Direct DNA Binding Research Articles

BRLF1 suppresses RNA Pol III-mediated RIG-I inflammasome activation in the early EBV lytic lifecycle.

Latent infection with herpesviruses constitutively activates inflammasomes, while lytic replication suppresses their activation through distinct mechanisms. However, how Epstein-Barr virus (EBV) lytic replication inhibits the activation of inflammasomes remains unknown. Here, we reveal that the EBV immediate-early protein BRLF1 inhibits inflammasome activation, and BRLF1 deficiency significantly increases the activation of inflammasomes and pyroptosis during early lytic lifecycle. BRLF1 interacts with RNA polymerase III subunits to suppress immunostimulatory small RNA transcription, RIG-I inflammasome activation, and antiviral responses. Consequently, BRLF1-deficient EBV primary infection induces robust T-cell and NK cell activation and killing through IL-1β and IL-18. A BRLF1-derived peptide that inhibits inflammasome activation is sufficient to suppress T-cell and NK cell responses during BRLF1-deficient EBV primary infection in lymphocytes. These results reveal a novel mechanism involved in the evasion of inflammasome activation and antiviral responses during EBV early lytic infection and provide a promising approach for the manipulation of inflammasomes against infection of oncogenic herpesviruses.

An immunochemistry-based screen for chemical inhibitors of DNA-protein interactions and its application to human CGGBP1

BackgroundInhibition of DNA-binding of proteins by small-molecule chemicals holds immense potential in manipulating the activities of DNA-binding proteins. Such a chemical inhibition of DNA-binding of proteins can be used to modulate processes such as replication, transcription, DNA repair and maintenance of epigenetic states. This prospect is currently challenged with the absence of robust and generic protocols to identify DNA-protein interactions. Additionally, much of the current approaches to designing inhibitors requires structural information of the target proteins.MethodsWe have developed a simple dot blot and immunodetection-based assay to screen chemical libraries for inhibitors of DNA-protein interactions. The assay has been applied to a library of 1685 FDA-approved chemicals to discover inhibitors of CGGBP1, a multifunctional DNA-binding protein with no known structure. Additional in vitro and in cellulo assays have been performed to verify and supplement the findings of the screen.ResultsOur primary screen has identified multiple inhibitors of direct or indirect interactions between CGGBP1 and genomic DNA. Of these, one inhibitor, Givinostat, was found to inhibit direct DNA-binding of CGGBP1 in the secondary screen using purified recombinant protein as the target. DNA and chromatin immunoprecipitation assays reinforced the findings of the screen that Givinostat inhibits CGGBP1-DNA binding.ConclusionsThe assay we have described successfully identifies verifiable inhibitors of DNA-binding of protein; in this example, the human CGGBP1. This assay is customizable for a wide range of targets for which primary antibodies are available. It works with different sources of the target protein, cell lysates or purified recombinant preparations and does not require special equipment, DNA modifications or protein structural data. This assay is scalable and highly adaptable with the potential to discover inhibitors of transcription factors with implications in cancer biology.

Unraveling the Hierarchy of cis and trans Factors That Determine the DNA Binding by Peroxisome Proliferator-Activated Receptor γ.

Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor essential for adipocyte development and the maintenance of the alternatively polarized macrophage phenotype. Biochemical studies have established that as an obligate heterodimer with retinoid X receptor (RXR), PPARγ binds directly repeated nuclear receptor half sites spaced by one nucleotide (direct repeat 1 [DR1]). However, it has not been analyzed systematically and genome-wide how cis factors such as the sequences of DR1s and adjacent sequences and trans factors such as cobinding lineage-determining transcription factors (LDTFs) contribute to the direct binding of PPARγ in different cellular contexts. We developed a novel motif optimization approach using sequence composition and chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) densities from macrophages and adipocytes to complement de novo motif enrichment analysis and to define and classify high-affinity binding sites. We found that approximately half of the PPARγ cistrome represents direct DNA binding; both half sites can be extended upstream, and these are typically not of equal strength within a DR1. Strategically positioned LDTFs have greater impact on PPARγ binding than the quality of DR1, and the presence of the extension of DR1 provides a remarkable synergy with LDTFs. This approach of considering not only nucleotide frequencies but also their contribution to protein binding in a cellular context is applicable to other transcription factors.

Strigolactones and Brassinosteroids Antagonistically Regulate the Stability of the D53–OsBZR1 Complex to Determine FC1 Expression in Rice Tillering

Rice tillering, a key architecture trait determining grain yield, is highly regulated by a class of newly identified phytohormones, strigolactones (SLs). However, the whole SL signaling pathway from the receptor to downstream transcription factors to finally inhibit tillering remains unrevealed. In this study, we first found that brassinosteroids (BRs) strongly enhance tillering by promoting bud outgrowth in rice, which is largely different from the function of BRs in Arabidopsis. Genetic and biochemical analyses indicated that both the SL and BR signaling pathways control rice tillering by regulating the stability of D53 and/or the OsBZR1–RLA1–DLT module, a transcriptional complex in the rice BR signaling pathway. We further found that D53 interacts with OsBZR1 to inhibit the expression of FC1, a local inhibitor of tillering, and that this inhibition depends on direct DNA binding by OsBZR1, which recruits D53 to the FC1 promoter in rice buds. Taken together, these findings uncover a mechanism illustrating how SLs and BRs coordinately regulate rice tillering via the early responsive gene FC1.

Nickel Carcinogenesis Mechanism: DNA Damage.

Nickel (Ni) is known to be a major carcinogenic heavy metal. Occupational and environmental exposure to Ni has been implicated in human lung and nasal cancers. Currently, the molecular mechanisms of Ni carcinogenicity remain unclear, but studies have shown that Ni-caused DNA damage is an important carcinogenic mechanism. Therefore, we conducted a literature search of DNA damage associated with Ni exposure and summarized known Ni-caused DNA damage effects. In vitro and vivo studies demonstrated that Ni can induce DNA damage through direct DNA binding and reactive oxygen species (ROS) stimulation. Ni can also repress the DNA damage repair systems, including direct reversal, nucleotide repair (NER), base excision repair (BER), mismatch repair (MMR), homologous-recombination repair (HR), and nonhomologous end-joining (NHEJ) repair pathways. The repression of DNA repair is through direct enzyme inhibition and the downregulation of DNA repair molecule expression. Up to now, the exact mechanisms of DNA damage caused by Ni and Ni compounds remain unclear. Revealing the mechanisms of DNA damage from Ni exposure may contribute to the development of preventive strategies in Ni carcinogenicity.

Transcription Factors Indirectly Regulate Genes through Nuclear Colocalization.

Various types of data, including genomic sequences, transcription factor (TF) knockout data, TF-DNA interaction and expression profiles, have been used to decipher TF regulatory mechanisms. However, most of the genes affected by knockout of a particular TF are not bound by that factor. Here, I showed that this interesting result can be partially explained by considering the nuclear positioning of TF knockout affected genes and TF bound genes. I found that a statistically significant number of TF knockout affected genes show nuclear colocalization with genes bound by the corresponding TF. Although these TF knockout affected genes are not directly bound by the corresponding TF; the TF tend to be in the same cellular component with the TFs that directly bind these genes. TF knockout affected genes show co-expression and tend to be involved in the same biological process with the spatially adjacent genes that are bound by the corresponding TF. These results demonstrate that TFs can regulate genes through nuclear colocalization without direct DNA binding, complementing the conventional view that TFs directly bind DNA to regulate genes. My findings will have implications in understanding TF regulatory mechanisms.

Pioneer Factor-Nucleosome Binding Events during Differentiation Are Motif Encoded.

Although the invitro structural and invivo spatial characteristics of transcription factor (TF) binding are well defined, TF interactions with chromatin and other companion TFs during development are poorly understood. To analyze such interactions invivo, we profiled several TFs across a time course of human embryonic stem cell differentiation and studied their interactions with nucleosomes and co-occurring TFs by enhanced chromatin occupancy (EChO), a computational strategy for classifying TF interactions with chromatin. EChO shows that multiple individual TFs can employ either direct DNA binding or "pioneer" nucleosome binding at different enhancer targets. Nucleosome binding is not exclusively confined to inaccessible chromatin but rather correlated with local binding of other TFs and degeneracy at key bases in the pioneer factor target motif responsible for direct DNA binding. Our strategy reveals a dynamic exchange of TFs at enhancers across developmental time that is aided by pioneer nucleosome binding.

OR26-4 Genome-Wide Binding Profile of Phosphorylated Estrogen Receptor Reveals Association with Direct DNA Binding

Post-translational modifications are key regulators of protein function, providing signals that alter protein interactions and activity. Phosphorylation of estrogen receptor-α (ER) at serine 118 (pS118-ER) occurs in response to multiple stimuli and is involved in modulating ER-dependent gene transcription. While the effects of pS118 on ER-DNA interactions have been investigated at specific genomic sites, the genome-wide binding profile (cistrome) of pS118-ER remains to be studied. To determine and characterize the pS118-ER cistrome, chromatin immunoprecipitation sequencing (ChIP-seq) was performed on both pS118-ER and ER and their binding profiles were compared. A subset of ER sites was found to be occupied by pS118-ER and although these sites were not found to be enriched in promoter regions relative to all ER sites, the pS118-ER sites were associated with the active enhancer mark H3K27ac as well as genes upregulated, but not downregulated, by estrogen. Additionally, pS118-ER sites were found to be enriched in the DNA binding motif for GRHL2 as well as the estrogen response element (ERE) relative to all ER sites. Utilizing a DNA binding microarray, pS118-ER was found to be more commonly associated with direct DNA binding events compared to indirect binding events. These results suggest a role for pS118-ER at active enhancers and as a regulator of direct ER-DNA interactions. Prevention of this phosphorylation event could serve as a potential treatment for ER-positive breast cancers by reducing the transcriptional activity of ER and decreasing its occupancy on DNA.

Transcriptional regulatory activity of the cereal grain bZip protein TaABF1 can be either stimulated or inhibited by phosphorylation

AbstractThe wheat bZip transcription factor TaABF1 mediates both abscisic acid (ABA)-induced and ABA-suppressed gene expression. As levels of TaABF1 protein do not change in response to ABA, and TaABF1 is in a phosphorylated statein vivo, we investigated whether TaABF1 could be regulated at the post-translational level. In bombarded aleurone cells, a TaABF1 protein carrying phosphomimetic mutations (serine to aspartate) at four sites (S36D, S37D, S113D, S115D) was three to five times more potent than wild-type TaABF1 in activatingHVA1, an ABA-responsive gene. The phosphomimetic mutations also increased the ability of TaABF1 to downregulate the ABA-suppressed geneAmy32b. These findings strongly suggest that phosphorylation at these sites increases the transcriptional regulatory activity of TaABF1. In contrast to the activation observed by the quadruple serine to aspartate mutation, a single S113D mutation completely eliminated the ability of TaABF1 to upregulateHVA1or downregulateAmy32b. Thus phosphorylation of TaABF1 can either stimulate or inhibit the activity of TaABF1 in regulating downstream genes, depending on the site and pattern of phosphorylation. Mutation of S318 and S322 (in the bZIP domain) eliminated the ability of TaABF1 to activateHVA1, but had no effect on the ability of TaABF1 to downregulateAmy32b, suggesting that TaABF1 repressesAmy32bexpression through a mechanism other than direct DNA binding. An important step towards understanding how ABA and gibberellin (GA) signals are integrated through TaABF1 phosphorylation to regulate downstream gene expression is to clarify the effects of those hormones on the expression of specific genes. In contrast to some other ABA-induced genes, we found thatHVA1induction by ABA or TaABF1 is not inhibited by GA.

The HMG box transcription factor HBP1: a cell cycle inhibitor at the crossroads of cancer signaling pathways.

HMG box protein 1 (HBP1) is a transcription factor and a potent cell cycle inhibitor in normal and cancer cells. HBP1 activates or represses the expression of different cell cycle genes (such as CDKN2A, CDKN1A, and CCND1) through direct DNA binding, cofactor recruitment, chromatin remodeling, or neutralization of other transcription factors. Among these are LEF1, TCF4, and MYC in the WNT/beta-catenin pathway. HBP1 also contributes to oncogenic RAS-induced senescence and terminal cell differentiation. Collectively, these activities suggest a tumor suppressor function. However, HBP1 is not listed among frequently mutated cancer driver genes. Nevertheless, HBP1 expression is lower in several tumor types relative to matched normal tissues. Several micro-RNAs, such as miR-155, miR-17-92, and miR-29a, dampen HBP1 expression in cancer cells of various origins. The phosphatidylinositol-3 kinase (PI3K)/AKT pathway also inhibits HBP1 transcription by preventing FOXO binding to the HBP1 promoter. In addition, AKT directly phosphorylates HBP1, thereby inhibiting its transcriptional activity. Taken together, these findings place HBP1 at the center of a network of micro-RNAs and oncoproteins that control cell proliferation. In this review, we discuss our current understanding of HBP1 function in human physiology and diseases.

Autoimmune Regulator Enhanced the Expression of Caspase-3 and Did Not Induce Massive Germ Cell Apoptosis in GC1-Spg Cells.

AIRE is known for its involvement in autoreactive T-cell deletion in thymic epithelium. Though extrathymic expression of AIRE is well documented, the functional relevance of AIRE in non-thymus tissues is emerging. AIRE is expressed in neonatal and adult testis, and has been implicated in sporadic germ cell apoptosis in developing testis. In this study we examined whether AIRE has any role in inducing apoptosis in cultured spermatogonial cells. We over-expressed AIRE or CARD domain of AIRE in GC1-spg cells and evaluated its impact on cell cycle using fluorescence activated cell sorting following Hoechst 33342 staining. Apoptosis was assayed using Annexin-V staining. Caspase-3 cleavage was assessed on western blots and caspase-3 expression was quantitated using realtime PCR. We report that C18-4 cells which are derived from Type A spermatogonia expressed AIRE, while GC1-spg which is closer to Type B spermatogonia was negative for AIRE expression. Overexpression of AIRE or CARD domain of AIRE induced Caspase-3 expression in GC1-spg cells. Silencing of AIRE in C18-4 cells inhibited Caspase-3 expression. When overexpressed, AIRE and CARD brought about a very negligible increase in germ cell death and resulted in altered cell cycle pattern with a reduction in G1 phase. This was not associated with any increase in activation of Caspase-3. We conclude that the CARD domain of AIRE enhances caspase-3 expression through possible direct DNA binding and triggers non-apoptotic downstream signaling in cultured spermatogonial cells.

NRF2 Mediates Epigenetic Changes in DNA and Chromatin Structure to Regulate γ-Globin Gene Expression in a Human βYAC Transgenic Mouse Model

NRF2 is the master regulator for the cellular anti-oxidative stress response and previously shown to activate γ-globin gene expression in human erythroid progenitor cells. The goal of this study was to expand on these findings by exploring the in vivo function of NRF2 using the human β-globin locus YAC transgenic (βYAC) mouse carrying the entire 248kb human β-globin locus (HBB). We observed that NRF2 activation by chronic dimethyl fumarate treatment of βYAC mice, induced human γ-globin gene expression, but had no effect on the adult β-globin gene. Subsequently in a novel βYAC/NRF2 knockout mouse model established in our laboratory, we demonstrated that NRF2 loss increased mouse erythroid CD71 levels while reducing human γ-globin gene expression during erythropoiesis in mouse embryonic E13.5 and E18.5 day fetal livers and peripheral blood. Furthermore, the ability of dimethyl fumarate to induce γ-globin gene expression was abolished after NRF2 loss. By western blot analysis of nuclear protein, we confirmed that part of the mechanism of globin gene regulation by NRF2 loss involves a decline of global histone H3 lysine 4 trimethylation levels, without changing histone acetylation. Interestingly, NRF2 loss decreased the protein levels of the DNA methylcytosine dioxygenases including TET1, TET2 and TET3. Analysis of DNA methylation/hydroxymethylation levels by DNA dot-blot assay of mouse E13.5 fetal livers isolated from βYAC/NRF2 knockout mice, showed inhibition of genome wide DNA hydroxymethylation, while DNA methylation was not affected. In addition, DNA immunoprecipitation confirmed decreased hydroxymethylation in the HBB locus control region (LCR) enhancer and γ-globin gene region. These data suggest an essential role of NRF2 in modifying chromatin structure and assembling transcription complexes to regulate γ-globin gene expression. ChIP assay to assess in vivo DNA-protein interactions showed decreased associations of histone H3 lysine 4 monomethylation and trimethylation, TATA-binding protein and RNA polymerase II to the LCR and γ-globin promoter after NRF2 loss. Final studies were conducted to evaluate long-range chromatin interactions between the LCR and individual globin genes by chromosome conformation capture assay. We observed decreased interactions between the LCR and γ-globin gene promoter region after NRF2 loss while interactions in the adult β-globin was not affected suggesting NRF2 preferentially mediates γ-globin gene regulation. In conclusion, our data suggest that NRF2 alters γ-globin expression through epigenetic DNA/histone modifications in addition to direct DNA binding. Therefore, activation of NRF2 expression using small chemical compounds is an innovative strategy to induce γ-globin gene transcription for the treatment of β-hemoglobinopathies. This work was supported by funding from the National Heart, Lung, and Blood Institute to XZ through the Hemoglobinopathy Translational Research Skills Core component of U01 grant HL117684 and R01 grant HL069234 to BSP. DisclosuresNo relevant conflicts of interest to declare.

Zeb1 potentiates genome-wide gene transcription with Lef1 to promote glioblastoma cell invasion.

Glioblastoma is the most common and aggressive brain tumor, with a subpopulation of stem-like cells thought to mediate its recurring behavior and therapeutic resistance. The epithelial-mesenchymal transition (EMT) inducing factor Zeb1 was linked to tumor initiation, invasion, and resistance to therapy in glioblastoma, but how Zeb1 functions at molecular level and what genes it regulates remain poorly understood. Contrary to the common view that EMT factors act as transcriptional repressors, here we show that genome-wide binding of Zeb1 associates with both activation and repression of gene expression in glioblastoma stem-like cells. Transcriptional repression requires direct DNA binding of Zeb1, while indirect recruitment to regulatory regions by the Wnt pathway effector Lef1 results in gene activation, independently of Wnt signaling. Amongst glioblastoma genes activated by Zeb1 are predicted mediators of tumor cell migration and invasion, including the guanine nucleotide exchange factor Prex1, whose elevated expression is predictive of shorter glioblastoma patient survival. Prex1 promotes invasiveness of glioblastoma cells invivo highlighting the importance of Zeb1/Lef1 gene regulatory mechanisms in gliomagenesis.

DNA activates the Nse2/Mms21 SUMO E3 ligase in the Smc5/6 complex.

Modification of chromosomal proteins by conjugation to SUMO is a key step to cope with DNA damage and to maintain the integrity of the genome. The recruitment of SUMO E3 ligases to chromatin may represent one layer of control on protein sumoylation. However, we currently do not understand how cells upregulate the activity of E3 ligases on chromatin. Here we show that the Nse2 SUMO E3 in the Smc5/6 complex, a critical player during recombinational DNA repair, is directly stimulated by binding to DNA Activation of sumoylation requires the electrostatic interaction between DNA and a positively charged patch in the ARM domain of Smc5, which acts as a DNA sensor that subsequently promotes a stimulatory activation of the E3 activity in Nse2. Specific disruption of the interaction between the ARM of Smc5 and DNA sensitizes cells to DNA damage, indicating that this mechanism contributes to DNA repair. These results reveal a mechanism to enhance a SUMO E3 ligase activity by direct DNA binding and to restrict sumoylation in the vicinity of those Smc5/6-Nse2 molecules engaged on DNA.

Proposed 3-D Structure of a Critical Protein-Protein Interaction Required for Transcription of the IL1B Gene Coding for Human Interleukin 1β

Abstract We previously reported that expression of the human IL1B gene coding for Interleukin 1β in LPS-activated macrophages requires long-range chromatin looping mediated by a mutual DNA binding domain (DBD) interaction between two transcription factors, Spi-1/PU.1 (Spi1) at the promoter and C/EBPβ at a far-upstream enhancer. We have now combined preexisting X-ray structures and advanced computational techniques with additional biochemical and cell-based studies in order to further investigate the structure of this critical interaction. In vitro binding was used to generate a surface interaction map for the two proteins that suggested an association similar to that of a C/EBPβ·Myb X-ray structure reported by others. The distinction is that, in addition to a C/EBPβ leucine zipper interaction similar to that with the Myb DBD, there is an interaction between a carboxyl-tail extension of one of the C/EBPβ leucine zipper monomers with Spi1. This is consistent with our previous report that the carboxyl-tail of C/EBPβ is critical for association with arginine 232 within a pocket at one end of the Spi1 DNA recognition helix. L-arginine, a known anti-inflammatory, was computationally identified to bind in this interaction pocket. In vivo studies of LPS-activated THP-1 macrophages treated with L-arginine decreased IL1B transcription in parallel with a reduction in C/EBPβ association with Spi1 on the IL1B promoter. No significant change in direct binding of Spi1 and C/EBPβ to DNA was observed. These results suggest that L-arginine inhibits IL1B gene expression by competitive inhibition of C/EBPβ binding to Spi1 in the absence of any effect on direct DNA binding for either factor, and support the predicted structure of the C/EBPβ·Spi1 interaction.

StpA and Hha stimulate pausing by RNA polymerase by promoting DNA-DNA bridging of H-NS filaments.

In enterobacteria, AT-rich horizontally acquired genes, including virulence genes, are silenced through the actions of at least three nucleoid-associated proteins (NAPs): H-NS, StpA and Hha. These proteins form gene-silencing nucleoprotein filaments through direct DNA binding by H-NS and StpA homodimers or heterodimers. Both linear and bridged filaments, in which NAPs bind one or two DNA segments, respectively, have been observed. Hha can interact with H-NS or StpA filaments, but itself lacks a DNA-binding domain. Filaments composed of H-NS alone can inhibit transcription initiation and, in the bridged conformation, slow elongating RNA polymerase (RNAP) by promoting backtracking at pause sites. How the other NAPs modulate these effects of H-NS is unknown, despite evidence that they help regulate subsets of silenced genes in vivo (e.g. in pathogenicity islands). Here we report that Hha and StpA greatly enhance H-NS-stimulated pausing by RNAP at 20°C. StpA:H-NS or StpA-only filaments also stimulate pausing at 37°C, a temperature at which Hha:H-NS or H-NS-only filaments have much less effect. In addition, we report that both Hha and StpA greatly stimulate DNA–DNA bridging by H-NS filaments. Together, these observations indicate that Hha and StpA can affect H-NS-mediated gene regulation by stimulating bridging of H-NS/DNA filaments.

Odd-Paired: The Drosophila Zic Gene.

Zinc finger in the cerebellum (Zic) proteins are a family of transcription factors with multiple roles during development, particularly in neural tissues. The founding member of the Zic family is the Drosophila odd-paired (opa) gene. The Opa protein has a DNA binding domain containing five Cys2His2-type zinc fingers and has been shown to act as a sequence-specific DNA binding protein. Opa has significant homology to mammalian Zic1, Zic2, and Zic3 within the zinc finger domain and in two other conserved regions outside that domain. opa was initially identified as a pair-rule gene, part of the hierarchy of genes that establish the segmental body plan of the early Drosophila embryo. However, its wide expression pattern during embryogenesis indicates it plays additional roles. Embryos deficient in opa die before hatching with aberrant segmentation but also with defects in larval midgut formation. Post-embryonically, opa plays important roles in adult head development and circadian rhythm. Based on extensive neural expression, opa is predicted to be involved in many aspects of neural development and behavior, like other proteins of the Zic family. Consensus DNA binding sites have been identified for Opa and have been shown to activate transcription in vivo. However, there is evidence Opa may serve as a transcriptional regulator in the absence of direct DNA binding, as has been seen for other Zic proteins.

Glucocorticoids Suppress the Browning of Adipose Tissue via miR-19b in Male Mice

Physiological levels of glucocorticoids (GCs) are required for proper metabolic control, and excessive GC action has been linked to a variety of pandemic metabolic diseases. MicroRNA (miRNA)-19b plays a critical role in the pathogenesis of GC-induced metabolic diseases. This study explored the potential of miRNA-based therapeutics targeting adipose tissue. Our results showed that overexpressed miR-19b in stromal vascular fraction (SVF) cells derived from subcutaneous adipose tissue had the same effects as dexamethasone (DEX) treatment on the inhibition of adipose browning and oxygen consumption rate. The inhibition of miR-19b blocked DEX-mediated suppression of the expression of browning marker genes as well as the oxygen consumption rate in differentiated SVF cells derived from subcutaneous and brown adipose tissue. Overexpressed miR-19b in SVF cells derived from brown adipose tissue had the same effects as DEX treatment on the inhibition of brown adipose differentiation and energy expenditure. Glucocorticoids transcriptionally regulate the expression of miR-19b via a GC receptor-mediated direct DNA binding mechanism. This study confirmed that miR-19b is an essential target for GC-mediated control of adipose tissue browning. It is hoped that the plasticity of the adipose organ can be exploited in the next generation of therapeutic strategies to combat the increasing incidence of metabolic diseases, including obesity and diabetes.

Retinoid X receptor suppresses a metastasis-promoting transcriptional program in myeloid cells via a ligand-insensitive mechanism

Retinoid X receptor (RXR) regulates several key functions in myeloid cells, including inflammatory responses, phagocytosis, chemokine secretion, and proangiogenic activity. Its importance, however, in tumor-associated myeloid cells is unknown. In this study, we demonstrate that deletion of RXR in myeloid cells enhances lung metastasis formation while not affecting primary tumor growth. We show that RXR deficiency leads to transcriptomic changes in the lung myeloid compartment characterized by increased expression of prometastatic genes, including important determinants of premetastatic niche formation. Accordingly, RXR-deficient myeloid cells are more efficient in promoting cancer cell migration and invasion. Our results suggest that the repressive activity of RXR on prometastatic genes is mediated primarily through direct DNA binding of the receptor along with nuclear receptor corepressor (NCoR) and silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) corepressors and is largely unresponsive to ligand activation. In addition, we found that expression and transcriptional activity of RXRα is down-modulated in peripheral blood mononuclear cells of patients with lung cancer, particularly in advanced and metastatic disease. Overall, our results identify RXR as a regulator in the myeloid cell-assisted metastatic process and establish lipid-sensing nuclear receptors in the microenvironmental regulation of tumor progression.

Abstract 2434: LEF1 negatively regulates differentiation of HCC by activation of Notch signaling pathway

Abstract Lymphoid enhancer binding factor-1 (LEF1), an essential regulatory protein, plays a crucial role on self-renewal and differentiation properties of several cancers, contributing to tumor malignancy degree and prognosis. However, its molecular mechanisms to promote tumor differentiation still keep unknown, especially in hepatocellular carcinoma (HCC). In this study, by analyzing the transcriptome datasets and our qPCR result of clinical HCC samples, we found that LEF is frequently overexpressed in HCCs and significantly associated with HCC differentiation (p=0.015). In addition, functional study showed that LEF1 could enhance the cell proliferation, matrigel invasion, transwell migration, as well as self-renewal abilities both in vivo and in vitro experiments. We also demonstrated LEF1 is closely related to Notch intracellular domain (NICD), and revealed several putative sites as the targets for LEF1 activating Notch signaling pathway. LEF1 could induce the Notch signaling via transcriptionally up-regulate Notch expression through direct DNA binding. The expressions of several downstream members in Notch signaling pathway including Notch1 and Notch2 were subsequently increased. Moreover, LEF1 could down-regulate various differentiation markers and over-express tumor stemness related genes. Together, LEF1 is a novel oncogene that show a crucial role in the tumorigenesis and progression of HCC via activating Notch signaling pathway and inhibiting the differentiation and stemness of HCC, resulting in poor pathologic differentiation and leads to high degree of malignant. All the findings suggest a novel treatment possibility and a new target for liver stem cell eradication. LEF1 could be investigated for clinical early diagnosis and prognosis prediction of HCC patients and improving the development of new therapy against HCC. Note: This abstract was not presented at the meeting. Citation Format: Shuo FANG, Lei LI, MIng LIU, Janice Tsang, Xinyuan GUAN. LEF1 negatively regulates differentiation of HCC by activation of Notch signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2434. doi:10.1158/1538-7445.AM2017-2434