Co-activation of WT1 and AP-1 proteins on WT1 gene promoter to induce WT1 gene expression in K562 cells.

Earlier studies have revealed one function of the inhibitory mechanism of curcumin. Activating PKCα induces WT1 gene expression via signalling through downstream JNK and c-JUN. In the present study, the effect of c-JUN/AP-1 binding and transcriptional regulation of the WT1 gene promoter was investigated in K562 leukaemic cells. The non-cytotoxic dose (IC20 values) of curcumin (WT1 and AP-1 inhibitors) was employed to examine its effect on WT1 gene-mediated WT1 and AP-1 protein expression. Non-cytotoxic doses of both tanshinone IIA (AP-1 DNA-binding inhibitor) and SP600125 (JNK inhibitor) were used to test the role of c-JUN/AP-1 in WT1 gene expression. Curcumin, tanshinone IIA, and SP600125 inhibited WT1 protein expression in a dose-dependent manner (5-15 µM) at 24 h as shown by immunoblotting. A ChIP assay showed that curcumin and tanshinone IIA inhibited AP-1 and WT1 binding to the proximal WT1 promoter (-301 bp), and a luciferase reporter assay showed that the WT1 luciferase gene reporter activity was decreased after curcumin, tanshinone IIA, and SP600126 treatments. Furthermore, depletion of c-JUN abrogated WT1 gene expression. In summary, AP-1 contributes to the WT1 autoregulation of WT1 gene expression in leukaemic K562 cells.

Coordination of m(6)A mRNA Methylation and Gene Transcription by ZFP217 Regulates Pluripotency and Reprogramming.

Epigenetic and epitranscriptomic networks have important functions in maintaining the pluripotency of embryonic stem cells (ESCs) and somatic cell reprogramming. However, the mechanisms integrating the actions of these distinct networks are only partially understood. Here we show that the chromatin-associated zinc finger protein 217 (ZFP217) coordinates epigenetic and epitranscriptomic regulation. ZFP217 interacts with several epigenetic regulators, activates the transcription of key pluripotency genes, and modulates N6-methyladenosine (m(6)A) deposition on their transcripts by sequestering the enzyme m(6)A methyltransferase-like 3 (METTL3). Consistently, Zfp217 depletion compromises ESC self-renewal and somatic cell reprogramming, globally increases m(6)A RNA levels, and enhances m(6)A modification of the Nanog, Sox2, Klf4, and c-Myc mRNAs, promoting their degradation. ZFP217 binds its own target gene mRNAs, which are also METTL3 associated, and is enriched at promoters of m(6)A-modified transcripts. Collectively, these findings shed light on how a transcription factor can tightly couple gene transcription to m(6)A RNA modification to ensure ESC identity.

Global analysis of ZNF217 chromatin occupancy in the breast cancer cell genome reveals an association with ERalpha.

BACKGROUND: The ZNF217 gene, encoding a C2H2 zinc finger protein, is located at 20q13 and found amplified and overexpressed in greater than 20% of breast tumors. Current studies indicate ZNF217 drives tumorigenesis, yet the regulatory mechanisms of ZNF217 are largely unknown. Because ZNF217 associates with chromatin modifying enzymes, we postulate that ZNF217 functions to regulate specific gene signaling networks. Here, we present a large-scale functional genomic analysis of ZNF217, which provides insights into the regulatory role of ZNF217 in MCF7 breast cancer cells. RESULTS: ChIP-seq analysis reveals that the majority of ZNF217 binding sites are located at distal regulatory regions associated with the chromatin marks H3K27ac and H3K4me1. Analysis of ChIP-seq transcription factor binding sites shows clustering of ZNF217 with FOXA1, GATA3 and ERalpha binding sites, supported by the enrichment of corresponding motifs for the ERalpha-associated cis-regulatory sequences. ERalpha expression highly correlates with ZNF217 in lysates from breast tumors (n = 15), and ERalpha co-precipitates ZNF217 and its binding partner CtBP2 from nuclear extracts. Transcriptome profiling following ZNF217 depletion identifies differentially expressed genes co-bound by ZNF217 and ERalpha; gene ontology suggests a role for ZNF217-ERalpha in expression programs associated with ER+ breast cancer studies found in the Molecular Signature Database. Data-mining of expression data from breast cancer patients correlates ZNF217 with reduced overall survival. CONCLUSIONS: Our genome-wide ZNF217 data suggests a functional role for ZNF217 at ERalpha target genes. Future studies will investigate whether ZNF217 expression contributes to aberrant ERalpha regulatory events in ER+ breast cancer and hormone resistance.

The transcription factor ZNF217 is a prognostic biomarker and therapeutic target during breast cancer progression.

UNLABELLED: The transcription factor ZNF217 is a candidate oncogene in the amplicon on chromosome 20q13 that occurs in 20% to 30% of primary human breast cancers and that correlates with poor prognosis. We show that Znf217 overexpression drives aberrant differentiation and signaling events, promotes increased self-renewal capacity, mesenchymal marker expression, motility, and metastasis, and represses an adult tissue stem cell gene signature downregulated in cancers. By in silico screening, we identified candidate therapeutics that at low concentrations inhibit growth of cancer cells expressing high ZNF217. We show that the nucleoside analogue triciribine inhibits ZNF217-induced tumor growth and chemotherapy resistance and inhibits signaling events [e.g., phospho-AKT, phospho-mitogen-activated protein kinase (MAPK)] in vivo. Our data suggest that ZNF217 is a biomarker of poor prognosis and a therapeutic target in patients with breast cancer and that triciribine may be part of a personalized treatment strategy in patients overexpressing ZNF217. Because ZNF217 is amplified in numerous cancers, these results have implications for other cancers. SIGNIFICANCE: This study finds that ZNF217 is a poor prognostic indicator and therapeutic target in patients with breast cancer and may be a strong biomarker of triciribine treatment efficacy in patients. Because previous clinical trials for triciribine did not include biomarkers of treatment efficacy, this study provides a rationale for revisiting triciribine in the clinical setting as a therapy for patients with breast cancer who overexpress ZNF217.

Lrig1 is an estrogen-regulated growth suppressor and correlates with longer relapse-free survival in ERα-positive breast cancer.

Lrig1 is the founding member of the Lrig family and has been implicated in the negative regulation of several oncogenic receptor tyrosine kinases including ErbB2. Lrig1 is expressed at low levels in several cancer types but is overexpressed in some prostate and colorectal tumors. Given this heterogeneity, whether Lrig1 functions to suppress or promote tumor growth remains a critical question. Previously, we found that Lrig1 was poorly expressed in ErbB2-positive breast cancer, suggesting that Lrig1 has a growth-inhibitory role in this tumor type. However, breast cancer is a complex disease, with ErbB2-positive tumors accounting for just 25% of all breast cancers. To gain a better understanding of the role of Lrig1 in breast cancer, we examined its expression in estrogen receptor α (ERα)-positive disease which accounts for the majority of breast cancers. We find that Lrig1 is expressed at significantly higher levels in ERα-positive disease than in ERα-negative disease. Our study provides a molecular rationale for Lrig1 enrichment in ERα-positive disease by showing that Lrig1 is a target of ERα. Estrogen stimulates Lrig1 accumulation and disruption of this induction enhances estrogen-dependent tumor cell growth, suggesting that Lrig1 functions as an estrogen-regulated growth suppressor. In addition, we find that Lrig1 expression correlates with prolonged relapse-free survival in ERα-positive breast cancer, identifying Lrig1 as a new prognostic marker in this setting. Finally, we show that ErbB2 activation antagonizes ERα-driven Lrig1 expression, providing a mechanistic explanation for Lrig1 loss in ErbB2-positive breast cancer. This work provides strong evidence for a growth-inhibitory role for Lrig1 in breast cancer.

Inhibitory mechanism of pure curcumin on Wilms' tumor 1 (WT1) gene expression through the PKCα signaling pathway in leukemic K562 cells.

The aim of this study was to investigate the inhibitory mechanism of pure curcumin on WT1 expression in leukemic K562 cells. Pure curcumin suppressed WT1 expression, independent of effects on protein degradation or WT1 mRNA stability. Chromatin immunoprecipitation and reporter gene assays indicate that pure curcumin treatment attenuates WT1 auto-regulation. Interestingly, PKCα inhibition mimicks the repressive effects of pure curcumin in K562 cells. Conversely, myristoylated PKCα over-expression increased WT1 expression and reversed the inhibitory effect of pure curcumin. Our study indicates that pure curcumin attenuates WT1 auto-regulatory function through inhibition of PKCα signaling in K562 cells.

N-Myc regulates a widespread euchromatic program in the human genome partially independent of its role as a classical transcription factor.

Myc proteins have long been modeled to operate strictly as classic gene-specific transcription factors; however, we find that N-Myc has a robust role in the human genome in regulating global cellular euchromatin, including that of intergenic regions. Strikingly, 90% to 95% of the total genomic euchromatic marks histone H3 acetylated at lysine 9 and methylated at lysine 4 is N-Myc-dependent. However, Myc regulation of transcription, even of genes it directly binds and at which it is required for the maintenance of active chromatin, is generally weak. Thus, Myc has a much more potent ability to regulate large domains of euchromatin than to influence the transcription of individual genes. Overall, Myc regulation of chromatin in the human genome includes both specific genes, but also expansive genomic domains that invoke functions independent of a classic transcription factor. These findings support a new dual model for Myc chromatin function with important implications for the role of Myc in cancer and stem cell biology, including that of induced pluripotent stem cells.

Identification of genes directly regulated by the oncogene ZNF217 using chromatin immunoprecipitation (ChIP)-chip assays.

It has been proposed that ZNF217, which is amplified at 20q13 in various tumors, plays a key role during neoplastic transformation. ZNF217 has been purified in complexes that contain repressor proteins such as CtBP2, suggesting that it acts as a transcriptional repressor. However, the function of ZNF217 has not been well characterized due to a lack of known target genes. Using a global chromatin immunoprecipitation (ChIP)-chip approach, we identified thousands of ZNF217 binding sites in three tumor cell lines (MCF7, SW480, and Ntera2). Further analysis of ZNF217 in Ntera2 cells showed that many promoters are bound by ZNF217 and CtBP2 and that a subset of these promoters are activated upon removal of ZNF217. Thus, our in vivo studies corroborate the in vitro biochemical analyses of ZNF217-containing complexes and support the hypothesis that ZNF217 functions as a transcriptional repressor. Gene ontology analysis showed that ZNF217 targets in Ntera2 cells are involved in organ development, suggesting that one function of ZNF217 may be to repress differentiation. Accordingly we show that differentiation of Ntera2 cells with retinoic acid led to down-regulation of ZNF217. Our identification of thousands of ZNF217 target genes will enable further studies of the consequences of aberrant expression of ZNF217 during neoplastic transformation.

Suz12 binds to silenced regions of the genome in a cell-type-specific manner.

Suz12 is a component of the Polycomb group complexes 2, 3, and 4 (PRC 2/3/4). These complexes are critical for proper embryonic development, but very few target genes have been identified in either mouse or human cells. Using a variety of ChIP-chip approaches, we have identified a large set of Suz12 target genes in five different human and mouse cell lines. Interestingly, we found that Suz12 target promoters are cell type specific, with transcription factors and homeobox proteins predominating in embryonal cells and glycoproteins and immunoglobulin-related proteins predominating in adult tumors. We have also characterized the localization of other components of the PRC complex with Suz12 and investigated the overall relationship between Suz12 binding and markers of active versus inactive chromatin, using both promoter arrays and custom tiling arrays. Surprisingly, we find that the PRC complexes can be localized to discrete binding sites or spread through large regions of the mouse and human genomes. Finally, we have shown that some Suz12 target genes are bound by OCT4 in embryonal cells and suggest that OCT4 maintains stem cell self-renewal, in part, by recruiting PRC complexes to certain genes that promote differentiation.

Gene-specific TCDD suppression of RARalpha- and RXR-mediated induction of tissue transglutaminase.

The malignant human keratinocyte line SCC4 provides a model system to study the mechanism by which 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suppresses retinoid induction of the tissue transglutaminase gene (TGM2). The current work explores the nature of TCDD suppression of retinoid action to determine whether it is gene specific, whether it is retinoid receptor isoform-dependent, and whether it requires close proximity of retinoid and TCDD response elements. First, two other retinoid-inducible genes were identified in SCC4 by microarray screening whose induction was unaffected by TCDD, clearly demonstrating the gene specificity of TCDD suppression. Second, the receptor isoform dependence of retinoid responsiveness in SCC4 was tested. TGM2 was found to be inducible by an RARalpha-specific but not by an RARgamma-selective agonist. A lack of responsiveness to RARgamma agonists was found to be characteristic of SCC4, however, inasmuch as transcription driven by a retinoid response element in transfections was also stimulated only by the alpha-specific agonist in these cells. Because SCC4 lacks expression of RARbeta, the gene specificity evidently was not attributable to differential TCDD targeting of retinoid receptor isoforms. Finally, the proximal 5 kb of the TGM2 promoter was found to be retinoid responsive in stable transfections, but the induction was not suppressed by TCDD. These results indicate that the suppressive action of TCDD occurs indirectly and through a separate DNA site likely located outside the 5-kb region, not by direct interference with retinoid action or at retinoid response elements.