DNA-binding Partner Research Articles

A steroid receptor coactivator acts as the DNA-binding partner of the methoprene-tolerant protein in regulating juvenile hormone response genes

Methoprene-tolerant (Met) protein is a juvenile hormone (JH) receptor in insects. JH-bound Met forms a complex with the βFtz-F1-interacting steroid receptor coactivator (FISC) and together they regulate JH response genes in mosquitoes. Both proteins contain basic helix–loop–helix (bHLH) and PAS motifs. Here we demonstrated that FISC is the obligatory partner of Met for binding to JH-response elements (JHREs). Met or FISC alone could not bind a previously characterized JHRE, while formation of the Met–FISC complex was necessary and sufficient to bind to the JHRE. This binding required participation of the DNA-binding domains of both Met and FISC. The optimal DNA sequence recognized by Met and FISC contained a core consensus sequence GCACGTG. While formation of the Met–FISC complex in mosquito cells was induced by JH, heterodimerization and DNA binding of bacterially expressed Met and FISC were JH-independent, implying that additional mosquito proteins were required to modulate formation of the receptor complex.

The core and conserved role of MAL is homeostatic regulation of actin levels.

The transcription cofactor MAL is regulated by free actin levels and thus by actin dynamics. MAL, together with its DNA-binding partner, SRF, is required for invasive cell migration and in experimental metastasis. Although MAL/SRF has many targets, we provide genetic evidence in both Drosophila and human cellular models that actin is the key target that must be regulated by MAL/SRF for invasive cell migration. By regulating MAL/SRF activity, actin protein feeds back on production of actin mRNA to ensure sufficient supply of actin. This constitutes a dedicated homeostatic feedback system that provides a foundation for cellular actin dynamics.

MYC interacts with the human STAGA coactivator complex via multivalent contacts with the GCN5 and TRRAP subunits

MYC is an oncogenic DNA-binding transcription activator of many genes and is often upregulated in human cancers. MYC has an N-terminal transcription activation domain (TAD) that is also required for cell transformation. Various MYC TAD-interacting coactivators have been identified, including the transcription/transformation-associated protein (TRRAP), a subunit of different histone acetyltransferase (HAT) complexes such as the human “SPT3–TAF9–GCN5 Acetyltransferase” (STAGA) complex involved in MYC transactivation of the TERT gene. However, it remains unclear whether TRRAP and/or other subunits are directly contacted by MYC within these macromolecular complexes. Here, we characterize the interactions of MYC TAD with the STAGA complex. By protein crosslinking we identify both TRRAP and the GCN5 acetyltransferase as MYC TAD-interacting subunits within native STAGA. We show that purified GCN5 binds to an N-terminal sub-domain of MYC TAD (residues 21–108) and that the interaction of GCN5 and STAGA with this sub-domain is dependent on two related sequence motifs: M2 within the conserved MYC homology box I (MBI), and M3 located between residues 100–106. Interestingly, specific substitutions within the M2/3 motifs that only moderately reduce the intracellular MYC–STAGA interaction and do not influence dimerization of MYC with its DNA-binding partner MAX, strongly inhibit MYC acetylation by GCN5 and reduce MYC binding and transactivation of the GCN5-dependent TERT promoter in vivo. Hence, we propose that MYC associates with STAGA through extended interactions of the TAD with both TRRAP and GCN5 and that the TAD–GCN5 interaction is important for MYC acetylation and MYC binding to certain chromatin loci.

P618 INFLAMMATORY RESPONSE OF PRIMARY MOUSE HEPATIC STELLATE CELLS TO LIPOPOLYSACCHARIDE IN VITRO

study demonstrates a role for mechano-sensitive signalling by the transcriptional regulators Yes associated protein (YAP) and WW domain-containing transcription regulator protein 1 (TAZ) during HSC activation. Methods: Expression was assayed by qPCR, immunoblot and immunocytochemistry of primary HSCs. Acrylamide gels were used to model altered matrix stiffness. Chromatin immunoprecipitation (ChIP) identified YAP binding sites. Results: During activation of HSCs, expression of Yap, Taz and their DNA binding partner Tead was increased. Yap was localised to the nucleus of HSCs positive for profibrotic markers alpha-smooth muscle actin, collagen 1 and Sox9. In contrast, HSCs cultured on soft substrates appeared quiescent-like (characterised by autofluorescence) with inactive Yap sequestered in the cytoplasm. Abrogation of Yap and Taz using siRNA reduced fibrotic gene expression. Hippo signalling (an alternative mechanism regulating Yap/Taz activation) via Lats1 and Lats2 has no effect on fibrotic gene expression in HSCs, confirming a role for mechano-transduction via Yap/Taz in HSC activation in vitro. Moreover, chemical inhibition of the Tead-Yap interaction caused reduced expression of Yap targets (Gli2, Ctgf ) and fibrotic markers (collagen, Sox9). ChIP identified Sox9 as a novel downstream Yap target. Conclusions: These data indicate a functional role for mechanotransduction by Yap/Taz during HSC activation. Capitalizing on this finding, particularly the ability to pharmacologically disrupt Yap target gene interaction may provide insight into novel therapeutic strategies.

Recent advances in the structural molecular biology of Ets transcription factors: interactions, interfaces and inhibition

The Ets family of eukaryotic transcription factors is based around the conserved Ets DNA-binding domain. Although their DNA-binding selectivity is biochemically and structurally well characterized, structures of homodimeric and ternary complexes point to Ets domains functioning as versatile protein-interaction modules. In the present paper, we review the progress made over the last decade to elucidate the structural mechanisms involved in modulation of DNA binding and protein partner selection during dimerization. We see that Ets domains, although conserved around a core architecture, have evolved to utilize a variety of interaction surfaces and binding mechanisms, reflecting Ets domains as dynamic interfaces for both DNA and protein interaction. Furthermore, we discuss recent advances in drug development for inhibition of Ets factors, and the roles structural biology can play in their future.

Mammalian Argonaute-DNA binding?

When a field shares the consensus that a particular phenomenon does NOT occur, this may reflect extensive experimental investigations with negative outcomes, or may represent the “common sense” position based on current knowledge and established ways of thinking. The current consensus of the RNA field is that eukaryotic Argonaute (Ago) proteins employ RNA guides and target other RNAs. The alternative -- that eukaryotic Ago has biologically important interactions with DNA in vivo – has not been seriously considered, in part because the only role contemplated for DNA was as a guide strand, and in part because it did not seem plausible that any natural source of suitable DNAs exists in eukaryotic cells. However, eukaryotic Argonaute domains bind DNA in the test tube, and several articles report that small inhibitory double-stranded DNAs do have the ability to silence target RNAs in a sequence-dependent (though poorly characterized) manner. A search of the literature identified potential DNA binding partners for Ago, including (among others) single-stranded DNAs residing in extracellular vesicles, and cytoplasmic satellite-repeat DNA fragments that are associated with the plasma membrane and transcribed by Pol II. It is interesting to note that both cytoplasmic and extracellular vesicle DNA are expressed at greatly elevated levels in cancer cells relative to normal cells. In such a pathological scenario, if not under normal conditions, there may be appreciable binding of Ago to DNA despite its lower affinity compared to RNA. If so, DNA might displace Ago from binding to its normal partners (miRNAs, siRNAs and other short ncRNAs), disrupting tightly controlled post-transcriptional gene silencing processes that are vital to correct functioning of a normal cell. The possible contribution to cancer pathogenesis is a strong motivator for further investigation of Ago-DNA binding. More generally, this case underscores the need for better informatics tools to allow investigators to analyze the state of a given scientific question at a high-level and to identify possible new research directions.Reviewers: This article was reviewed by Eugene Koonin, Kira S. Makarova, Alexander Maxwell Burroughs (nominated by L Aravind), and Isidore Rigoutsos.Open peer review: Reviewed by Eugene Koonin, Kira S. Makarova, Alexander Maxwell Burroughs (nominated by L Aravind), and Isidore Rigoutsos. For the full reviews, please go to the Reviewers’ comments section.

Monitoring Notch activation in cultured mammalian cells: luciferase complementation imaging assays.

Notch activation and cleavage releases the Notch intracellular domain (NICD), which translocates to the nucleus, where it associates with its DNA-binding partner CSL to recruit the coactivator MAML and additional cofactors to ultimately activate target gene expression. Taking advantage of the specific interaction between NICD and these factors, we have developed a luciferase complementation imaging (LCI)-based reporter system to quantitatively monitor Notch activation in real time in live cells. In this chapter, we describe the use of Notch LCI reporters for measuring protein interactions and performing detailed kinetic analyses of receptor activation and its responses to various stimuli.

Notch signaling is required for the formation of mesangial cells from a stromal mesenchyme precursor during kidney development

Mesangial cells are specialized pericyte/smooth muscle cells that surround and constrain the vascular network within the glomerulus of the kidney. They are derived from the stromal mesenchyme, a progenitor population distinct from nephron stem cells. Whether mesangial cells have a distinct origin from vascular smooth muscle cells (VSMCs) and the pathways that govern their specification are unknown. Here we show that Notch signaling in stromal progenitors is essential for mesangial cell formation but is dispensable for the smooth muscle and interstitial cell lineages. Deletion of RBPjk, the common DNA-binding partner of all active Notch receptors, with Foxd1(tgCre) results in glomerular aneurysm and perinatal death from kidney failure. This defect occurs early in glomerular development as stromal-derived, desmin-positive cells fail to coalesce near forming nephrons and thus do not invade the vascular cleft of the S-shaped body. This is in contrast to other mutants in which the loss of the mesangium was due to migration defects, and suggests that loss of Notch signaling results in a failure to specify this population from the stroma. Interestingly, Pdgfrb-positive VSMCs do not enter the vascular cleft and cannot rescue the mesangial deficiency. Notch1 and Notch2 act redundantly through γ-secretase and RBPjk in this process, as individual mutants have mesangial cells at birth. Together, these data demonstrate a unique origin of mesangial cells and demonstrate a novel, redundant function for Notch receptors in mesangial cell specification, proliferation or survival during kidney development.

Switch Enhancers Interpret TGF-β and Hippo Signaling to Control Cell Fate in Human Embryonic Stem Cells

A small toolkit of morphogens is used repeatedly to direct development, raising the question of how context dictates interpretation of the same cue. One example is the transforming growth factor β (TGF-β) pathway that in human embryonic stem cells fulfills two opposite functions: pluripotency maintenance and mesendoderm (ME) specification. Using proteomics coupled to analysis of genome occupancy, we uncover a regulatory complex composed of transcriptional effectors of the Hippo pathway (TAZ/YAP/TEAD), the TGF-β pathway (SMAD2/3), and the pluripotency regulator OCT4 (TSO). TSO collaborates with NuRD repressor complexes to buffer pluripotency gene expression while suppressing ME genes. Importantly, the SMAD DNA binding partner FOXH1, a major specifier of ME, is found near TSO elements, and upon fate specification we show that TSO is disrupted with subsequent SMAD-FOXH1 induction of ME. These studies define switch-enhancer elements and provide a framework to understand how cellular context dictates interpretation of the same morphogen signal in development.

Combination Therapy In Mast Cell Neoplasms: Co-Targeting KIT and NFAT Signaling Pathways

Activating KIT mutations are a hallmark of human systemic mast cell disease and have also been identified in gastrointestinal stromal tumors, AML, melanoma, and seminoma. Traditionally, constitutively active KIT mutations have been targeted by tyrosine kinase inhibitors; however, currently available inhibitors have limited activity against resistant mutations and sustainable responses are often not achieved. Our goal has been to identify a synergistic drug combination that increases the effectiveness of KIT inhibitors. A recent study in chronic myelogenous leukemia reported enhanced anti-proliferative/pro-apoptotic effects when combining a BCR-ABL inhibitor with inhibitors targeting a novel Wnt/Ca++/Calcineurin/NFAT pathway. Based on shared downstream signaling mechanisms between BCR-ABL and KIT kinases we hypothesized that a similar combination would work to target KIT mutant kinases.We found that combining a KIT inhibitor with a calcineurin phosphatase inhibitor led to a synergistic decrease (average combination index (CI) values less than 1.0) in cell viability as well as an increase in apoptosis in KIT-mutant cell lines including six mastocytosis cell lines. For example, we found that 1uM of cyclosporine A (CSA) plus 20nM of dasatinib in the p815 cell line consistently reduced cell viability by 50% compared to dasatinib alone. This decrease in cell viability corresponded to a 40 fold increase in capsase 3/7 activity (Figure 1). Similar results were obtained using the other five mastocytosis cell lines. [Display omitted] The activity of NFAT transcription factors is known to be regulated by the phosphatase activity of calcineurin. To confirm our results were calcineurin- and NFAT-dependent, we repeated the previous combination experiments with the NFAT specific inhibitors, rocaglamide and tributylhexadecylphosphonium bromide (THPB). We observed a synergistic decrease (average CI value 0.81 for rocaglamide and 0.43 for THPB) in cell viability and increase in apoptosis when combining these NFAT-specific and KIT kinase inhibitors, indicating NFAT as an important mediator of the observed synergy. Next we characterized NFAT in KIT mutant mast cells and found constitutive NFAT activation in each cell line, as indicated by nuclear localization and dephosphorylation under unstimulated conditions. NFAT-dependent transcriptional activity, as measured by a luciferase reporter system, decreased following treatment with either calcineurin or NFAT inhibitors. Unexpectedly, NFAT-dependent transcriptional activity also decreased following treatment with a number of KIT inhibitors. However, only calcineurin inhibitors and not KIT inhibitors, completely blocked NFAT activation following calcium influx, suggesting a unique mechanism by which KIT inhibition affects NFAT-dependent transcriptional activity. To determine downstream effectors of the observed synergy, we measured changes in RNA expression following mono- and combination therapy. RNA-Seq revealed synergistic decreases in a number of genes including members of the JAK-STAT and MAPK pathways, which are druggable targets downstream of this transcriptional program. Further validation studies of these targets are currently underway and will be reported later.The constitutive activation of NFAT has not been previously reported in KIT mutant cell lines. Further, our data suggest a point of convergence between KIT and NFAT signaling pathways, possibly through the interaction of NFAT with a DNA binding partner. Importantly, our results indicate that combination therapy using an NFAT inhibitor and a KIT kinase inhibitor results in synergistic killing of KIT mutant mast cells. However, existing calcineurin phosphatase inhibitors such as CSA have known chronic long-term toxicities, including immunosuppression. To identify alternative drug targets we used RNA-Seq and found that simultaneous calcineurin and KIT inhibition modulated members of the JAK-STAT and MAPK pathways. Preliminary data indicates that targeting these pathways in combination with dasatinib achieves the same synergistic effect on KIT mutant cancer cells. We are currently testing the potential of these alternative combination therapies for the treatment of advanced mast cell disease. Disclosures:Heinrich: Onyx: Consultancy; Pfizer: Consultancy; Novartis: Consultancy, Patents & Royalties, Research Funding; MolecularMD: Consultancy, Equity Ownership; AROG: Research Funding.

Regulation of Gγ-Globin Gene by ATF2 and Its Associated Proteins through the cAMP-Response Element

The upstream Gγ-globin cAMP-response element (G-CRE) plays an important role in regulating Gγ-globin expression through binding of ATF2 and its DNA-binding partners defined in this study. ATF2 knockdown resulted in a significant reduction of γ-globin expression accompanied by decreased ATF2 binding to the G-CRE. By contrast, stable ATF2 expression in K562 cells increased γ-globin transcription which was reduced by ATF2 knockdown. Moreover, a similar effect of ATF2 on γ-globin expression was observed in primary erythroid progenitors. To understand the role of ATF2 in γ-globin expression, chromatographically purified G-CRE/ATF2-interacting proteins were subjected to mass spectrometry analysis; major binding partners included CREB1, cJun, Brg1, and histone deacetylases among others. Immunoprecipitation assays demonstrated interaction of these proteins with ATF2 and in vivo GCRE binding in CD34+ cells undergoing erythroid differentiation which was correlated with γ-globin expression during development. These results suggest synergism between developmental stage-specific recruitments of the ATF2 protein complex and expression of γ-globin during erythropoiesis. Microarray studies in K562 cells support ATF2 plays diverse roles in hematopoiesis and chromatin remodeling.

A recurrent dominant negative E47 mutation causes agammaglobulinemia and BCR– B cells

Approximately 90% of patients with isolated agammaglobulinemia and failure of B cell development have mutations in genes required for signaling through the pre–B cell and B cell receptors. The nature of the gene defect in the majority of remaining patients is unknown. We recently identified 4 patients with agammaglobulinemia and markedly decreased numbers of peripheral B cells. The B cells that could be detected had an unusual phenotype characterized by the increased expression of CD19 but the absence of a B cell receptor. Genetic studies demonstrated that all 4 patients had the exact same de novo mutation in the broadly expressed transcription factor E47. The mutant protein (E555K) was stable in patient-derived EBV-transformed cell lines and cell lines transfected with expression vectors. E555K in the transfected cells localized normally to the nucleus and resulted in a dominant negative effect when bound to DNA as a homodimer with wild-type E47. Mutant E47 did permit DNA binding by a tissue-specific heterodimeric DNA-binding partner, myogenic differentiation 1 (MYOD). These findings document a mutational hot-spot in E47 and represent an autosomal dominant form of agammaglobulinemia. Further, they indicate that E47 plays a critical role in enforcing the block in development of B cell precursors that lack functional antigen receptors.

The Human RVB Complex Is Required for Efficient Transcription of Type I Interferon-Stimulated Genes

Type I interferons (IFNs) stimulate transcription through a latent heterotrimeric transcription factor composed of tyrosine-phosphorylated STAT1 and STAT2 and the DNA binding partner IRF9, with STAT2 contributing a critical transactivation domain. Human RVB1 and RVB2, which are highly conserved AAA(+) ATP binding proteins contained in chromatin-remodeling complexes such as Ino80, SNF2-related CBP activator protein (SRCAP), and Tip60/NuA4, interacted with the transactivation domain of STAT2 in the nuclei of IFN-stimulated cells. RNA interference (RNAi) experiments demonstrated that RVB proteins were required for robust activation of IFN-α-stimulated genes (ISGs). The requirement for RVB proteins was specific to IFN-α/STAT2 signaling; transcription of tumor necrosis factor alpha (TNF-α)- and IFN-γ-driven genes was not affected by RVB1 depletion. Using RNAi-based depletion, we assessed the involvement of catalytic subunits of the RVB-containing Tip60, BRD8, Ino80, SRCAP, and URI complexes. No component other than RVB1/2 was uniquely required for ISG induction, suggesting that RVB1/2 functions as part of an as yet unidentified complex. Chromatin immunoprecipitation assays indicated that RVB1/2 was required for recruitment of RNA polymerase II (Pol II) to ISG promoters but was dispensable for STAT2 recruitment to chromatin. We hypothesize that an RVB1/2 chromatin-remodeling complex is required for efficient Pol II recruitment and initiation at ISG promoters and is recruited through interaction with the STAT2 transactivation domain.

Abstract IA34: Hippo signaling in development and cancer

Abstract The Hippo signaling pathway regulates organ size and tissue homeostasis in diverse species from Drosophila to mammals. Central to the Hippo pathway is a functionally conserved kinase cascade leading from the Ste20-like protein kinase Hippo (Hpo) (Mst1/2 in mammals) and the NDR family kinase Warts (Lats1/2 in mammals) to the transcription factor complex formed by the coactivator Yorkie (Yki) (YAP/TAZ in mammals) and its major DNA-binding partner Scalloped (Sd) (TEAD1/2/3/4 in mammals). Inactivation of the Hippo pathway tumor suppressors or overexpression/activation of the YAP oncoprotein results in uncontrolled growth and tumorigenesis in transgenic mouse models and has been implicated in diverse human cancers. Compared to the core kinase cascade leading from Hpo to Yki, signaling events upstream of the Hippo kinase cascade are less well understood. My presentation will focus on Merlin/NF2, which encodes a member of the ezrin, radixin, moesin (ERM) family of membrane-cytoskeleton adapter proteins. Merlin/NF2 was discovered twenty years ago as a tumor suppressor underlying Neurofibromatosis type II, an autosomal dominant disorder characterized by the development of benign schwannomas, meningiomas and ependymomas. Although it is generally believed that Merlin/NF2 impacts cell signaling as a membrane-cytoskeleton scaffold, its precise molecular mechanism remains poorly understood. Recent studies in Drosophila revealed a potential link between Merlin and the Hippo pathway by placing Merlin genetically upstream of the kinase Hpo. These genetic findings have led to the prevailing view in the field that Merlin functions upstream of Hpo in a linear biochemical pathway, even though the genetic epistasis is equally compatible with Merlin and Hpo acting in parallel to promote Hippo signaling output. Using a combination of genetics, cell biology and biochemistry, we investigated the molecular relationship between Merlin/NF2 and the Hippo pathway in both Drosophila and mammals. In contrast to the commonly assumed linear pathway of Merlin/NF2 functioning through Hpo, we show that in both Drosophila and mammals, Merlin/NF2 promotes downstream Hippo signaling without activating the intrinsic kinase activity of Hpo. Instead, Merlin/NF2 directly binds and recruits the effector kinase Wts to plasma membrane. Membrane recruitment, in turn, promotes Wts phosphorylation and activation by the membrane-associated Hpo-Sav kinase complex. Accordingly, disease-causing mutations in Merlin/NF2 impair its ability to bind/recruit Wts to plasma membrane. Conversely, gain-of-function mutations in Merlin/NF2 potentiate its ability to bind/recruit Wts to plasma membrane. Our studies therefore uncover a critical function of Merlin in spatial organization of Hippo signaling at the plasma membrane. Citation Format: Duojia Pan. Hippo signaling in development and cancer. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr IA34.

Biochemistry of the tale transcription factors PREP, MEIS, and PBX in vertebrates

TALE (three amino acids loop extension) homeodomain transcription factors are required in various steps of embryo development, in many adult physiological functions, and are involved in important pathologies. This review focuses on the PREP, MEIS, and PBX sub-families of TALE factors and aims at giving information on their biochemical properties, i.e., structure, interactors, and interaction surfaces. Members of the three sets of protein form dimers in which the common partner is PBX but they can also directly interact with other proteins forming higher-order complexes, in particular HOX. Finally, recent advances in determining the genome-wide DNA-binding sites of PREP1, MEIS1, and PBX1, and their partial correspondence with the binding sites of some HOX proteins, are reviewed. These studies have generated a few general rules that can be applied to all members of the three gene families. PREP and MEIS recognize slightly different consensus sequences: PREP prefers to bind to promoters and to have PBX as a DNA-binding partner; MEIS prefers HOX as partner, and both PREP and MEIS drive PBX to their own binding sites. This outlines the clear individuality of the PREP and MEIS proteins, the former mostly devoted to basic cellular functions, the latter more to developmental functions. Developmental Dynamics 243:59–75, 2014. © 2013 Wiley Periodicals, Inc.

The aryl hydrocarbon receptor interacts with nuclear factor erythroid 2-related factor 2 to mediate induction of NAD(P)H:quinoneoxidoreductase 1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin

NAD(P)H:quinoneoxidoreductase 1 (NQO1) belongs to a group of the aryl hydrocarbon receptor (AhR) battery of drug-metabolizing enzymes that are characteristically induced by both AhR agonists and nuclear factor erythroid 2-related factor 2 (Nrf2) activators. We have previously reported that induction of Nqo1 by the AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in hepa1c1c7 cells involves Nrf2 (Ma et al., Biochem J 377, 205–213, 2004). Here we analyzed the molecular mechanism of induction. Induction required AhR and its DNA-binding partner Arnt because induction was not observed in AhR or Arnt-defective cells, but induction was restored upon reconstitution of the variant cells with functional AhR or Arnt. Induction also required Nrf2, as induction by benzo[a]pyrene was lost in the liver of Nrf2 knockout mice similarly to induction by butyl hydroxyanisol, demonstrating a cross-interaction between the AhR and Nrf2 pathways for induction in vivo. TCDD increased the protein level and induced the nuclear accumulation of Nrf2 with a delayed kinetics compared with activation of AhR. Chromatin immunoprecipitation revealed that TCDD recruited both AhR and Nrf2 to the Nqo1 promoter enhancer region containing a DRE and an ARE in time-dependent manners. Co-immunoprecipitation experiments revealed that, in addition to AhR-Arnt binding, TCDD induced an interaction between AhR and Nrf2 as well as Keap1. The findings reveal that TCDD induces multi protein complexes to mediate cross-interaction between the AhR and Nrf2 pathways, uncovering a novel mechanistic aspect of gene regulation by environmental chemicals through AhR and Nrf2.

The Hippo Effector Yorkie Controls Normal Tissue Growth by Antagonizing Scalloped-Mediated Default Repression

The Hippo tumor suppressor pathway restricts tissue growth by inactivating the transcriptional coactivator Yki. Although Sd has been implicated as a DNA-binding transcription factor partner for Yki and can genetically account for gain-of-function Yki phenotypes, how Yki regulates normal tissue growth remains a long-standing puzzle because Sd, unlike Yki, is dispensable for normal growth in most Drosophila tissues. Here we show that the yki mutant phenotypes in multiple developmental contexts are rescued by inactivation of Sd, suggesting that Sd functions as a default repressor and that Yki promotes normal tissue growth by relieving Sd-mediated default repression. We further identify Tgi as a cofactor involved in Sd's default repressor function and demonstrate that the mammalian ortholog of Tgi potently suppresses the YAP oncoprotein in transgenic mice. These findings fill a major gap in Hippo-mediated transcriptional regulation and open up possibilities for modulating the YAP oncoprotein in cancer and regenerative medicine.

Abstract 2742: Activation of Wnt/β-catenin signaling accelerates progression of Kras-induced pancreatic cancer.

Abstract Pancreatic ductal adenocarcinoma (PDAC), a poor prognostic cancer, commonly develops following activating mutations in the KRAS oncogene. Activation of the Wnt signaling pathway is also commonly observed in PDAC. To ascertain the impact of postnatal activation of the Wnt signaling pathways in PDAC development, we combined the elastase-tva-based RCAS-TVA pancreatic cancer model with the established LSL-KrasG12D, Ptf1a-cre model. Delivery of RCAS viruses encoding β-cateninS37A and Wnt1 stimulated the progression of premalignant PanIN and PDAC development. Moreover, mice injected with RCAS-β-cateninS37A and Wnt1 had reduced survival relative to RCAS-GFP controls (log-rank test; p < 0.05). Meanwhile, active β-catenin or its DNA-binding partner TCF4 enhanced PDAC cell proliferation, soft-agar colony formation, as well as migration and invasion activity. In contrast, these phenotypes were significantly blocked by the introduction of Icat, an inhibitor of the β-catenin/TCF4 interaction. Interestingly, Id2 (inhibitor of differentiation/DNA binding2) was significantly up-regulated by induction of β-catenin and TCF4, whereas Id2 expression was inhibited by Icat. Furthermore, nuclear β-catenin and Id2 were mainly observed in poorly differentiated PDACs and sarcomatoid tumors. Together, these data suggest that the Wnt/β-catenin signaling pathway stimulates pancreatic tumor development and progression through the activation of Id2. Citation Format: Makoto Sano, David R. Driscoll, Wilfredo E. DeJesus-Monge, David S. Klimstra, Brian C. Lewis. Activation of Wnt/β-catenin signaling accelerates progression of Kras-induced pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2742. doi:10.1158/1538-7445.AM2013-2742

FANCM and FAAP24 Maintain Genome Stability via Cooperative as Well as Unique Functions

The DNA remodeling enzyme FANCM and its DNA-binding partner, FAAP24, constitute a complex involved in the activation of Fanconi anemia (FA) DNA damage response mechanism, but neither gene has distinct patient mutants. In this study, we created isogenic models for both FANCM and FAAP24 and investigated their integrated functions in DNA damage response. We found that FANCM and FAAP24 coordinately facilitate FA pathway activation and suppress sister chromatid exchange. Importantly, we show that FANCM and FAAP24 possess nonoverlapping functions such that FAAP24 promotes ATR-mediated checkpoint activation particularly in response to DNA crosslinking agents, whereas FANCM participates in recombination-independent interstrand crosslink repair by facilitating recruitment of lesion incision activities, which requires its translocase activity. Our data suggest that FANCM and FAAP24 play multiple, while not fully epistatic, roles in maintaining genomic integrity.

Genome-wide analysis of functional sirtuin chromatin targets in yeast

BackgroundThe sirtuins are a conserved family of NAD+-dependent histone/protein deacetylases that regulate numerous cellular processes, including heterochromatin formation and transcription. Multiple sirtuins are encoded by each eukaryotic genome, raising the possibility of cooperativity or functional overlap. The scope and variety of chromatin binding sites of the sirtuins in any specific organism remain unclear.ResultsHere we utilize the ChIP-seq technique to identify and functionally characterize the genome-wide targets of the sirtuins, Sir2, Hst1 to Hst4, and the DNA binding partner of Hst1, Sum 1, in Saccharomyces cerevisiae. Unexpectedly, Sir2, Hst1 and Sum1, but not the other sirtuins, exhibit co-enrichment at several classes of chromatin targets. These include telomeric repeat clusters, tRNA genes, and surprisingly, the open reading frames (ORFs) of multiple highly expressed RNA polymerase II-transcribed genes that function in processes such as fermentation, glycolysis, and translation. Repression of these target genes during the diauxic shift is specifically dependent on Sir2/Hst1/Sum1 binding to the ORF and sufficiently high intracellular NAD+ concentrations. Sir2 recruitment to the ORFs is independent of the canonical SIR complex and surprisingly requires Sum1. The shared Sir2/Hst1/Sum1 targets also significantly overlap with condensin and cohesin binding sites, where Sir2, Hst1, and Sum1 were found to be important for condensin and cohesin deposition, suggesting a possible mechanistic link between metabolism and chromatin architecture during the diauxic shift.ConclusionsThis study demonstrates the existence of overlap in sirtuin function, and advances our understanding of conserved sirtuin-regulated functions, including the regulation of glycolytic gene expression and condensin loading.