger (Dof) domain proteins are a multigene family of plant-specific transcription factors involved in numerous aspects of plant growth and development. In this study, we report a [...]

Introduction Heritable and acquired syndromes affecting fast conduction in the atria and ventricular conduction system (VCS) produce a broad spectrum of arrhythmic disease, including atrial fibrillation, ventricular tachyarrhythmias, and heart [...], Rapid impulse propagation in the heart is a defining property of pectinated atrial myocardium (PAM) and the ventricular conduction system (VCS) and is essential for maintaining normal cardiac rhythm and optimal cardiac output. Conduction defects in these tissues produce a disproportionate burden of arrhythmic disease and are major predictors of mortality in heart failure patients. Despite the clinical importance, little is known about the gene regulatory network that dictates the fast conduction phenotype. Here, we have used signal transduction and transcriptional profiling screens to identify a genetic pathway that converges on the NRG1-responsive transcription factor ETV1 as a critical regulator of fast conduction physiology for PAM and VCS cardiomyocytes. Etv1 was highly expressed in murine PAM and VCS cardiomyocytes, where it regulates expression of Nkx2-5, Gja5, and Scn5a, key cardiac genes required for rapid conduction. Mice deficient in Etv1 exhibited marked cardiac conduction defects coupled with developmental abnormalities of the VCS. Loss of ETV1 resulted in a complete disruption of the normal sodium current heterogeneity that exists between atrial, VCS, and ventricular myocytes. Lastly, a phenome-wide association study identified a link between ETV1 and bundle branch block and heart block in humans. Together, these results identify ETV1 as a critical factor in determining fast conduction physiology in the heart.

Introduction The intestine has a mucosal surface that directly interacts with large quantities of food antigens and microorganisms daily. As a consequence, the gut is the accessible entry site for [...], The transcription factor NR4A3 (also known as NOR-1) is a member of the Nr4a family of nuclear receptors and is expressed in myeloid and lymphoid cells. Here, we have shown that Nr4a3 is essential for the migration of [CD103.sup.+] dendritic cells (DCs) to lymph nodes (LNs). Nr4a3-deficient mice had very few [CD103.sup.+] migratory DCs (mDCs) present in LNs, and mixed- chimera studies revealed that this migratory defect was cell intrinsic. We further found that [CD103.sup.+] DCs from Nr4a3- deficient mice displayed a marked loss of surface expression of the chemokine CCR7. This defect in CCR7 expression was confined to [CD103.sup.+] DCs, as CCR7 expression on T lymphocytes was unaffected. Moreover, CCR7 was not induced on [CD103.sup.+] DCs from Nr4a3-deficient mice in response to either administration of the TLR7 agonist R848 or infection with Citrobacter rodentium in vivo. The transcription factor FOXO1 has been shown to regulate CCR7 expression. We found that FOXO1 protein was reduced in Nr4a3-deficient DCs through an AKT-dependent mechanism. Further, we found a requirement for NR4A3 in the maintenance of homeostatic mitochondrial function in [CD103.sup.+] DCs, although this is likely independent of the NR4A3/FOXO1/CCR7 axis in the regulation of DC migration. Thus, NR4A3 plays an important role in the regulation of [CD103.sup.+] mDCs by regulating CCR7-dependent cell migration.

Introduction Progressive tissue fibrosis is a major cause of morbidity and mortality with limited therapeutic choices. Severe pulmonary fibrosis is the integrative result of repeated epithelial injuries and recruitment and [...], Progressive tissue fibrosis is a major cause of the morbidity and mortality associated with repeated epithelial injuries and accumulation of myofibroblasts. Successful treatment options are limited by an incomplete understanding of the molecular mechanisms that regulate myofibroblast accumulation. Here, we employed in vivo lineage tracing and real-time gene expression transgenic reporting methods to analyze the early embryonic transcription factor T-box gene 4 (TBX4), and determined that TBX4-lineage mesenchymal progenitors are the predominant source of myofibroblasts in injured adult lung. In a murine model, ablation of TBX4-expressing cells or disruption of TBX4 signaling attenuated lung fibrosis after bleomycin-induced injury. Furthermore, TBX4 regulated hyaluronan synthase 2 production to enable fibroblast invasion of matrix both in murine models and in fibroblasts from patients with severe pulmonary fibrosis. These data identify TBX4 as a mesenchymal transcription factor that drives accumulation of myofibroblasts and the development of lung fibrosis. Targeting TBX4 and downstream factors that regulate fibroblast invasiveness could lead to therapeutic approaches in lung fibrosis.

Vascular and haematopoietic cells organize into specialized tissues during early embryogenesis to supply essential nutrients to all organs and thus play critical roles in development and disease. At the top [...]

Stringent control is a pleiotropic response to the failure of amino acid availability to keep up with the demands of protein synthesis (1). It is mediated by a hyperphosphorylated nucleotide [...], In order to survive, bacteria continually sense, and respond to, environmental fluctuations. Stringent control represents a key bacterial stress response to nutrient starvation (1,2) that leads to rapid and comprehensive reprogramming of metabolic and transcriptional patterns3. In general, transcription of genes for growth and proliferation is downregulated, while those important for survival and virulence are upregulated (4). Amino acid starvation is sensed by depletion of the aminoacylated tRNA pools (5), and this results in accumulation of ribosomes stalled with non-aminoacylated (uncharged) tRNA in the ribosomal A site (6,7). RelA is recruited to stalled ribosomes and activated to synthesize a hyperphosphorylated guanosine analogue, (p)ppGpp (8), which acts as a pleiotropic secondary messenger. However, structural information about how RelA recognizes stalled ribosomes and discriminates against aminoacylated tRNAs is missing. Here we present the cryo-electron microscopy structure of RelA bound to the bacterial ribosome stalled with uncharged tRNA. The structure reveals that RelA utilizes a distinct binding site compared to the translational factors, with a multi-domain architecture that wraps around a highly distorted A-site tRNA. The TGS (ThrRS, GTPase and SpoT) domain of RelA binds the CCA tail to orient the free 3' hydroxyl group of the terminal adenosine towards a β-strand, such that an aminoacylated tRNA at this position would be sterically precluded. The structure supports a model in which association of RelA with the ribosome suppresses auto-inhibition to activate synthesis of (p)ppGpp and initiate the stringent response. Since stringent control is responsible for the survival of pathogenic bacteria under stress conditions, and contributes to chronic infections and antibiotic tolerance, RelA represents a good target for the development of novel antibacterial therapeutics.

Introduction SOX9 belongs to the SOX (SRY-related HMG box) family of transcription factors and regulates many developmental processes. SOX9 mutations cause campomelic dysplasia, a disease that is characterized by extreme [...], The transcription factor SOX9 is critical for prostate development, and dysregulation of SOX9 is implicated in prostate cancer (PCa). However, the SOX9-dependent genes and pathways involved in both normal and neoplastic prostate epithelium are largely unknown. Here, we performed SOX9 ChIP sequencing analysis and transcriptome profiling of PCa cells and determined that SOX9 positively regulates multiple WNT pathway genes, including those encoding WNT receptors (frizzled [FZD] and lipoprotein receptor-related protein [LRP] family members) and the downstream β-catenin effector TCF4. Analyses of PCa xenografts and clinical samples both revealed an association between the expression of SOX9 and WNT pathway components in PCa. Finally, treatment of SOX9-expressing PCa cells with a WNT synthesis inhibitor (LGK974) reduced WNT pathway signaling in vitro and tumor growth in murine xenograft models. Together, our data indicate that SOX9 expression drives PCa by reactivating the WNT/β-catenin signaling that mediates ductal morphogenesis in fetal prostate and define a subgroup of patients who would benefit from WNT-targeted therapy.

Initiation of transcription by Pol II represents a major control point for eukaryotic cells, and its regulation is the primary means of differential gene expression in metazoans (1). A prerequisite [...], The general transcription factor IID (TFIID) plays a central role in the initiation of RNA polymerase II (Pol II)-dependent transcription by nucleating pre-initiation complex (PIC) assembly at the core promoter. TFIID comprises the TATA-binding protein (TBP) and 13 TBP-associated factors (TAF1-13), which specifically interact with a variety of core promoter DNA sequences. Here we present the structure of human TFIID in complex with TFIIA and core promoter DNA, determined by single-particle cryo-electron microscopy at sub-nanometre resolution. All core promoter elements are contacted by subunits of TFIID, with TAF1 and TAF2 mediating major interactions with the downstream promoter. TFIIA bridges the TBP-TATA complex with lobe B of TFIID. We also present the cryo-electron microscopy reconstruction of a fully assembled human TAF-less PIC. Superposition of common elements between the two structures provides novel insights into the general role of TFIID in promoter recognition, PIC assembly, and transcription initiation.

Introduction The independent lineages of mature hematopoietic cells are initially generated from stem cells that are extrinsically and intrinsically regulated to traverse multiple, distinct developmental stages. A host of tissue- [...], The transcription factor GATA3 is essential for the genesis and maturation of the T cell lineage, and GATA3 dysregulation has pathological consequences. Previous studies have shown that GATA3 function in T cell development is regulated by multiple signaling pathways and that the Notch nuclear effector, RBP-J, binds specifically to the Gata3 promoter. We previously identified a T cell-specific Gata3 enhancer (Tce1) lying 280 kb downstream from the structural gene and demonstrated in transgenic mice that Tce1 promoted T lymphocyte-specific transcription of reporter genes throughout T cell development; however, it was not clear if Tce1 is required for Gata3 transcription in vivo. Here, we determined that the canonical Gata3 promoter is insufficient for Gata3 transcriptional activation in T cells in vivo, precluding the possibility that promoter binding by a host of previously implicated transcription factors alone is responsible for Gata3 expression in T cells. Instead, we demonstrated that multiple lineage-affiliated transcription factors bind to Tce1 and that this enhancer confers T lymphocyte-specific Gata3 activation in vivo, as targeted deletion of Tce1 in a mouse model abrogated critical functions of this T cell-regulatory element. Together, our data show that Tce1 is both necessary and sufficient for critical aspects of Gata3 T cell-specific transcriptional activity.

Author(s): Yong Cheng [1]; Zhihai Ma [1]; Bong-Hyun Kim [2]; Weisheng Wu [3, 4]; Philip Cayting [1]; Alan P. Boyle [1]; Vasavi Sundaram [5]; Xiaoyun Xing [5]; Nergiz Dogan [3]; [...], To broaden our understanding of the evolution of gene regulation mechanisms, we generated occupancy profiles for 34 orthologous transcription factors (TFs) in human-mouse erythroid progenitor, lymphoblast and embryonic stem-cell lines. By combining the genome-wide transcription factor occupancy repertoires, associated epigenetic signals, and co-association patterns, here we deduce several evolutionary principles of gene regulatory features operating since the mouse and human lineages diverged. The genomic distribution profiles, primary binding motifs, chromatin states, and DNA methylation preferences are well conserved for TF-occupied sequences. However, the extent to which orthologous DNA segments are bound by orthologous TFs varies both among TFs and with genomic location: binding at promoters is more highly conserved than binding at distal elements. Notably, occupancy-conserved TF-occupied sequences tend to be pleiotropic; they function in several tissues and also co-associate with many TFs. Single nucleotide variants at sites with potential regulatory functions are enriched in occupancy-conserved TF-occupied sequences.

Drosophila embryogenesis proceeds very rapidly, taking 18 h from egg lay to completion. Underlying this dynamic developmental program are marked changes in transcription, which are in turn regulated by characterized [...], Developmental enhancers initiate transcription and are fundamental to our understanding of developmental networks, evolution and disease. Despite their importance, the properties governing enhancer-promoter interactions and their dynamics during embryogenesis remain unclear. At the β-globin locus, enhancer-promoter interactions appear dynamic and cell-type specific (1,2), whereas at the HoxD locus they are stable and ubiquitous, being present in tissues where the target genes are not expressed (3,4). The extent to which preformed enhancer-promoter conformations exist at other, more typical, loci and how transcription is eventually triggered is unclear. Here we generated a high-resolution map of enhancer three-dimensional contacts during Drosophila embryogenesis, covering two developmental stages and tissue contexts, at unprecedented resolution. Although local regulatory interactions are common, long-range interactions are highly prevalent within the compact Drosophila genome. Each enhancer contacts multiple enhancers, and promoters with similar expression, suggesting a role in their co-regulation. Notably, most interactions appear unchanged between tissue context and across development, arising before gene activation, and are frequently associated with paused RNA polymerase. Our results indicate that the general topology governing enhancer contacts is conserved from flies to humans and suggest that transcription initiates from preformed enhancer-promoter loops through release of paused polymerase.

In an effort to discover new inhibitors of kinases that regulate gene transcription, we performed cell-based screening and kinase selectivity profiling of a library of known and novel ATP-site-directed kinase [...], Tumour oncogenes include transcription factors that co-opt the general transcriptional machinery to sustain the oncogenic state (1), but direct pharmacological inhibition of transcription factors has so far proven difficult (2). However, the transcriptional machinery contains various enzymatic cofactors that can be targeted for the development of new therapeutic candidates (3), including cyclin-dependent kinases (CDKs) (4). Here we present the discovery and characterization of a covalent CDK7 inhibitor, THZ1, which has the unprecedented ability to target a remote cysteine residue located outside of the canonical kinase domain, providing an unanticipated means of achieving selectivity for CDK7. Cancer cell-line profiling indicates that a subset of cancer cell lines, including human T-cell acute lymphoblastic leukaemia (T-ALL), have exceptional sensitivity to THZ1. Genome-wide analysis in Jurkat T-ALL cells shows that THZ1 disproportionally affects transcription of RUNX1 and suggests that sensitivity to THZ1 may be due to vulnerability conferred by the RUNX1 super-enhancer and the key role of RUNX1 in the core transcriptional regulatory circuitry of these tumour cells. Pharmacological modulation of CDK7 kinase activity may thus provide an approach to identify and treat tumour types that are dependent on transcription for maintenance of the oncogenic state.

Cancer stem cells (CSCs) have been reported in various cancers, including in skin squamous-cell carcinoma (SCC) (1-4). The molecular mechanisms regulating tumour initiation and stemness are still poorly characterized. Here [...]