Identification of a small-molecule compound that inhibits homodimerization of oncogenic NAC1 protein and sensitizes cancer cells to anticancer agents.

Nucleus accumbens-associated protein-1 (NAC1) is a transcriptional repressor encoded by the NACC1 gene, which is amplified and overexpressed in various human cancers and plays critical roles in tumor development, progression, and drug resistance. NAC1 has therefore been explored as a potential therapeutic target for managing malignant tumors. However, effective approaches for effective targeting of this nuclear protein remain elusive. In this study, we identified a core unit consisting of Met7 and Leu90 in NAC1's N-terminal domain (amino acids 1-130), which is critical for its homodimerization and stability. Furthermore, using a combination of computational analysis of the NAC1 dimerization interface and high-throughput screening (HTS) for small molecules that inhibit NAC1 homodimerization, we identified a compound (NIC3) that selectively binds to the conserved Leu-90 of NAC1 and prevents its homodimerization, leading to proteasomal NAC1 degradation. Moreover, we demonstrate that NIC3-mediated down-regulation of NAC1 protein sensitizes drug-resistant tumor cells to conventional chemotherapy and enhances the antimetastatic effect of the antiangiogenic agent bevacizumab both in vitro and in vivo These results suggest that small-molecule inhibitors of NAC1 homodimerization may effectively sensitize cancer cells to some anticancer agents and that NAC1 homodimerization could be further explored as a potential therapeutic target in the development of antineoplastic agents.

Copper levels affect targeting of hypoxia-inducible factor 1α to the promoters of hypoxia-regulated genes.

Hypoxia-inducible factor 1α (HIF-1α) is a transcription factor that regulates cellular responses to hypoxia. It controls the expression of both BCL2/adenovirus E1B 19-kDa protein-interacting protein 3 (BNIP3) and insulin-like growth factor 2 (IGF2). Previous studies have demonstrated that in hypoxia, copper is required for the expression of BNIP3 but not for that of IGF2 Here, using ChIP assays, computational analyses, luciferase reporter assays, and real-time quantitative RT-PCR, we sought to better understand how copper regulates the differential target gene selectivity of HIF-1α. Human umbilical vein endothelial cells (HUVECs) were exposed to CoCl2 or hypoxia conditions to increase HIF-1α accumulation. The binding of HIF-1α to hypoxia-responsive element (HRE) sites in the BNIP3 or IGF2 gene promoter in high- or low-copper conditions was examined. Our analyses revealed three and two potential HRE sites in the BNIP3 and IGF2 promoters, respectively. We identified that HRE (-412/-404) in the BNIP3 promoter and HRE (-354/-347) in the IGF2 promoter are the critical binding sites of HIF-1α. Tetraethelenepentamine (TEPA)-mediated reduction in copper concentration did not affect hypoxia- or CoCl2-induced HIF-1α accumulation. However, the copper reduction did suppress the binding of HIF-1α to the HRE (-412/-404) in BNIP3 but not the binding of HIF-1α to the HRE (-354/-347) in IGF2 In summary, our findings uncovered the mechanistic basis for differential HIF-1α-mediated regulation of BNIP3 and IGF2, indicating that copper regulates target gene selectivity of HIF-1α at least in part by affecting HIF-1α binding to its cognate HRE in the promoters of these two genes.

Transcription factor Sp2 potentiates binding of the TALE homeoproteins Pbx1:Prep1 and the histone-fold domain protein Nf-y to composite genomic sites.

Different transcription factors operate together at promoters and enhancers to regulate gene expression. Transcription factors either bind directly to their target DNA or are tethered to it by other proteins. The transcription factor Sp2 serves as a paradigm for indirect genomic binding. It does not require its DNA-binding domain for genomic DNA binding and occupies target promoters independently of whether they contain a cognate DNA-binding motif. Hence, Sp2 is strikingly different from its closely related paralogs Sp1 and Sp3, but how Sp2 recognizes its targets is unknown. Here, we sought to gain more detailed insights into the genomic targeting mechanism of Sp2. ChIP-exo sequencing in mouse embryonic fibroblasts revealed genomic binding of Sp2 to a composite motif where a recognition sequence for TALE homeoproteins and a recognition sequence for the trimeric histone-fold domain protein nuclear transcription factor Y (Nf-y) are separated by 11 bp. We identified a complex consisting of the TALE homeobox protein Prep1, its partner PBX homeobox 1 (Pbx1), and Nf-y as the major partners in Sp2-promoter interactions. We found that the Pbx1:Prep1 complex together with Nf-y recruits Sp2 to co-occupied regulatory elements. In turn, Sp2 potentiates binding of Pbx1:Prep1 and Nf-y. We also found that the Sp-box, a short sequence motif close to the Sp2 N terminus, is crucial for Sp2's cofactor function. Our findings reveal a mechanism by which the DNA binding-independent activity of Sp2 potentiates genomic loading of Pbx1:Prep1 and Nf-y to composite motifs present in many promoters of highly expressed genes.

Proteasomal Degradation of the EWS-FLI1 Fusion Protein Is Regulated by a Single Lysine Residue.

E-26 transformation-specific (ETS) proteins are transcription factors directing gene expression through their conserved DNA binding domain. They are implicated as truncated forms or interchromosomal rearrangements in a variety of tumors including Ewing sarcoma, a pediatric tumor of the bone. Tumor cells express the chimeric oncoprotein EWS-FLI1 from a specific t(22;11)(q24;12) translocation. EWS-FLI1 harbors a strong transactivation domain from EWSR1 and the DNA-binding ETS domain of FLI1 in the C-terminal part of the protein. Although Ewing cells are crucially dependent on continuous expression of EWS-FLI1, its regulation of turnover has not been characterized in detail. Here, we identify the EWS-FLI1 protein as a substrate of the ubiquitin-proteasome system with a characteristic polyubiquitination pattern. Using a global protein stability approach, we determined the half-life of EWS-FLI1 to lie between 2 and 4 h, whereas full-length EWSR1 and FLI1 were more stable. By mass spectrometry, we identified two ubiquitin acceptor lysine residues of which only mutation of Lys-380 in the ETS domain of the FLI1 part abolished EWS-FLI1 ubiquitination and stabilized the protein posttranslationally. Expression of this highly stable mutant protein in Ewing cells while simultaneously depleting the endogenous wild type protein differentially modulates two subgroups of target genes to be either EWS-FLI1 protein-dependent or turnover-dependent. The majority of target genes are in an unaltered state and cannot be further activated. Our study provides novel insights into EWS-FLI1 turnover, a critical pathway in Ewing sarcoma pathogenesis, and lays new ground to develop novel therapeutic strategies in Ewing sarcoma.

EVI1 Interferes with Myeloid Maturation via Transcriptional Repression of Cebpa, via Binding to Two Far Downstream Regulatory Elements.

One mechanism by which oncoproteins work is through perturbation of cellular maturation; understanding the mechanisms by which this occurs can lead to the development of targeted therapies. EVI1 is a zinc finger oncoprotein involved in the development of acute myeloid leukemia; previous work has shown it to interfere with the maturation of granulocytes from immature precursors. Here we investigate the mechanism by which that occurs, using an immortalized hematopoietic progenitor cell line, EML-C1, as a model system. We document that overexpression of EVI1 abrogates retinoic acid-induced maturation of EML cells into committed myeloid cells, a process that can be documented by the down-regulation of stem cell antigen-1 and acquisition of responsiveness to granulocyte-macrophage colony-stimulating factor. We show that this requires DNA binding capacity of EVI1, suggesting that downstream target genes are involved. We identify the myeloid regulator Cebpa as a target gene and identify two EVI1 binding regions within evolutionarily conserved enhancer elements at +35 and +37 kb relative to the gene. EVI1 can strongly suppress Cebpa transcription, and add-back of Cebpa into EVI1-expressing EML cells partially corrects the block in maturation. We identify the DNA sequences to which EVI1 binds at +35 and +37 kb and show that mutation of one of these releases Cebpa from EVI1-induced suppression. We observe a more complex picture in primary bone marrow cells, where EVI1 suppresses Cebpa in stem cells but not in more committed progenitors. Our data thus identify a regulatory node by which EVI1 contributes to leukemia, and this represents a possible therapeutic target for treatment of EVI1-expressing leukemia.

Mitochondrial Activity in Human White Adipocytes Is Regulated by the Ubiquitin Carrier Protein 9/microRNA-30a Axis.

The acquisition of beige adipocyte features by white fat cells corresponds to protection against obesity-induced metabolic diseases in humans and animal models of type 2 diabetes. In adipose tissue, expression of the E2 small ubiquitin-like modifier ligase ubiquitin carrier protein 9 (Ubc9) is positively correlated with markers of insulin resistance and corresponds with impaired browning of human white adipocytes. However, the molecular regulation of Ubc9 expression in adipocytes and other cells remains unclear. In this study, we demonstrate that the mRNA and protein expression of Ubc9 are regulated by the microRNA miRNA-30a (miR-30a) in human subcutaneous adipocytes. Ubc9 and miR-30a exhibit inverse expression in adipose tissue, with miR-30a robustly elevated in brown fat. Depletion of Ubc9 by siRNA or enforced expression of a miR-30a mimic augments mitochondrial volume and respiration in human white adipocytes, reflecting features of brown fat cells. Furthermore, Ubc9 depletion induces a brown fat gene program in human subcutaneous adipocytes. Induction of the beige-selective gene program corresponds to stabilization of the PR domain-containing 16 (PRDM16) protein, an obligate transcriptional regulator of the brown/beige fat metabolic program in white adipocytes that interacts with Ubc9. Taken together, our data demonstrate a previously unappreciated molecular axis that controls browning of human white adipocytes.

DNA Methylation Affects the SP1-regulated Transcription of FOXF2 in Breast Cancer Cells.

FOXF2 (forkhead box F2) is a mesenchyme-specific transcription factor that plays a critical role in tissue homeostasis through the maintenance of epithelial polarity. In a previous study, we demonstrated that FOXF2 is specifically expressed in basal-like breast cancer (BLBC) cells and functions as an epithelial-mesenchymal transition suppressor. FOXF2 deficiency enhances the metastatic ability of BLBC cells through activation of the epithelial-mesenchymal transition program, but reduces cell proliferation. In this study, we demonstrate that CpG island methylation of the FOXF2 proximal promoter region is involved in the regulatory mechanism of the subtype-specific expression of FOXF2 in breast cancer cells. DNMT1, DNMT3A, and DNMT3B commonly or individually contributed to this DNA methylation in different breast cancer cells. SP1 regulated the transcriptional activity of FOXF2 through direct binding to the proximal promoter region, whereas this binding was abrogated through DNA methylation. FOXF2 mediated the SP1-regulated suppression of progression and promotion of proliferation of non-methylated BLBC cells. Thus, we conclude that the subtype-specific expression and function of FOXF2 in breast cancer cells are regulated through the combined effects of DNA methylation and SP1 transcriptional regulation.

Structural Basis for Small Molecule NDB (N-Benzyl-N-(3-(tert-butyl)-4-hydroxyphenyl)-2,6-dichloro-4-(dimethylamino) Benzamide) as a Selective Antagonist of Farnesoid X Receptor α (FXRα) in Stabilizing the Homodimerization of the Receptor.

Farnesoid X receptor α (FXRα) as a bile acid sensor plays potent roles in multiple metabolic processes, and its antagonist has recently revealed special interests in the treatment of metabolic disorders, although the underlying mechanisms still remain unclear. Here, we identified that the small molecule N-benzyl-N-(3-(tert-butyl)-4-hydroxyphenyl)-2,6-dichloro-4-(dimethylamino) benzamide (NDB) functioned as a selective antagonist of human FXRα (hFXRα), and the crystal structure of hFXRα ligand binding domain (hFXRα-LBD) in complex with NDB was analyzed. It was unexpectedly discovered that NDB induced rearrangements of helix 11 (H11) and helix 12 (H12, AF-2) by forming a homodimer of hFXRα-LBD, totally different from the active conformation in monomer state, and the binding details were further supported by the mutation analysis. Moreover, functional studies demonstrated that NDB effectively antagonized the GW4064-stimulated FXR/RXR interaction and FXRα target gene expression in primary mouse hepatocytes, including the small heterodimer partner (SHP) and bile-salt export pump (BSEP); meanwhile, administration of NDB to db/db mice efficiently decreased the gene expressions of phosphoenolpyruvate carboxykinase (PEPCK), glucose 6-phosphatase (G6-pase), small heterodimer partner, and BSEP. It is expected that our first analyzed crystal structure of hFXRα-LBD·NDB will help expound the antagonistic mechanism of the receptor, and NDB may find its potential as a lead compound in anti-diabetes research.

The Active Form of Vitamin D Transcriptionally Represses Smad7 Signaling and Activates Extracellular Signal-regulated Kinase (ERK) to Inhibit the Differentiation of a Inflammatory T Helper Cell Subset and Suppress Experimental Autoimmune Encephalomyelitis.

The ability of the active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), to transcriptionally modulate Smads to inhibit Th17 differentiation and experimental autoimmune encephalomyelitis (EAE) has not been adequately studied. This study reports modulation of Smad signaling by the specific binding of the VDR along with its heterodimeric partner RXR to the negative vitamin D response element on the promoter of Smad7, which leads to Smad7 gene repression. The vitamin D receptor-mediated increase in Smad3 expression partially explains the IL10 augmentation seen in Th17 cells. Furthermore, the VDR axis also modulates non-Smad signaling by activating ERK during differentiation of Th17 cells, which inhibits the Th17-specific genes il17a, il17f, il22, and il23r. In vivo EAE experiments revealed that, 1,25(OH)2D3 suppression of EAE correlates with the Smad7 expression in the spleen and lymph nodes. Furthermore, Smad7 expression also correlates well with IL17 and IFNγ expression in CNS infiltered inflammatory T cells. We also observed similar gene repression of Smad7 in in vitro differentiated Th1 cells when cultured in presence of 1,25(OH)2D3. The above canonical and non-canonical pathways in part address the ability of 1,25(OH)2D3-VDR to inhibit EAE.

Steroid receptor coactivator-2 is a dual regulator of cardiac transcription factor function.

We have previously demonstrated the potential role of steroid receptor coactivator-2 (SRC-2) as a co-regulator in the transcription of critical molecules modulating cardiac function and metabolism in normal and stressed hearts. The present study seeks to extend the previous information by demonstrating SRC-2 fulfills this role by serving as a critical coactivator for the transcription and activity of critical transcription factors known to control cardiac growth and metabolism as well as in their downstream signaling. This knowledge broadens our understanding of the mechanism by which SRC-2 acts in normal and stressed hearts and allows further investigation of the transcriptional modifications mediating different types and degrees of cardiac stress. Moreover, the genetic manipulation of SRC-2 in this study is specific for the heart and thereby eliminating potential indirect effects of SRC-2 deletion in other organs. We have shown that SRC-2 is critical to transcriptional control modulated by MEF2, GATA-4, and Tbx5, thereby enhancing gene expression associated with cardiac growth. Additionally, we describe SRC-2 as a novel regulator of PPARα expression, thus controlling critical steps in metabolic gene expression. We conclude that through regulation of cardiac transcription factor expression and activity, SRC-2 is a critical transcriptional regulator of genes important for cardiac growth, structure, and metabolism, three of the main pathways altered during the cardiac stress response.

FLI1 mediates the selective expression of hypoxia-inducible factor 1 target genes in endothelial cells under hypoxic conditions.

The selective expression of hypoxia-inducible factor (HIF) target genes in different physiological and pathological environments forms the basis for cellular adaptation to hypoxia in development and disease. Several E26 transformation-specific (ETS) transcription factors have been shown to specifically regulate the expression of a subset of HIF-2 target genes. However, it is unknown whether there are ETS factors that specifically regulate hypoxia-induced HIF-1 target genes. The present study was undertaken to explore whether friend leukemia integration 1 (FLI1), an ETS transcription factor, regulates the expression of HIF-1 target genes. To investigate this possibility, EA.hy926 cells were exposed to 20% O2 (normoxia) or 1% O2 (hypoxia). Western blotting, immunofluorescence staining, and RT-qPCR revealed that FLI1 mRNA and protein levels increased slightly and that the FLI1 protein co-localized with HIF-1α in the nucleus under hypoxic conditions. Further analysis showed that, in the absence of FLI1, the hypoxia-mediated induction of HIF-1 target genes was selectively inhibited. The results from immunoprecipitation and luciferase reporter assays indicated that FLI1 cooperates with HIF-1α and is required for the transcriptional activation of a subset of HIF-1 target genes with a core promoter region containing FBS in proximity to a functional hypoxia response element (HRE). Furthermore, ChIP analysis further confirmed the direct interaction between FLI1 and the promoter region of FLI1-dependent HIF-1 target genes under hypoxia. Together, this study demonstrates that FLI1 is involved in the transactivation of certain HIF-1 target genes in endothelial cells under hypoxic conditions.