17-371 Sigma-AldrichEZ-ChIP™

The aberrant overexpression of Wilms' tumor-1 gene (WT1) plays an important role in blast cell survival and resistance to chemotherapy in acute myeloid leukemia (AML). Here, we found in chemotherapeutic drug etoposide-induced apoptosis, WT1 protein was cleaved into smaller fragment by caspase-3 in leukemic cells. The cleavage was blocked by pan-caspase inhibitor and special caspase-3 inhibitor, suggesting that caspase-3 might cleave WT1 protein. Furthermore, recombinant active caspase-3 cleaved the Flag-WT1 and GST-WT1 proteins in vitro. However, site-directed mutagenesis analyses failed to identify caspase-3-targeted sites in WT1 protein, indicating that caspase-3 cleaved uncommon sites but not classical motifs (DXXD) and non-classical motifs (XXXD). Finally, Eto decreased c-Myc and Bcl-2 expression via reducing the binding of WT1 to the promoter and Eto-induced apoptosis was partially prevented by overexpression of WT1. Collectively, we identify a new substrate for caspase-3 and shed new light on understanding the complicated biology of WT1 in leukemia.

Lsh is a chromatin remodeling factor that regulates DNA methylation and chromatin function in mammals. The dynamics of these chromatin changes and whether they are directly controlled by Lsh remain unclear. To understand the molecular mechanisms of Lsh chromatin controlled regulation of gene expression, we established a tethering system that recruits a Gal4-Lsh fusion protein to an engineered Oct4 locus through Gal4 binding sites in murine embryonic stem (ES) cells. We examined the molecular epigenetic events induced by Lsh binding including: histone modification, DNA methylation and chromatin accessibility to determine nucleosome occupancy before and after embryonic stem cell differentiation. Our results indicate that Lsh assists gene repression upon binding to the Oct4 promoter region. Furthermore, we detected less chromatin accessibility and reduced active histone modifications at the tethered site in undifferentiated ES, while GFP reporter gene expression and DNA methylation patterns remained unchanged at this stage. Upon differentiation, association of Lsh promotes transcriptional repression of the reporter gene accompanied by the increase of repressive histone marks and a gain of DNA methylation at distal and proximal Oct4 enhancer sites. Taken together, this approach allowed us to examine Lsh mediated epigenetic regulation as a dynamic process and revealed chromatin accessibility changes as the primary consequence of Lsh function.

Transcription factors of nuclear receptor 5A (Nr5a) subfamily play pivotal roles in regulation of steroidogenic enzymes in vertebrates including teleosts. In the orange-spotted grouper, the expression of Nr5a1a was only detectable in the ovary, spleen, and head kidney in the female. The immunoreactive Nr5a1a was present in ovarian follicular and germ cells. In the ovarian follicular cells surrounding vitellogenic oocytes, Nr5a1a was detected both in the nucleus and cytoplasm, and co-localized with Cyp19a1a and Nr5a2. In the ovarian follicular cells surrounding fully grown oocytes, Nr5a1a was localized almost exclusively to the cytoplasm together with Nr5a2. Nr5a1a could up-regulate cyp19a1a promoter activities through Nr5a sites, and further increase the responses elicited by Nr5a2 at sub-maximal doses. Chromatin immunoprecipitation analysis showed that Nr5a1a bound to cyp19a1a promoter in the vitellogenic but not fully grown ovary. Taken together, Nr5a1a up-regulates cyp19a1a additively with Nr5a2 during vitellogenesis, and its cytoplasmic sequestration may also contribute to the down-regulation of cyp19a1a in the fully grown ovary.

N-Myc interactor (Nmi) is reported to participate in many activities, such as signaling transduction, transcription regulation, and antiviral responses. As Nmi may play important roles in interferon (IFN)-induced responses, we investigated the mechanism how Nmi protein is regulated. We identified and cloned the promoter of Nmi gene. Sequence analysis and luciferase assays shown that an IFN-stimulated response element (ISRE) and a GC box in the promoter were essential for the basal transcription activity of Nmi gene. We also found that interferon regulatory factor 1 (IRF-1) could activate transcription of Nmi by binding to the ISRE in the promoter. Knockdown of IRF-1 decreases IFN-induced Nmi transcription. These results revealed that IRF-1 is involved in the IFN-inducible expression of Nmi.

Inhibitors against poly (ADP-ribose) polymerase (PARP) are promising targeted agents currently used to treat BRCA-mutant ovarian cancer and are in clinical trials for other cancer types, including BRCA-mutant breast cancer. To enhance the clinical response to PARP inhibitors (PARPis), understanding the mechanisms underlying PARPi sensitivity is urgently needed. Here, we show enhancer of zeste homolog 2 (EZH2), an enzyme that catalyzes H3 lysine trimethylation and associates with oncogenic function, contributes to PARPi sensitivity in breast cancer cells. Mechanistically, upon oxidative stress or alkylating DNA damage, PARP1 interacts with and attaches poly-ADP-ribose (PAR) chains to EZH2. PARylation of EZH2 by PARP1 then induces PRC2 complex dissociation and EZH2 downregulation, which in turn reduces EZH2-mediated H3 trimethylation. In contrast, inhibition of PARP by PARPi attenuates alkylating DNA damage-induced EZH2 downregulation, thereby promoting EZH2-mediated gene silencing and cancer stem cell property compared with PARPi-untreated cells. Moreover, the addition of an EZH2 inhibitor sensitizes the BRCA-mutant breast cells to PARPi. Thus, these results may provide a rationale for combining PARP and EZH2 inhibition as a therapeutic strategy for BRCA-mutated breast and ovarian cancers.

Cushing's syndrome (CS) caused by hypercortisolism is occasionally accompanied by metabolic disorders such as hypertension, diabetes mellitus (DM), dyslipidemia, and central obesity. Thus morbidity and mortality, observed in cardiovascular disease, are elevated in patients with CS. We hypothesized that HDAC inhibition (HDACi) decreased transcriptional activity of glucocorticoid receptor (GR), which ameliorates hypertension and hyperglycemia in patients with CS. To establish an animal model of hypercortisolism, Sprague-Dawley rats were infused with adrenocorticotropic hormone (ACTH, 40 ng/day) or dexamethasone (Dex, 10 μg/day) via osmotic minipumps for 4 wk. Expression of GR target genes was determined by quantitative real-time PCR (qRT-PCR). GR enrichment on specific loci, and across the whole genome, was analyzed by chromatin immunoprecipitation (ChIP) and ChIPseq, respectively. HDACi decreased blood pressure and expression of ion regulators in the kidneys of ACTH-infused rats. Additionally, HDACi reduced deposition of polysaccharide, fasting blood glucose level, glucose intolerance, and expression of gluconeogenesis genes in the livers and kidneys of ACTH- and Dex-infused rats. Among class I HDACs, HDAC1 and HDAC3 interacted with GR. HDAC1 knockdown resulted in increased level of acetylation and decreased transcriptional activity of GR. GR recruitment on the promoters of 2,754 genes, which include ion transporters, channels, and gluconeogenic genes, was significantly decreased by MS-275, a class I HDAC inhibitor. These results indicate that HDACi ameliorates hypertension and hyperglycemia in a model of CS by decreasing the transcriptional activity of GR via elevating its level of acetylation.

DNA methylation plays a critical role in diverse biological processes of plants. Arabidopsis DNA METHYLTRANSFERASE1 (MET1) represses shoot regeneration by inhibiting WUSCHEL (WUS) expression, which is essential for shoot initiation. However, the upstream signals regulating MET1 expression during this process are unclear. We analyzed the signals regulating MET1 expression using a number of established strategies, such as genetic analysis, confocal microscopy, quantitative real-time PCR and chromatin immunoprecipitation. MET1 expression patterns underwent dynamic changes with the initiation of WUS during shoot regeneration. The cell cycle regulator E2FA was characterized as an upstream factor directly promoting MET1 expression. Moreover, cytokinin promoted MET1 expression partially by enhancing CYCD3 expression. Our findings reveal that MET1-mediated shoot regeneration is regulated by the cytokinin-induced cell cycle, and provide new insights into the regulation of DNA methylation in shoot regeneration.

Targeting apoptosis of vascular smooth muscle cells (VSMCs) represents an attractive approach to diminish the occurrence of restenosis. Neddylation is a highly conserved post-translational modification process and inhibition of neddylation has been shown to regulate apoptosis of other cells. However, the impacts of neddylation inhibition on VSMCs and neointimal hyperplasia have not been studied. In our present study, we have shown that MLN4924, a selective inhibitor of NEDD8-activating enzyme (NAE), markedly inhibited neointimal hyperplasia and accumulation of VSMCs, whereas increased apoptosis in the vascular wall. In vitro studies revealed that MLN4924 induced G2/M arrest and apoptosis of human VSMCs. Knockdown of NAE1 had similar effects. MLN4924 upregulated p53 and p62 in human VSMCs. Knockdown of either p53 or p62 mitigated the impacts of MLN4924 on G2/M arrest and apoptosis. Moreover, p53 knockdown abolished MLN4924-induced upregulation of p62. Finally, smooth muscle p53 knockout mice were generated and subjected to femoral artery injury and MLN4924 treatment. Deficiency of p53 in smooth muscle blocked the effects of MLN4924 on neointimal hyperplasia and apoptosis. Together, our results revealed that neddylation inhibition induces apoptosis through p53 and p62 in VSMCs and improves neointimal hyperplasia mainly by promoting apoptosis through smooth muscle p53 in mice. These pre-clinical data provide strong translational implications for targeting restenosis by perturbation of neddylation using MLN4924.

Pulpitis is a multi-factorial disease that could be caused by complex interactions between genetics, epigenetics and environmental factors. We aimed to evaluate the role of Enhancer of Zeste Homolog 2 (EZH2) in the inflammatory response of human dental pulp cells (HDPCs) and dental pulp tissues.The expressions of inflammatory cytokines in HDPCs treated by EZH2 complex or EZH2 siRNA with or without rhTNF-α were examined by quantitative real-time polymerase chain reaction (q-PCR). The levels of secreted inflammatory cytokines including IL-6, IL-8, IL-15, CCL2 and CXCL12 in culture supernatants were measured by Luminex assay. In rat pulpitis model, the effects of EZH2 on dental pulp tissues were verified by histology. We invested the mechanisms of the effect of EZH2 on the inflammatory factors by ChIP assay.EZH2 down-regulation inhibited the expression of inflammatory factors, including IL-6, IL-8, IL-15, CCL2 and CXCL12 in HDPCs. EZH2 complex promoted the expression and secretion of these inflammatory factors in HDPCs, while EZH2 silencing could attenuate the promotion of inflammatory factors that were induced by rhTNF-α. In pulpitis models of rats, EZH2 down-regulation inhibited the inflammatory process of dental pulp while EZH2 complex showed no significant facilitation of pulpal inflammation. In addition, EZH2 could bind on the promoters of IL-6, IL-8 and CCL2, but not IL-15 and CXCL12, to affect the transcription of these proinflammatory cytokines.In HDPCs, EZH2 could induce inflammation, while EZH2 down-regulation could attenuate the inflammatory responses. EZH2 plays an important role in this inflammatory process of dental pulp.

The transcriptional regulation of the human angiotensin receptor subtype 1 (AT1R) gene in pathophysiologies, like the metabolic syndrome, is poorly understood. The human AT1R gene has polymorphisms in its promoter that can be arranged in 2 haplotypes. Variants -810T, -713T, -214A, and -153A always occur together (Hap-I) and variants -810A, -713G, -214C, and -153G form Hap-II. We have hypothesized that high fat diet will alter cellular transcriptional milieu and increase hAT1R gene expression in a haplotype-dependent manner. This will set up an AT1R-mediated feed-forward loop promoting inflammation, oxidative stress, and hypertension in Hap-I mice.Since Hap-I of the human AT1R gene is associated with hypertension in Caucasians, we generated transgenic (TG) mice with Hap-I and Hap-II and studied the physiological significance of high fat diet (HFD) on haplotype specific gene expression. Animals were fed with HFD for 20 weeks followed by blood pressure (BP) analysis and collection of their tissues for molecular and biochemical studies.After HFD treatment, as compared to Hap-II, TG mice with Hap-I show increased expression of hAT1R gene and higher BP; suppression of antioxidant defenses (HO1, SOD1) and increased expression of IL-6, TNFα, IL-1β, NOX1. In vivo ChIP assay has shown that transcription factors CEBPβ, STAT3, and USF bind more strongly to the chromatin obtained from Hap-I TG mice.Taken together, our results suggest, that after HFD treatment, as compared to Hap-II, the TG mice with Hap-I overexpress the AT1R gene due to the stronger transcriptional activity, thus resulting in an increase in their BP.