Master Regulatory Transcription Factor Research Articles

Hormone-dependent control of developmental timing through regulation of chromatin accessibility.

Specification of tissue identity during development requires precise coordination of gene expression in both space and time. Spatially, master regulatory transcription factors are required to control tissue-specific gene expression programs. However, the mechanisms controlling how tissue-specific gene expression changes over time are less well understood. Here, we show that hormone-induced transcription factors control temporal gene expression by regulating the accessibility of DNA regulatory elements. Using the Drosophila wing, we demonstrate that temporal changes in gene expression are accompanied by genome-wide changes in chromatin accessibility at temporal-specific enhancers. We also uncover a temporal cascade of transcription factors following a pulse of the steroid hormone ecdysone such that different times in wing development can be defined by distinct combinations of hormone-induced transcription factors. Finally, we show that the ecdysone-induced transcription factor E93 controls temporal identity by directly regulating chromatin accessibility across the genome. Notably, we found that E93 controls enhancer activity through three different modalities, including promoting accessibility of late-acting enhancers and decreasing accessibility of early-acting enhancers. Together, this work supports a model in which an extrinsic signal triggers an intrinsic transcription factor cascade that drives development forward in time through regulation of chromatin accessibility.

Vasoactive intestinal peptide induced epithelial to mesenchymal transition activities in PC3 prostate cancer cells

Abstract Epithelial to mesenchymal transition (EMT) is a fundamental cellular process preceding movement. Malignant transformation of epithelial cells also undergo EMT leading to metastasis and a worse prognosis for human cancer patients. Understanding molecular pathways that are linked to EMT may provide additional drug targets to treat epithelial cancers like prostate cancer. Vasoactive intestinal peptide signaling through one of its naturally occurring G-protein coupled receptors, termed VPAC1, induces non-tumor and tumorigenic epithelial cell lines (RWPE-1, LnCaP and PC3 cells) to undergo EMT and downregulate E-cadherin, a hallmark for EMT. Curcumin, an anti-inflammatory molecule, blocked these VIP EMT effects suggesting a role for NF-kB signaling. Our group has uncovered EMT-associated molecular mRNA targets induced by VIP treatment of primary, resting CD4 T cells, including the EMT master regulator transcription factor, SNAIL1. We hypothesize that EMT transition effects by VIP signaling may also involve the upregulation of SNAIL1 protein. To this end, we plan on treating PC3 cells with varying levels of VIP over time (0–24 hr) and measure by immunoblots the extent to which Snail1 protein, and up to 10 additional EMT-associated proteins, are differentially altered. Successful completion of this research is expected to uncover important VIP protein targets that is expected to better explain its epithelial tumorigenic potential.

Pax7 remodels the chromatin landscape in skeletal muscle stem cells.

Pluripotent stem cells (PSC) hold great promise for the treatment of human skeletal muscle diseases. However, it remains challenging to convert PSC to skeletal muscle cells, and the mechanisms by which the master regulatory transcription factor, Pax7, promotes muscle stem (satellite) cell identity are not yet understood. We have taken advantage of PSC-derived skeletal muscle precursor cells (iPax7), wherein the induced expression of Pax7 robustly initiates the muscle program and enables the in vitro generation of precursors that seed the satellite cell compartment upon transplantation. Remarkably, we found that chromatin accessibility in myogenic precursors pre-figures subsequent activation of myogenic differentiation genes. We also found that Pax7 binding is generally restricted to euchromatic regions and excluded from H3K27 tri-methylated regions in muscle cells, suggesting that recruitment of this factor is circumscribed by chromatin state. Further, we show that Pax7 binding induces dramatic, localized remodeling of chromatin characterized by the acquisition of histone marks associated with enhancer activity and induction of chromatin accessibility in both muscle precursors and lineage-committed myoblasts. Conversely, removal of Pax7 leads to rapid reversal of these features on a subset of enhancers. Interestingly, another cluster of Pax7 binding sites is associated with a durably accessible and remodeled chromatin state after removal of Pax7, and persistent enhancer accessibility is associated with subsequent, proximal binding by the muscle regulatory factors, MyoD1 and myogenin. Our studies provide new insights into the epigenetic landscape of skeletal muscle stem cells and precursors and the role of Pax7 in satellite cell specification.

A novel statistical approach for identification of the master regulator transcription factor

BackgroundTranscription factors are known to play key roles in carcinogenesis and therefore, are gaining popularity as potential therapeutic targets in drug development. A ‘master regulator’ transcription factor often appears to control most of the regulatory activities of the other transcription factors and the associated genes. This ‘master regulator’ transcription factor is at the top of the hierarchy of the transcriptomic regulation. Therefore, it is important to identify and target the master regulator transcription factor for proper understanding of the associated disease process and identifying the best therapeutic option.MethodsWe present a novel two-step computational approach for identification of master regulator transcription factor in a genome. At the first step of our method we test whether there exists any master regulator transcription factor in the system. We evaluate the concordance of two ranked lists of transcription factors using a statistical measure. In case the concordance measure is statistically significant, we conclude that there is a master regulator. At the second step, our method identifies the master regulator transcription factor, if there exists one.ResultsIn the simulation scenario, our method performs reasonably well in validating the existence of a master regulator when the number of subjects in each treatment group is reasonably large. In application to two real datasets, our method ensures the existence of master regulators and identifies biologically meaningful master regulators. An R code for implementing our method in a sample test data can be found in http://www.somnathdatta.org/software.ConclusionWe have developed a screening method of identifying the ‘master regulator’ transcription factor just using only the gene expression data. Understanding the regulatory structure and finding the master regulator help narrowing the search space for identifying biomarkers for complex diseases such as cancer. In addition to identifying the master regulator our method provides an overview of the regulatory structure of the transcription factors which control the global gene expression profiles and consequently the cell functioning.

From Gene Expression To Disease Association

Overall Abstract Association studies have achieved significant progress in mapping hundreds of genetic variants to major psychiatric disorders. But the underlying molecular mechanisms regarding specific genes, pathways involved, and interactions among the genes remain largely unknown. Gene expression and its regulation systems are critical for unraveling the causal relationships. This symposium presents cutting edge studies of brain gene expression regulation and its relevance to disease associations. Hermona Soreq from the Hebrew University of Jerusalem will present a study of microRNA (miR) and pseudogene expression and their roles in psychiatry. A selected group of non-coding pseudogenes (PSGs) carry micro RNA (miR) recognition elements (PSG+MRE) compete with brain-expressed genes for the available miR regulators. Furthermore, SNPs surrounding these coding genes tend to be more abundant in psychiatric patients compared to controls. They used transfection-mediated over-expression of PSG+MRE and GapmeR-directed suppression of selected cholinergic transcripts sharing MREs with them in cultured human cells, and demonstrated bi-directional modulation of the interaction of these transcripts with miRs. The study illustrated functional roles of PSGs+MRE in cholinergic functioning, with potential impact on mental disorders. Seth Ament from the Institute for Systems Biology will present a study of transcription factor (TF) binding targets in the brain. They reconstructed a model for the genomic binding sites and functional target genes of 778 TFs in the human brain by integrating large-scale epigenomics and transcriptomics datasets. Using this model, they predicted master regulator transcription factors and functional non-coding variants associated with risk for bipolar disorder and schizophrenia. They experimentally validated the prediction that a master regulator transcription factor, POU3F2, interacts with a risk-associated SNP to modulate the VRK2 promoter. Chunyu Liu from the University of Illinois at Chicago will present a study of co-expression networks involving noncoding RNAs. They retrieved lncRNAs mapped to the seven major CNV deletion regions known to increase risks of developing schizophrenia and carried out weighted gene co-expression network analysis (WGCNA) using data from Genotype-Tissue Expression (GTEx) and BrainSpan projects to look for co-expression modules that harbor CNV-lncRNAs. They identified coexpression modules that were associated with male reproduction in male individuals and associated with neuronal functions in both male and female individuals. The findings suggest that lncRNAs inside those rare CNVs might play significant temporal and spatial roles in regulating other protein-coding genes and subsequently contribute to schizophrenia risk. Hae Kyung Im from the University of Chicago will present the PrediXcan method, which was developed by him, Nancy Cox, and colleagues. By using genotype to predict expression (or other molecular traits) and correlating them with the trait of interest. They have developed prediction models for gene expression in 40 human tissues using the GTEx and Depression Genes Network data. They also extended the method so that only summary statistics are needed to infer PrediXcan results so that they can perform large meta analysis. Using this new method called MetaXcan they have generated results for 117 phenotypes with publicly available GWAS meta-analysis results.

Regulatory T Cells: Molecular and Cellular Basis for Immunoregulation.

CD4+ regulatory T cells (Tregs) are a highly immune-suppressive subset of CD4+ T cells, characterized by expression of the master regulatory transcription factor FOXP3. Tregs are proven to play central roles in the maintenance of self-tolerance in healthy individuals. Tregs are involved in maintaining immune homeostasis: they protect hosts from developing autoimmune diseases and allergy, whereas in malignancies, they promote tumor progression by suppressing anti-tumor immunity. Elucidating factors influencing Treg homeostasis and function have important implications for understanding disease pathogenesis and identifying therapeutic opportunities. Thus, the manipulating Tregs for up- or down-regulation of their suppressive function is a new therapeutic strategy for treating various diseases including autoimmune disorders and cancer. This review will focus on recent advances in how Tregs integrate extracellular and intracellular signals to control their survival and stability. Deeper mechanistic understanding of disease-specific Treg development, maintenance, and function could make disease-specific Treg-targeted therapy more effective, resulting in an increase of efficacy and decrease of side effects related to manipulating Tregs.

Abstract B44: Emerging targets from Cancer Dependency Map v0.1

Abstract Precision Cancer Medicine requires the identification of vulnerabilities linked to genetic features of tumors. Recent studies utilizing highly annotated small molecule collections to assess dependencies across hundreds of genomically annotated cell lines have demonstrated the potential for such large-scale preclinical “Dependency Map” projects. We have undertaken a complementary approach using genetic perturbation tools (RNAi and CRISPR-Cas9 based loss-of-function viability screens), to systematically catalog preferential genetic dependencies and markers that predict response. These efforts are providing a foundation for the discovery of novel targets poised for early therapeutic discovery projects together with patient populations that may be enriched for responders to such therapies. Here, we present results from our initial Cancer Dependency Map consisting of RNAi loss-of-function screens across 503 cell lines, including both solid and hematopoietic tumors. We discovered 43 genes whose mutation or copy number creates a cancer dependency (oncogene addiction) including a novel dependency on the small GTPase, GNAI2 in Diffuse large B-cell Lymphoma. We discovered 142 genes in which elevated levels of expression create a dependency (gene addiction), a group of genes highly enriched for master regulator transcription factors such as SOX10, SPDEF, PAX8 and HNF1B. We discovered 474 genes for which hemizygous copy number creates a dependency (CYCLOPS genes), a group of genes highly enriched for members of macromolecular protein complexes including the spliceosome and proteasome. Finally, we discovered 171 genes that become a dependency when a redundant functional paralog is lost in cancer cells (redundant essentials). We demonstrate the mechanistic basis behind one such redundant essential dependency relationship in which promoter methylation of the UBB ubiquitin gene eliminates a compensatory mechanism leading to a novel vulnerability on the suppression of the UBC ubiquitin gene. These observations begin to provide an initial census, categorization and prioritization of robust cancer dependencies and support the potential impact for expanding early efforts to develop dependency maps of cancer. Citation Format: Francisca Vazquez, Aviad Tsherniak, Barbara Weir, Phil Montgomery, Glenn Cowley, Stanley Gill, Gregory Kryukov, Sasha Pantel, Will Harrington, Mike Burger, Robin Meyers, Levi Ali, Amy Goodale, Yenarae Lee, Levi Garraway, Jesse Boehm, David Root, Todd Golub, William Hahn. Emerging targets from Cancer Dependency Map v0.1. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr B44.

Chromatin Accessibility Identifies CTCF As a Gatekeeper of Stemness Functions in Human Hematopoietic Development

Hematopoietic stem cells (HSC), at the apex of the blood hierarchy, generate a series of increasingly committed progenitors and ultimately produce terminally differentiated cells at the bottom of the hierarchy. Although HSC and their immediate downstream, multipotent progenitors (MPP) have full multilineage differentiation capacity, only HSC have the capacity for long term self-renewal. Thus comparison of HSC with downstream progenitors that exhibit gradual fate restriction by assuming the identity of a mature blood cell is central to defining how the stemness state is established and how lineage commitment is executed. Interestingly, few genes are differentially expressed between human HSC and MPP, suggesting that some important stem cell mechanisms are in a permissive state and therefore difficult to identify simply by differential gene expression analysis. We hypothesized that these permissive genetic regions could be identified by chromatin analysis. To identify permissive genomic regions, we delineated regions of accessible chromatin through ATAC-seq assays in HSC, MPP, committed progenitors and seven mature lineages from human Cord Blood. DNA recognition motif analysis across accessible chromatin in each developmental stage identified both known and unknown candidate master regulatory transcription factors for each step of lineage development.The DNA recognition motif for CTCF, a crucial effector of chromatin interactions, is enriched in accessible chromatin associated with progenitors but not in HSC, even though CTCF expression is not significantly different among stem and progenitor populations. Knockdown of CTCF in HSC by lentiviral vector system increased the proportion of HSC in G0 cell cycle and consequently reduced the number of mature cells generated in both conventional and high sensitivity single cell assays. The development of differentiated lymphoid and megakaryocytic lineages was affected. Few effects were seen when CTCF was silenced in MPP or more downstream progenitors. Collectively, our results suggest that CTCF is a "gate keeper factor" that regulates transition from dormant to active stages in HSC, and might control stem cell pool throughout life.In parallel to normal blood hierarchies, AML is also composed of a hierarchy with leukemia stem cells and non-stem-like leukemia cells; LSC and non-LSC fractions sorted from 24 AML patients have also been subjected to ATAC-seq. Currently we are comparing HSC and non-HSC with LSC and non-LSC fractions to identify the similarity and differences between normal HSC and LSC. Overall, our study is revealing insights into the stemness state of normal and leukemic stem cells in primary human tissues. DisclosuresTakayanagi:Kyowa Hakko Kirin: Employment.

Artemisinins Target GABAA Receptor Signaling and Impair α Cell Identity

SummaryType 1 diabetes is characterized by the destruction of pancreatic β cells, and generating new insulin-producing cells from other cell types is a major aim of regenerative medicine. One promising approach is transdifferentiation of developmentally related pancreatic cell types, including glucagon-producing α cells. In a genetic model, loss of the master regulatory transcription factor Arx is sufficient to induce the conversion of α cells to functional β-like cells. Here, we identify artemisinins as small molecules that functionally repress Arx by causing its translocation to the cytoplasm. We show that the protein gephyrin is the mammalian target of these antimalarial drugs and that the mechanism of action of these molecules depends on the enhancement of GABAA receptor signaling. Our results in zebrafish, rodents, and primary human pancreatic islets identify gephyrin as a druggable target for the regeneration of pancreatic β cell mass from α cells.

Abstract A072: Glucose metabolism and O-linked GlcNAcylation in the tissue repair function of Treg cells

Abstract Regulatory T (Treg) cells dampen immune responses to avoid excessive, deleterious inflammation in the context of autoimmunity, allergy, and infection. Treg cells also impede anti-tumor immunity. Therefore, modulation of Treg cell activity is of great therapeutic potential for treating many debilitating inflammatory diseases and cancer. Recent studies have suggested that in addition to their prominent immunosuppressive function, Treg cells have a distinct and non-redundant role in tissue maintenance and remodeling. These Treg cells exhibit a unique transcriptional signature represented by high expression of the EGF family member amphiregulin (Areg). Importantly, tumor infiltrating Treg cells highly express Areg. Since Areg is known to exert pleiotropic oncogenic activities, Areg producing Treg cells may be crucial for establishing the tumor microenvironment. It has been increasingly appreciated that lymphocyte activation and differentiation are coupled to specific metabolic pathways. Metabolic reprogramming is dictated by cytokine and growth factor signals, as well as the availability of nutrients. In addition, metabolic products provide substrates that can alter the functional status of a cell through posttranslational modifications (PTMs). Among the various nutrients, glucose is a key metabolic substrate for T cells. Aerobic glycolysis in activated T cells is required for the acquisition of full effector function. Treg cells in the secondary lymphoid organs and the tissue have distinct glucose availability and metabolic needs. We thus hypothesized that glucose sensing and glucose metabolism play essential roles in shaping the unique phenotype and function of tissue resident Treg cells. O-linked GlcNAcylation is a PTM dynamically regulated by glucose levels and controls the function of many proteins, including transcription factors that play key roles in T cell activation such as NF-κB, CREB, and NFAT. To understand the mechanism by which glucose metabolism controls the function of tissue-resident Treg cells, we began to characterize protein O-GlcNAcylation in Treg cells, and found that both human and mouse Foxp3, the master regulator transcription factor of Treg cells, undergoes O-GlcNAcylation. We are in the process of mapping the site of O-GlcNAcylation of Foxp3, and understanding the physiological significance of this PTM. Citation Format: Wei Hu, Nicholas Arpaia, Jesse A. Green, Ronald C. Hendrickson, Alexander Y. Rudensky. Glucose metabolism and O-linked GlcNAcylation in the tissue repair function of Treg cells [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A072.

Reactive electrophilic oxylipins trigger a heat stress-like response through HSFA1 transcription factors.

Abiotic and biotic stresses are often characterized by an induction of reactive electrophile species (RES) such as the jasmonate 12-oxo-phytodienoic acid (OPDA) or the structurally related phytoprostanes. Previously, RES oxylipins have been shown massively to induce heat-shock-response (HSR) genes including HSP101 chaperones. Moreover, jasmonates have been reported to play a role in basal thermotolerance. We show that representative HSR marker genes are strongly induced by RES oxylipins through the four master regulator transcription factors HSFA1a, b, d, and e essential for short-term adaptation to heat stress in Arabidopsis. When compared with Arabidopsis seedlings treated at the optimal acclimation temperature of 37 °C, the exogenous application of RES oxylipins at 20 °C induced a much weaker induction of HSP101 at both the gene and protein expression levels which, however, was not sufficient to confer short-term acquired thermotolerance. Moreover, jasmonate-deficient mutant lines displayed a wild-type-like HSR and were not compromised in acquiring thermotolerance. Hence, the OPDA- and RES oxylipin-induced HSR is not sufficient to protect seedlings from severe heat stress but may help plants to cope better with stresses associated with protein unfolding by inducing a battery of chaperones in the absence of heat.

Mechanism of allele specific assembly and disruption of master regulator transcription factor complexes of NF-KBp50, NF-KBp65 and HIF1a on a non-coding FAS SNP

A challenging question in genetics is to understand the molecular function of non-coding variants of the genome. By using differential EMSA, ChIP and functional genome analysis, we have found that changes in transcription factors (TF) apparent binding affinity and dissociation rates are responsible for allele specific assembly or disruption of master TFs: we observed that NF-KBp50, NF-KBp65 and HIF1a bind with an affinity of up to 10 fold better to the C-allele than to the T-allele of rs7901656 both in vivo and in vitro. Furthermore, we showed that NF-KBp50, p65 and HIF1a form higher order heteromultimeric complexes overlapping rs7901656, implying synergism of action among TFs governing cellular response to infection and hypoxia. With rs7901656 on the FAS gene as a paradigm, we show how allele specific transcription factor complex assembly and disruption by a causal variant contributes to disease and phenotypic diversity. This finding provides the highly needed mechanistic insight into how the molecular etiology of regulatory SNPs can be understood in functional terms.

Glucolipotoxicity diminishes cardiomyocyte TFEB and inhibits lysosomal autophagy during obesity and diabetes

Impaired cardiac metabolism in the obese and diabetic heart leads to glucolipotoxicity and ensuing cardiomyopathy. Glucolipotoxicity causes cardiomyocyte injury by increasing energy insufficiency, impairing proteasomal-mediated protein degradation and inducing apoptosis. Proteasome-evading proteins are degraded by autophagy in the lysosome, whose metabolism and function are regulated by master regulator transcription factor EB (TFEB). Limited studies have examined the impact of glucolipotoxicity on intra-lysosomal signaling proteins and their regulators. By utilizing a mouse model of diet-induced obesity, type-1 diabetes (Akita) and ex-vivo model of glucolipotoxicity (H9C2 cells and NRCM, neonatal rat cardiomyocyte), we examined whether glucolipotoxicity negatively targets TFEB and lysosomal proteins to dysregulate autophagy and cause cardiac injury. Despite differential effects of obesity and diabetes on LC3B-II, expression of proteins facilitating autophagosomal clearance such as TFEB, LAMP-2A, Hsc70 and Hsp90 were decreased in the obese and diabetic heart. In-vivo data was recapitulated in H9C2 and NRCM cells, which exhibited impaired autophagic flux and reduced TFEB content when exposed to a glucolipotoxic milieu. Notably, overloading myocytes with a saturated fatty acid (palmitate) but not an unsaturated fatty acid (oleate) depleted cellular TFEB and suppressed autophagy, suggesting a fatty acid specific regulation of TFEB and autophagy in the cardiomyocyte. The effect of glucolipotoxicity to reduce TFEB content was also confirmed in heart tissue from patients with Class-I obesity. Therefore, during glucolipotoxicity, suppression of lysosomal autophagy was associated with reduced lysosomal content, decreased cathepsin-B activity and diminished cellular TFEB content likely rendering myocytes susceptible to cardiac injury.

Transcription of the mating-type-regulated lncRNA IRT1 is governed by TORC1 and PKA

Cell fate decisions are controlled by multiple cell-intrinsic and -extrinsic factors. In budding yeast, the decision to enter gametogenesis or sporulation is dictated by nutrient availability and mating type. Recently, we showed that in diploid cells harbouring opposite mating types (MATa and MATα), the protein kinase A (PKA) and target of rapamycin complex I (TORC1) signalling pathways integrate at the promoter of the master regulatory transcription factor IME1 to control sporulation via nutrient availability (Weidberg, et al. 2016). In cells with a single mating type (MATa or MATα), however, IME1 is repressed by transcription through the IME1 promoter of a long non-coding RNA called IRT1, which prevents this cell type from undergoing sporulation. Here, we investigated the role of nutrient signalling in mating-type control of IME1. We find that expression of IRT1, like IME1 itself, depends on nutrient availability and the activities of PKA and TORC1. IRT1 transcription is repressed when nutrients are ample and TORC1 and PKA are active. In contrast, inhibition of PKA and TORC1 is sufficient to recruit Rme1 to the IRT1 promoter and induce IRT1-mediated repression of IME1. Finally, we provide evidence that IRT1 and IME1 are co-repressed by the Tup1–Cyc8 complex when nutrients are ample. Thus, in cells with a single mating-type nutrient availability regulates mating-type repression of IME1 and sporulation. Our results indicate that there is a hierarchy between nutrient and mating-type signals in controlling the decision to enter sporulation.

GATA Factor-Dependent Positive-Feedback Circuit in Acute Myeloid Leukemia Cells

The master regulatory transcription factor GATA-2 triggers hematopoietic stem and progenitor cell generation. GATA2 haploinsufficiency is implicated in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), and GATA2 overexpression portends a poor prognosis for AML. However, the constituents of the GATA-2-dependent genetic network mediating pathogenesis are unknown. We described a p38-dependent mechanism that phosphorylates GATA-2 and increases GATA-2 target gene activation. We demonstrate that this mechanism establishes a growth-promoting chemokine/cytokine circuit in AML cells. p38/ERK-dependent GATA-2 phosphorylation facilitated positive autoregulation of GATA2 transcription and expression of target genes, including IL1B and CXCL2. IL-1β and CXCL2 enhanced GATA-2 phosphorylation, which increased GATA-2-mediated transcriptional activation. p38/ERK-GATA-2 stimulated AML cell proliferation via CXCL2 induction. As GATA2 mRNA correlated with IL1B and CXCL2 mRNAs in AML-M5 and high expression of these genes predicted poor prognosis of cytogenetically normal AML, we propose that the circuit is functionally important in specific AML contexts.

Abstract 4700: BET bromodomain inhibition is a promising treatment strategy for distinct subsets of lethal castration-resistant prostate cancer

Abstract Background: While treatment options for patients with castration-resistant prostate cancer (CRPC) are expanding, one American man is still predicted to die every 19 minutes from this disease this year. Moreover, more widespread use of novel and more potent AR-targeting agents has led to increased clinical frequency of virulent androgen and AR-independent CRPC subsets(Small, Huang et al. 2015). Currently, there are limited treatment options for men with CRPC who are resistant to these AR-targeting agents, clearly demonstrating an urgent need to develop more effective therapies for CRPC patients. Recent published reports demonstrate an important role for BET bromodomain chromatin reader proteins in prostate cancer models that are androgen or AR-dependent (Gao, Schwartzman et al. 2013; Wyce, Degenhardt et al. 2013; Asangani, Dommeti et al. 2014; Chan, Selth et al. 2015). However, there was limited information about the anti-tumor activity of this class of drugs in androgen-independent, enzalutamide-resistant, or AR-independent CRPC models. The studies reported herein were designed to address that deficit. Methods: We treated a panel of CRPC cell lines with dose escalation of the BET bromodomain inhibitor JQ1 and measured cell viability. To clarify molecular mechanisms that contribute to the anti-tumor effect, we treated CRPC cell lines with JQ1 and measured gene expression changes with RNA-sequencing. Finally, to determine the anti-tumor activity of JQ1 in vivo, we implanted enzalutamide resistant or AR-null CRPC xenografts in immunocompromised mice and treated them with JQ1. Results: All cell lines were sensitive to JQ1 with similar GI50 values (all < 1μM). Our analysis of RNA-seq data after JQ1 treatment identified a BET bromodomain response signature that was conserved across the population of cell lines. Further, we applied Virtual Inference of Protein-activity by Enriched Regulon (VIPER) analysis to identify novel Master Regulator transcription factors that mediate response to JQ1(Aytes, Mitrofanova et al. 2014). Finally, JQ1 treatment of enzalutamide resistant or AR-null CRPC xenografts implanted in vivo suppressed tumor growth without appreciable toxicity. Conclusions: BET bromodomain inhibition is a promising treatment strategy for distinct subsets of lethal CRPC. Our work using a broad panel of CRPC models sheds further light on pharmacodynamic markers of response and target proteins whose function is impacted by BET bromodomain inhibition. These findings may have implications for the design of BET bromodomain inhibitor clinical trials in men with CRPC and interpretation of on-target effects in those trials. Citation Format: Lina Gao, Daniel J. Coleman, Carly J. King, Jacob Schwartzman, Nicholas Wang, Amanda Esch, Joshua Urrutia, Archana Sehrawat, Laura M. Heiser, Joshi J. Alumkal. BET bromodomain inhibition is a promising treatment strategy for distinct subsets of lethal castration-resistant prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4700.

Grainyhead-like 2 inhibits the coactivator p300, suppressing tubulogenesis and the epithelial-mesenchymal transition.

Developmental morphogenesis and tumor progression require a transient or stable breakdown of epithelial junctional complexes to permit programmed migration, invasion, and anoikis resistance, characteristics endowed by the epithelial-mesenchymal transition (EMT). The epithelial master-regulatory transcription factor Grainyhead-like 2 (GRHL2) suppresses and reverses EMT, causing a mesenchymal-epithelial transition to the default epithelial phenotype. Here we investigated the role of GRHL2 in tubulogenesis of Madin-Darby canine kidney cells, a process requiring transient, partial EMT. GRHL2 was required for cystogenesis, but it suppressed tubulogenesis in response to hepatocyte growth factor. Surprisingly, GRHL2 suppressed this process by inhibiting the histone acetyltransferase coactivator p300, preventing the induction of matrix metalloproteases and other p300-dependent genes required for tubulogenesis. A 13-amino acid region of GRHL2 was necessary for inhibition of p300, suppression of tubulogenesis, and interference with EMT. The results demonstrate that p300 is required for partial or complete EMT occurring in tubulogenesis or tumor progression and that GRHL2 suppresses EMT in both contexts through inhibition of p300.

EBNA2 Drives Formation of New Chromosome Binding Sites and Target Genes for B-Cell Master Regulatory Transcription Factors RBP-jκ and EBF1.

Epstein-Barr Virus (EBV) transforms resting B-lymphocytes into proliferating lymphoblasts to establish latent infections that can give rise to malignancies. We show here that EBV-encoded transcriptional regulator EBNA2 drives the cooperative and combinatorial genome-wide binding of two master regulators of B-cell fate, namely EBF1 and RBP-jκ. Previous studies suggest that these B-cell factors are statically bound to target gene promoters. In contrast, we found that EBNA2 induces the formation of new binding for both RBP-jκ and EBF1, many of which are in close physical proximity in the cellular and viral genome. These newly induced binding sites co-occupied by EBNA2-EBF1-RBP-jκ correlate strongly with transcriptional activation of linked genes that are important for B-lymphoblast function. Conditional expression or repression of EBNA2 leads to a rapid alteration in RBP-jκ and EBF1 binding. Biochemical and shRNA depletion studies provide evidence for cooperative assembly at co-occupied sites. These findings reveal that EBNA2 facilitate combinatorial interactions to induce new patterns of transcription factor occupancy and gene programming necessary to drive B-lymphoblast growth and survival.

Navigating Transcriptional Coregulator Ensembles to Establish Genetic Networks: A GATA Factor Perspective.

Complex developmental programs require orchestration of intrinsic and extrinsic signals to control cell proliferation, differentiation, and survival. Master regulatory transcription factors are vital components of the machinery that transduce these stimuli into cellular responses. This is exemplified by the GATA family of transcription factors that establish cell type-specific genetic networks and control the development and homeostasis of systems including blood, vascular, adipose, and cardiac. Dysregulated GATA factor activity/expression underlies anemia, immunodeficiency, myelodysplastic syndrome, and leukemia. Parameters governing the capacity of a GATA factor expressed in multiple cell types to generate cell type-specific transcriptomes include selective coregulator usage and target gene-specific chromatin states. As knowledge of GATA-1 mechanisms in erythroid cells constitutes a solid foundation, we will focus predominantly on GATA-1, while highlighting principles that can be extrapolated to other master regulators. GATA-1 interacts with ubiquitous and lineage-restricted transcription factors, chromatin modifying/remodeling enzymes, and other coregulators to activate or repress transcription and to maintain preexisting transcriptional states. Major unresolved issues include: how does a GATA factor selectively utilize diverse coregulators; do distinct epigenetic landscapes and nuclear microenvironments of target genes dictate coregulator requirements; and do gene cohorts controlled by a common coregulator ensemble function in common pathways. This review will consider these issues in the context of GATA factor-regulated hematopoiesis and from a broader perspective.

The transcription factors Slug (SNAI2) and Snail (SNAI1) regulate phospholipase D (PLD) promoter in opposite ways towards cancer cell invasion

Slug (SNAI2) and Snail (SNAI1) are master regulatory transcription factors for organogenesis and wound healing, and they are involved in the epithelial to mesenchymal transition (EMT) of cancer cells. We found that the activity of phospholipase D isoform 2 (PLD2) is highly increased in cancers with larger size and poor prognosis (MDA-MB-231 versus MCF-7 cells), so we determined if Snail or Slug were responsible for PLD2 gene transcription regulation. Unexpectedly, we found that PLD2 expression was positively regulated by Slug but negatively regulated by Snail. The differential effects are amplified in breast cancer cells over normal cells and with MDA-MB-231 more robustly than MCF-7. Slug putatively binds to the PLD2 promoter and transactivates it, which is negated when Slug and Snail compete with each other. Meanwhile, PLD2 has a negative effect on Snail expression and a positive effect on Slug, thus closing a feedback loop between the lipase and the transcription factors. Further, PA, the product of PLD2 enzymatic reaction, has profound effects on its own and it further regulates the transcription factors. Thus, we show for the first time that the overexpressed PLD2 in human breast tumors is regulated by Slug and Snail transcription factors. The newly uncovered feedback loops in highly invasive cancer cells have important implications in the process of EMT.