Transcriptional Partners Research Articles

Exploiting the Gal4/UAS System as Plant Orthogonal Molecular Toolbox to Control Reporter Expression in Arabidopsis Protoplasts.

The ability of protein domains to fold independently from the rest of the polypeptide is the principle governing the generation of fusion proteins with customized functions. A clear example is the split transcription factor system based on the yeast GAL4 protein and its cognate UAS enhancer. The rare occurrence of the UAS element in the transcriptionally sensitive regions of the Arabidopsis genome makes this transcription factor an ideal orthogonal platform to control reporter induction. Moreover, heterodimeric transcriptional complexes can be generated by exploiting posttranslational modifications hampering or promoting the interaction between GAL4-fused transcriptional partners, whenever this leads to the reconstitution of a fully functional GAL4 factor.The assembly of multiple engineered proteins into a synthetic transcriptional complex requires preliminary testing, before its components can be stably introduced into the plant genome. Mesophyll protoplast transformation represents a fast and reliable technique to test and optimize synthetic regulatory modules. Remarkable properties are the possibility to transform different combinations of plasmids (co-transformation) and the physiological resemblance of these isolated cells with the original tissue.Here we describe an extensive protocol to produce and exploit Arabidopsis mesophyll protoplasts to investigate the transcriptional output of GAL4/UAS-based complexes that are sensitive to posttranslational protein modifications.

Redundant Functions of ERK1 and ERK2 Maintain Mouse Liver Homeostasis Through Down-Regulation of Bile Acid Synthesis.

Activation of extracellular signal–regulated kinase (ERK) 1/2 promotes hepatocyte proliferation in response to growth stimuli, but whether constitutive hepatocyte ERK1/2 signaling functions in liver physiology is unknown. To examine the role of ERK1/2 in hepatic homeostasis, the effects of a knockout of Erk1 and/or Erk2 in mouse liver were examined. The livers of mice with a global Erk1 knockout or a tamoxifen‐inducible, hepatocyte‐specific Erk2 knockout were normal. In contrast, Erk1/2 double‐knockout mice developed hepatomegaly and hepatitis by serum transaminases, histology, terminal deoxynucleotide transferase‐mediated deoxyuridine triphosphate nick end‐labeling, and assays of hepatic inflammation. Liver injury was associated with biochemical evidence of cholestasis with increased serum and hepatic bile acids and led to hepatic fibrosis and mortality. RNA sequencing and polymerase chain reaction analysis of double‐knockout mouse livers revealed that the rate‐limiting bile acid synthesis gene Cyp7a1 (cholesterol 7α‐hydroxylase) was up‐regulated in concert with decreased expression of the transcriptional repressor short heterodimer partner. Elevated bile acids were the mechanism of liver injury, as bile acid reduction by SC‐435, an inhibitor of the ileal apical sodium–dependent bile acid transporter, prevented liver injury. Conclusion: Constitutive ERK1 and ERK2 signaling has a redundant but critical physiological function in the down‐regulation of hepatic bile acid synthesis to maintain normal liver homeostasis.

Mineralocorticoid receptor actions in cardiovascular development and disease.

Mineralocorticoid receptors (MRs) are transcriptional regulators that mediate the diverse physiological and pathophysiological actions of corticosteroid hormones across many tissues. In the kidney aldosterone control of sodium/water resorption via DNA-binding actions of the MR is established. MRs also regulate tissues not involved in electrolyte homeostasis such as the heart, adipose tissue, brain, and inflammatory cells where the MRs can respond to both aldosterone and cortisol. The pathology of inappropriate MR activation in non-epithelial tissues are well-described, and steroidal antagonists of the MR have been clinically beneficial in the management of heart failure and blood pressure for decades. However, the role of cortisol-dependent MR activation in the physiological setting is less well defined. Like other steroid hormone receptors, the MR also regulates non-DNA-binding pathways including MAPK pathways and G protein coupled receptors to provide diversity to MR signaling. Whether nonDNA binding pathways are more relevant for MR activation in non-epithelial, versus epithelial, tissues remain unclear. This review will focus on molecular regulation of ligand-dependent MR activation and the physiology and pathophysiology of MR actions in the heart with a focus on the cardiomyocyte and provide a discussion of relevant genomic and non-genomic MR pathways and potential new transcriptional partners for the MR and their relevance for health and disease. Understanding MR actions in the heart will provide new insights into cell-selective mechanisms that underpin the therapeutic benefits of MRAs, and are a critical step towards developing next-generation tissue selective MR modulators with improved safety profiles.

Zfhx4 regulates endochondral ossification as the transcriptional platform of Osterix in mice

Endochondral ossification is regulated by transcription factors that include SRY-box transcription factor 9, runt-related protein 2 (Runx2), and Osterix. However, the sequential and harmonious regulation of the multiple steps of endochondral ossification is unclear. This study identified zinc finger homeodomain 4 (Zfhx4) as a crucial transcriptional partner of Osterix. We found that Zfhx4 was highly expressed in cartilage and that Zfhx4 deficient mice had reduced expression of matrix metallopeptidase 13 and inhibited calcification of cartilage matrices. These phenotypes were very similar to impaired chondrogenesis in Osterix deficient mice. Coimmunoprecipitation and immunofluorescence indicated a physical interaction between Zfhx4 and Osterix. Notably, Zfhx4 and Osterix double mutant mice showed more severe phenotype than Zfhx4 deficient mice. Additionally, Zfhx4 interacted with Runx2 that functions upstream of Osterix. Our findings suggest that Zfhx4 coordinates the transcriptional network of Osterix and, consequently, endochondral ossification.

A pathogenic DYT-THAP1 dystonia mutation causes hypomyelination and loss of YY1 binding.

Dystonia is a disabling disease that manifests as prolonged involuntary twisting movements. DYT-THAP1 is an inherited form of isolated dystonia caused by mutations in THAP1 encoding the transcription factor THAP1. The phe81leu (F81L) missense mutation is representative of a category of poorly understood mutations that do not occur on residues critical for DNA binding. Here, we demonstrate that the F81L mutation (THAP1F81L) impairs THAP1 transcriptional activity and disrupts CNS myelination. Strikingly, THAP1F81L exhibits normal DNA binding but causes a significantly reduced DNA binding of YY1, its transcriptional partner that also has an established role in oligodendrocyte lineage progression. Our results suggest a model of molecular pathogenesis whereby THAP1F81L normally binds DNA but is unable to efficiently organize an active transcription complex.

Au carrefour de la différenciation et des pathologies ovariennes, FOXL2, le grand intercesseur/Genomic and proteomic exploration of FOXL2 in an ovarian context : An update of its partners and targets

FOXL2 is a transcription factor critical for ovarian function. It has been implicated in diverse cellular processes, including apoptosis, the control of cell cycle or the regulation of steroid hormone synthesis. Our recent studies show that FOXL2 is involved in a large number of regulatory actions essential for the maintenance of granulosa cell fate. We have also identified hundreds of direct and indirect targets of estrogen receptor beta, of which one third are also targets of FOXL2. Accumulating evidence shows that both factors modulate, through a coherent feed-forward loop, a number of common targets. Many of the FOXL2/ERbeta targets are involved in cell migration, invasion and adhesion. Beyond the study of its transcriptional targets, the identification of the various interactors of FOXL2 is crucial to understand its diverse cellular roles. To reliably identify partners of endogenous FOXL2, we performed a proteome-wide analysis using co-immunoprecipitation in the murine granulosa cell-derived AT29c and the pituitary-derived alpha-T3 cell lines. Following a stringent selection, we identified a core set of about 250 (direct and indirect) partners common to both cell lines. Their analysis showed that FOXL2 belongs to complexes involved in mRNA processing, chromatin remodeling and DNA replication and repair. We further validated interactions with selected partners, suggesting an unexpected role for FOXL2. Our analysis expands the number of direct and indirect transcriptional targets and partners of both FOXL2, which deserve investigation in the context of adult-type granulosa cell tumors whose molecular diagnostic hallmark is the presence of the C134 W FOXL2 pathogenic variant.

TGF-β/activin signaling promotes CDK7 inhibitor resistance in triple-negative breast cancer cells through upregulation of multidrug transporters

Cyclin-dependent kinase 7 (CDK7) is a master regulatory kinase that drives cell cycle progression and stimulates expression of oncogenes in a myriad of cancers. Inhibitors of CDK7 (CDK7i) are currently in clinical trials; however, as with many cancer therapies, patients will most likely experience recurrent disease due to acquired resistance. Identifying targets underlying CDK7i resistance will facilitate prospective development of new therapies that can circumvent such resistance. Here we utilized triple-negative breast cancer as a model to discern mechanisms of resistance as it has been previously shown to be highly responsive to CDK7 inhibitors. After generating cell lines with acquired resistance, high-throughput RNA sequencing revealed significant upregulation of genes associated with efflux pumps and transforming growth factor-beta (TGF-β) signaling pathways. Genetic silencing or pharmacological inhibition of ABCG2, an efflux pump associated with multidrug resistance, resensitized resistant cells to CDK7i, indicating a reliance on these transporters. Expression of activin A (INHBA), a member of the TGF-β family of ligands, was also induced, whereas its intrinsic inhibitor, follistatin (FST), was repressed. In resistant cells, increased phosphorylation of SMAD3, a downstream mediator, confirmed an increase in activin signaling, and phosphorylated SMAD3 directly bound the ABCG2 promoter regulatory region. Finally, pharmacological inhibition of TGF-β/activin receptors or genetic silencing of SMAD4, a transcriptional partner of SMAD3, reversed the upregulation of ABCG2 in resistant cells and phenocopied ABCG2 inhibition. This study reveals that inhibiting the TGF-β/Activin-ABCG2 pathway is a potential avenue for preventing or overcoming resistance to CDK7 inhibitors.

The emerging role of transcription factor FOXP3 in thyroid cancer.

Transcription factor FOXP3 is a crucial regulator in the development and function of regulatory T cells (Treg) that are essential for immunological tolerance and homeostasis. Numerous studies have indicated the correlation of tumor infiltrating FOXP3+ Treg upregulation with poor prognostic parameters in thyroid cancer, including lymph node metastases, extrathyroidal extension, and multifocality. Most immune-checkpoint molecules are expressed in Treg. The blockage of such signals with checkpoint inhibitors has been approved for several solid tumors, but not yet for thyroid cancer. Thyroid abnormalities may be induced by checkpoint inhibitors. For example, hypothyroidism, thyrotoxicosis, painless thyroiditis, or even thyroid storm are more frequently associated with anti-PD-1 antibodies (pembrolizumab and nivolumab). Therefore, Targeting FOXP3+ Treg may have impacts on checkpoint molecules and the growth of thyroid cancer. Several factors may impact the role and stability of FOXP3, such as alternative RNA splicing, mutations, and post-translational modification. In addition, the role of FOXP3+ Treg in the tumor microenvironment is also affected by the complex regulatory network formed by FOXP3 and its transcriptional partners. Here we discussed how the expression and function of FOXP3 were regulated and how FOXP3 interacted with its targets in Treg, aiming to help the development of FOXP3 as a potential therapeutic target for thyroid cancer.

The transcriptional co-regulator LDB1 is required for brown adipose function

ObjectiveBrown adipose tissue (BAT) is critical for thermogenesis and glucose/lipid homeostasis. Exploiting the energy uncoupling capacity of BAT may reveal targets for obesity therapies. This exploitation requires a greater understanding of the transcriptional mechanisms underlying BAT function. One potential regulator of BAT is the transcriptional co-regulator LIM domain-binding protein 1 (LDB1), which acts as a dimerized scaffold, allowing for the assembly of transcriptional complexes. Utilizing a global LDB1 heterozygous mouse model, we recently reported that LDB1 might have novel roles in regulating BAT function. However, direct evidence for the LDB1 regulation of BAT thermogenesis and substrate utilization has not been elucidated. We hypothesize that brown adipocyte-expressed LDB1 is required for BAT function. MethodsLDB1-deficient primary cells and brown adipocyte cell lines were assessed via qRT-PCR and western blotting for altered mRNA and protein levels to define the brown adipose-specific roles. We conducted chromatin immunoprecipitation with primary BAT tissue and immortalized cell lines. Potential transcriptional partners of LDB1 were revealed by conducting LIM factor surveys via qRT-PCR in mouse and human brown adipocytes. We developed a Ucp1-Cre-driven LDB1-deficiency mouse model, termed Ldb1ΔBAT, to test LDB1 function in vivo. Glucose tolerance and uptake were assessed at thermoneutrality via intraperitoneal glucose challenge and glucose tracer studies. Insulin tolerance was measured at thermoneutrality and after stimulation with cold or the administration of the β3-adrenergic receptor (β3-AR) agonist CL316,243. Additionally, we analyzed plasma insulin via ELISA and insulin signaling via western blotting. Lipid metabolism was evaluated via BAT weight, histology, lipid droplet morphometry, and the examination of lipid-associated mRNA. Finally, energy expenditure and cold tolerance were evaluated via indirect calorimetry and cold challenges. ResultsReducing Ldb1 in vitro and in vivo resulted in altered BAT-selective mRNA, including Ucp1, Elovl3, and Dio2. In addition, there was reduced Ucp1 induction in vitro. Impacts on gene expression may be due, in part, to LDB1 occupying Ucp1 upstream regulatory domains. We also identified BAT-expressed LIM-domain factors Lmo2, Lmo4, and Lhx8, which may partner with LDB1 to mediate activity in brown adipocytes. Additionally, we observed LDB1 enrichment in human brown adipose. In vivo analysis revealed LDB1 is required for whole-body glucose and insulin tolerance, in part through reduced glucose uptake into BAT. In Ldb1ΔBAT tissue, we found significant alterations in insulin-signaling effectors. An assessment of brown adipocyte morphology and lipid droplet size revealed larger and more unilocular brown adipocytes in Ldb1ΔBAT mice, particularly after a cold challenge. Alterations in lipid handling were further supported by reductions in mRNA associated with fatty acid oxidation and mitochondrial respiration. Finally, LDB1 is required for energy expenditure and cold tolerance in both male and female mice. ConclusionsOur findings support LDB1 as a regulator of BAT function. Furthermore, given LDB1 enrichment in human brown adipose, this co-regulator may have conserved roles in human BAT.

A PGE2-MEF2A axis enables context-dependent control of inflammatory gene expression

SummaryTight control of inflammatory gene expression by antagonistic environmental cues is key to ensure immune protection while preventing tissue damage. Prostaglandin E2 (PGE2) modulates macrophage activation during homeostasis and disease, but the underlying mechanisms remain incompletely characterized. Here we dissected the genomic properties of lipopolysaccharide (LPS)-induced genes whose expression is antagonized by PGE2. The latter molecule targeted a set of inflammatory gene enhancers that, already in unstimulated macrophages, displayed poorly permissive chromatin organization and were marked by the transcription factor myocyte enhancer factor 2A (MEF2A). Deletion of MEF2A phenocopied PGE2 treatment and abolished type I interferon (IFN I) induction upon exposure to innate immune stimuli. Mechanistically, PGE2 interfered with LPS-mediated activation of ERK5, a known transcriptional partner of MEF2. This study highlights principles of plasticity and adaptation in cells exposed to a complex environment and uncovers a transcriptional circuit for IFN I induction with relevance for infectious diseases or cancer.

SPOP and OTUD7A Control EWS-FLI1 Protein Stability to Govern Ewing Sarcoma Growth.

Chromosomal translocation results in development of an Ewing sarcoma breakpoint region 1‐Friend leukemia integration 1 (EWS–FLI1) fusion oncogene in the majority of Ewing sarcoma. The persistent dependence of the tumor for this oncoprotein points to EWS–FLI1 as an ideal drug target. Although EWS–FLI1 transcriptional targets and binding partners are evaluated, the mechanisms regulating EWS–FLI1 protein stability remain elusive. Speckle‐type POZ protein (SPOP) and OTU domain‐containing protein 7A (OTUD7A) are identified as the bona fide E3 ligase and deubiquitinase, respectively, that control EWS–FLI1 protein turnover in Ewing sarcoma. Casein kinase 1‐mediated phosphorylation of the VTSSS degron in the FLI1 domain enhances SPOP activity to degrade EWS–FLI1. Opposing this process, OTUD7A deubiquitinates and stabilizes EWS–FLI1. Depletion of OTUD7A in Ewing sarcoma cell lines reduces EWS–FLI1 protein abundance and impedes Ewing sarcoma growth in vitro and in mice. Performing an artificial‐intelligence‐based virtual drug screen of a 4‐million small molecule library, 7Ai is identified as a potential OTUD7A catalytic inhibitor. 7Ai reduces EWS–FLI1 protein levels and decreases Ewing sarcoma growth in vitro and in a xenograft mouse model. This study supports the therapeutic targeting of OTUD7A as a novel strategy for Ewing sarcoma bearing EWS–FLI1 and related fusions, and may also be applicable to other cancers dependent on aberrant FLI1 expression.

The Diarylheptanoid Curcumin Induces MYC Inhibition and Cross-Links This Oncoprotein to the Coactivator TRRAP.

The c-Myc protein (MYC) is a transcription factor with strong oncogenic potential controlling fundamental cellular processes. In most human tumors, MYC is overexpressed by enhanced transcriptional activation, gene amplification, chromosomal rearrangements, or increased protein stabilization. To pharmacologically suppress oncogenic MYC functions, multiple approaches have been applied either to inhibit transcriptional activation of the endogenous MYC gene, or to interfere with biochemical functions of aberrantly activated MYC. Other critical points of attack are targeted protein modification, or destabilization leading to a non-functional MYC oncoprotein. It has been claimed that the natural compound curcumin representing the principal curcumoid of turmeric (Curcuma longa) has anticancer properties although its specificity, efficacy, and the underlying molecular mechanisms have been controversially discussed. Here, we have tested curcumin’s effect on MYC-dependent cell transformation and transcriptional activation, and found that this natural compound interferes with both of these MYC activities. Furthermore, in curcumin-treated cells, the endogenous 60-kDa MYC protein is covalently and specifically cross-linked to one of its transcriptional interaction partners, namely the 434-kDa transformation/transcription domain associated protein (TRRAP). Thereby, endogenous MYC levels are strongly reduced and cells stop to proliferate. TRRAP is a multidomain adaptor protein of the phosphoinositide 3-kinase-related kinases (PIKK) family and represents an important component of many histone acetyltransferase (HAT) complexes. TRRAP is important to mediate transcriptional activation executed by the MYC oncoprotein, but on the other hand TRRAP also negatively regulates protein stability of the tumor suppressor p53 (TP53). Curcumin-mediated covalent binding of MYC to TRRAP reduces the protein amounts of both interaction partners but does not downregulate TP53, so that the growth-arresting effect of wild type TP53 could prevail. Our results elucidate a molecular mechanism of curcumin action that specifically and irreversibly targets two crucial multifunctional cellular players. With regard to their broad impact in cancer, our findings contribute to explain the pleiotropic functions of curcumin, and suggest that this natural spice, or more bioavailable derivatives thereof, may constitute useful adjuvants in the therapy of MYC-dependent and TRRAP-associated human tumors.

Molecular Actions Underlying Wolffian Duct Regression in Sexual Differentiation of Murine Reproductive Tracts

Sexually dimorphic establishment of the reproductive tract system requires sex-specific regression of the Wolffian duct and Müllerian duct in the mesonephros. In an XX embryo, the Wolffian duct regresses under the control of the mesenchymal transcription factor COUP-TFII. To understand cellular and molecular actions underlying Wolffian duct regression, we performed transcriptomic analyses of XX mesonephroi with or without Coup-tfII and genome-wide analysis of COUP-TFII chromatin occupancy in XX mesonephroi. The integrative analysis of COUP-TFII genome-wide binding and transcriptomic analysis revealed the suppression of muscle differentiation and extracellular matrix genes by COUP-TFII and identified a group of potential transcriptional partners of COUP-TFII in the mesenchyme that potentially facilitate Wolffian duct regression. These findings provide insights into the molecular action of COUP-TFII in the Wolffian duct mesenchyme and identify a list of biologically relevant candidate genes and pathways for future functional analyses in sexual differentiation of reproductive tracts.

Targeting the Hippo Pathway in Prostate Cancer: What's New?

Simple SummaryProstate cancer is the most commonly diagnosed cancer in men in the UK, accounting for the deaths of over 11,000 men per year. A major problem in this disease are tumours which no longer respond to available treatments. Understanding how this occurs will reveal new ways to treat these patients. In this review, the latest findings regarding a particular group of cellular factors which make up a signalling network called the Hippo pathway will be described. Accumulating evidence suggests that this network contributes to prostate cancer progression and resistance to current treatments. Identifying how this pathway can be targeted with drugs is a promising area of research to improve the treatment of prostate cancer.Identifying novel therapeutic targets for the treatment of prostate cancer (PC) remains a key area of research. With the emergence of resistance to androgen receptor (AR)-targeting therapies, other signalling pathways which crosstalk with AR signalling are important. Over recent years, evidence has accumulated for targeting the Hippo signalling pathway. Discovered in Drosophila melanogasta, the Hippo pathway plays a role in the regulation of organ size, proliferation, migration and invasion. In response to a variety of stimuli, including cell–cell contact, nutrients and stress, a kinase cascade is activated, which includes STK4/3 and LATS1/2 to inhibit the effector proteins YAP and its paralogue TAZ. Transcription by their partner transcription factors is inhibited by modulation of YAP/TAZ cellular localisation and protein turnover. Trnascriptional enhanced associate domain (TEAD) transcription factors are their classical transcriptional partner but other transcription factors, including the AR, have been shown to be modulated by YAP/TAZ. In PC, this pathway can be dysregulated by a number of mechanisms, making it attractive for therapeutic intervention. This review looks at each component of the pathway with a focus on findings from the last year and discusses what knowledge can be applied to the field of PC.

Genome-wide chromatin occupancy of BRDT and gene expression analysis suggest transcriptional partners and specific epigenetic landscapes that regulate gene expression during spermatogenesis.

BRDT, a member of the BET family of double bromodomain-containing proteins, is essential for spermatogenesis in the mouse and has been postulated to be a key regulator of transcription in meiotic and post-meiotic cells. To understand the function of BRDT in these processes, we first characterized the genome-wide distribution of the BRDT binding sites, in particular within gene units, by ChIP-Seq analysis of enriched fractions of pachytene spermatocytes and round spermatids. In both cell types, BRDT binding sites were mainly located in promoters, first exons, and introns of genes. BRDT binding sites in promoters overlapped with several histone modifications and histone variants associated with active transcription, and were enriched for consensus sequences for specific transcription factors, including MYB, RFX, ETS, and ELF1 in pachytene spermatocytes, and JunD, c-Jun, CRE, and RFX in round spermatids. Subsequent integration of the ChIP-seq data with available transcriptome data revealed that stage-specific gene expression programs are associated with BRDT binding to their gene promoters, with most of the BDRT-bound genes being upregulated. Gene Ontology analysis further identified unique sets of genes enriched in diverse biological processes essential for meiosis and spermiogenesis between the two cell types, suggesting distinct developmentally stage-specific functions for BRDT. Taken together, our data suggest that BRDT cooperates with different transcription factors at distinctive chromatin regions within gene units to regulate diverse downstream target genes that function in male meiosis and spermiogenesis.

NEK1 Phosphorylation of YAP Promotes Its Stabilization and Transcriptional Output.

Simple SummaryWe earlier described the involvement of the TLK1>NEK1>ATR>Chk1 axis as a key determinant of cell cycle arrest in androgen-dependent prostate cancer (PCa) cells after androgen deprivation. We now report that the TLK1>NEK1 axis is also involved in stabilization of yes-associated protein 1 (YAP1), the transcriptional co-activator in the Hippo pathway, presumably facilitating reprogramming of the cells toward castration-resistant PCa (CRPC). NEK1 interacts with YAP1 physically resulting in its phosphorylation of 6 residues, which enhance its stability and activity. Analyses of cancer Protein Atlas and TCGA expression panels revealed a link between activated NEK1 and YAP1 expression and several YAP transcription targets.Most prostate cancer (PCa) deaths result from progressive failure in standard androgen deprivation therapy (ADT), leading to metastatic castration-resistant PCa (mCRPC); however, the mechanism and key players leading to this are not fully understood. While studying the role of tousled-like kinase 1 (TLK1) and never in mitosis gene A (NIMA)-related kinase 1 (NEK1) in a DNA damage response (DDR)-mediated cell cycle arrest in LNCaP cells treated with bicalutamide, we uncovered that overexpression of wt-NEK1 resulted in a rapid conversion to androgen-independent (AI) growth, analogous to what has been observed when YAP1 is overexpressed. We now report that overexpression of wt-NEK1 results in accumulation of YAP1, suggesting the existence of a TLK1>NEK1>YAP1 axis that leads to adaptation to AI growth. Further, YAP1 is co-immunoprecipitated with NEK1. Importantly, NEK1 was able to phosphorylate YAP1 on six residues in vitro, which we believe are important for stabilization of the protein, possibly by increasing its interaction with transcriptional partners. In fact, knockout (KO) of NEK1 in NT1 PCa cells resulted in a parallel decrease of YAP1 level and reduced expression of typical YAP-regulated target genes. In terms of cancer potential implications, the expression of NEK1 and YAP1 proteins was found to be increased and correlated in several cancers. These include PCa stages according to Gleason score, head and neck squamous cell carcinoma, and glioblastoma, suggesting that this co-regulation is imparted by increased YAP1 stability when NEK1 is overexpressed or activated by TLK1, and not through transcriptional co-expression. We propose that the TLK1>NEK1>YAP1 axis is a key determinant for cancer progression, particularly during the process of androgen-sensitive to -independent conversion during progression to mCRPC.

Abstract 16557: Hemodynamic Stress Activates a Comprehensive Adaptive Program in Murine Aortic Cells That Protects the Aortic Wall and Maintains Aortic Homeostasis

Background: The aorta adapts to changes in its biomechanical environment and insufficient adaptation results in aortic failure. However, the molecular and cellular processes underlying this adaptive response are poorly understood. In this study, we performed single-cell transcriptomic and single-cell epigenomic analyses to investigate gene expression and epigenetic regulation responses in aortas subjected to hemodynamic stress. Methods and Results: Angiotensin II infusion in wild-type mice (C57BL/6J) increased ascending aorta wall thickness that was associated with smooth muscle cell (SMC) proliferation and extracellular matrix (ECM) production. ScRNA-seq revealed an adaptive response in several SMC clusters that was characterized by upregulation of genes in processes such as proliferation, cell junction organization, ECM production and organization, wound healing, as well as DNA repair and anti-oxidation. ScATAC-seq showed increased chromatin accessibility at the locus of these adaptive genes. Motif analysis of the accessible regions showed that several transcription factors and chromatin architecture proteins may be involved in regulating these adaptive response genes. Among these, Yap1 was significantly activated in response to the aortic challenge. YAP1-based ChIP-seq in aortic SMCs indicated that YAP1 and its transcription partners can directly bind to genes involved in the adaptive response to biomechanical stretch. Finally, SMC-specific knockout of Yap1 resulted in a compromised adaptive response and increased expression of genes involved in protein disassembly or degradation, and cell death in all SMC clusters, leading to contractile dysfunction, aortic aneurysm, dissection, and rupture. Conclusion: In mice, hemodynamic stress induces a comprehensive and coordinated adaptive response in SMCs that is critical for maintaining aortic homeostasis. YAP1 in SMCs play an important role in promoting this adaptive response in aortic wall.

Negative regulation of IL-8 in human astrocytes depends on β-catenin while positive regulation is mediated by TCFs/LEF/ATF2 interaction

Expression of cytokines/chemokines is tightly regulated at the transcription level. This is crucial in the central nervous system to maintain neuroimmune homeostasis. IL-8 a chemoattractant, which recruits neutrophils, T cells, and basophils into the brain in response to inflammation and/or injury is secreted predominantly by neurons, microglia, and astrocytes. Here, we investigated the mechanism by which astrocytes regulate IL-8 expression. We demonstrate that while β-catenin negatively regulated IL-8 transcription, its canonical transcriptional partners, members of the TCF/LEF transcription factors (TCF1, TCF3, TCF4 and LEF1) and Activating transcription factor 2 (ATF2) positively regulated IL-8 transcription. We further identified a putative TCF/LEF binding site at −175nt close to the minimal transcription region on the IL-8 promoter, mutation of which caused a significant reduction in IL-8 promoter activity. Chromatin immunoprecipitation demonstrated binding of TCF1, TCF4, LEF1 and ATF2 on the IL-8 promoter suggesting that TCFs/LEF partner with ATF2 to induce IL-8 transcription. These findings demonstrate a novel role for β-catenin in suppression of IL-8 expression and for TCFs/LEF/ATF2 in inducing IL-8. These findings reveal a unique mechanism by which astrocytes tightly regulate IL-8 expression.

Abstract B37: Genomic view of YAP1 dependent transcription

Abstract YAP1 is a transcriptional coactivator regulated by the Hippo pathway, mechano-transduction, G-protein coupled receptors, and WNT signaling. YAP1 activity depends on its stabilization and its nuclear localization. Once in the nucleus, YAP1 can regulate gene expression by interacting with transcription factors with DNA binding capability. While YAP1 main transcriptional partners belong to the TEAD family, several reports indicate that YAP1 can also associate with SMADs, RUNX, and other transcription factors. YAP1 acts as a broad regulator of cell growth and differentiation, thus playing a pivotal role in regeneration, reprogramming, and cancer. How YAP1 may control such a large number of cellular processes is still to be fully understood. In the adult mouse liver, activation of YAP1 supports proficient cell proliferation and triggers dedifferentiation of hepatocytes towards a bipotent progenitor-like state. Overall, this results in abnormal liver growth, which, upon prolonged YAP1 activation, may lead to the emergence of focal hepatocellular carcinomas. In order to gain further insight into the role of YAP1 in these processes, we conducted a genome-wide investigation in the mouse liver, to identify how YAP1 controls transcription and to define gene expression programs directly regulated by YAP1. YAP1 binding to the genome was exclusively mediated by TEAD, yet given the preponderance of TEAD bound loci, only a subset of these sites was bound by YAP1. Such loci showed a defined pattern of epigenetic bookmarking and selective binding of transcription factors, thus indicating that pre-existing chromatin modifications and transcription factors are pivotal in biasing YAP1 binding to a defined set of TEAD bound genomic regions. Integrated analyses of ChIP-seq and RNA-seq data indicated that YAP1 can act both as an activator and as a repressor in a context-dependent way and allowed the dissection of transcriptional mechanisms. Longitudinal analysis by ChIP-seq and RNA-seq highlighted differences in genome binding and transcriptional control, which allowed the identification of acute and tonic YAP1 dependent responses. While during acute activation YAP1 triggered cell cycle and signal transduction programs, its prolonged activation sustained a feed-forward loop that potentiated and expanded YAP1-dependent transcription, thus leading to the direct engagement of cell identity programs. Overall this study provides a genome-scale understanding of how YAP1 controls gene expression and reveals principles in gene regulation that account for YAP1 ability to orchestrate development, regeneration, and cell reprogramming. Citation Format: Francesca Biagioni, Ottavio Croci, Elisa Donato, Silvia Sberna, Serena De Fazio, Arianna Sabò, Bruno Amati, Stefano Campaner. Genomic view of YAP1 dependent transcription [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B37.

The aryl hydrocarbon receptor pathway controls matrix metalloproteinase-1 and collagen levels in human orbital fibroblasts

Thyroid eye disease (TED) affects 25–50% of patients with Graves’ Disease. In TED, collagen accumulation leads to an expansion of the extracellular matrix (ECM) which causes destructive tissue remodeling. The purpose of this study was to investigate the therapeutic potential of activating the aryl hydrocarbon receptor (AHR) to limit ECM accumulation in vitro. The ability of AHR to control expression of matrix metalloproteinase-1 (MMP1) was analyzed. MMP1 degrades collagen to prevent excessive ECM. Human orbital fibroblasts (OFs) were treated with the pro-scarring cytokine, transforming growth factor beta (TGFβ) to induce collagen production. The AHR ligand, 6-formylindolo[3,2b]carbazole (FICZ) was used to activate the AHR pathway in OFs. MMP1 protein and mRNA levels were analyzed by immunosorbent assay, Western blotting and quantitative PCR. MMP1 activity was detected using collagen zymography. AHR and its transcriptional binding partner, ARNT were depleted using siRNA to determine their role in activating expression of MMP1. FICZ induced MMP1 mRNA, protein expression and activity. MMP1 expression led to a reduction in collagen 1A1 levels. Furthermore, FICZ-induced MMP1 expression required both AHR and ARNT, demonstrating that the AHR-ARNT transcriptional complex is necessary for expression of MMP1 in OFs. These data show that activation of the AHR by FICZ increases MMP1 expression while leading to a decrease in collagen levels. Taken together, these studies suggest that AHR activation could be a promising target to block excessive collagen accumulation and destructive tissue remodeling that occurs in fibrotic diseases such as TED.