Dana Bashari

Bar Ilan University, Gan, Tel Aviv, Israel

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Publications (3)13.34 Total impact

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    ABSTRACT: Objective: Chromatin immunoprecipitation (ChIP) has become a central method when studying in-vivo protein-DNA interactions, with the major challenge being the hope to .capture "authentic" interactions. While ChIP protocols have been optimized for use with specific cell types and tissues, and adipose tissue-derived cells have been studied by ChIP, a working ChIP protocol addressing the challenges imposed by fresh whole human adipose tissue has not been described. Design: Utilizing human paired omental and subcutaneous adipose tissue, we have carefully identified and optimized individual steps in the ChIP protocol employed directly on fresh tissue fragments. Subjects: Were recruited prior to undergoing elective abdominal surgeries. Results: We describe a complete working protocol for using ChIP on whole adipose tissue fragments. We identified specific steps that required adaptation of the ChIP protocol to human whole adipose tissue. In particular, crosslinking step was performed directly on fresh small tissue fragments; Nuclei were isolated before releasing chromatin, allowing better management of fat content; Sonication protocol to obtain fragmented chromatin was optimized. We also demonstrate the high sensitivity of immunoprecipitated chromatin from adipose tissue to freezing. Conclusions: We describe the development of a ChIP protocol optimized for use in studying whole human adipose tissue, providing solutions for the unique challenges imposed by this tissue. Unraveling protein-DNA interaction in whole human adipose tissue will likely contribute to elucidating molecular pathways contributing to common human diseases such as obesity and type 2 diabetes.
    AJP Endocrinology and Metabolism 09/2013; · 4.51 Impact Factor
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    ABSTRACT: The E2F family of transcription factors plays a pivotal role in the regulation of cell proliferation in higher eukaryotes and is a critical downstream target of the tumor suppressor pRB. The pRB/E2F pathway is defective in most human tumors, resulting in deregulated E2F activity that induces uncontrolled cell proliferation, a hallmark of tumor cells. The RNA-binding protein RBM38, also named RNPC1, induces cell-cycle arrest in G(1), at least in part, via binding to and stabilizing the mRNA of the cyclin-dependent kinase inhibitor p21. RBM38 levels are altered in human cancer. Generally, RBM38 is overexpressed in various tumors; however, RBM38 mRNA levels are reduced in some breast tumors due to increased methylation of its promoter region. We show here that expression of RBM38 is regulated by E2F1. Specifically, RBM38 mRNA and protein levels are elevated upon activation of either exogenous E2F1 or endogenous E2Fs. Moreover, endogenous E2F1 binds the human RBM38 promoter and E2F1 knockdown reduces RBM38 levels. Our data raise the possibility that E2F1 together with E2F1-regulated RBM38 constitute a negative feedback loop that modulates E2F1 activity. In support of this, inhibition of RBM38 expression increases E2F1-mediated cell-cycle progression. Moreover, in human ovarian cancer, high correlation between expression of E2F1 and RBM38 is associated with increased survival. Overall, our data identify RBM38 as novel transcriptional target of E2F1 that restricts E2F1-induced proliferation. Furthermore, this negative feedback loop seems to restrict tumor aggressiveness, thereby promoting survival of patients with cancer. Mol Cancer Res; 10(9); 1169-77. ©2012 AACR.
    Molecular Cancer Research 07/2012; 10(9):1169-77. · 4.35 Impact Factor
  • Dana Bashari, Dalia Hacohen, Doron Ginsberg
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    ABSTRACT: Members of the E2F transcription factor family are critical downstream targets of the tumor suppressor RB and are often deregulated and hyperactive in human tumors. E2F regulates a diverse array of cellular functions including cell proliferation and apoptosis. Recent studies indicate that E2F also regulates expression of upstream components of pivotal signal transduction pathways, thereby modulating the activity of these pathways. We show here that E2F modulates the activity of the JNK pathway via E2F-induced upregulation of JNK phosphorylation. Accordingly, downregulating E2F1and E2F3 inhibits sustained UV-induced JNK phosphorylation and ectopic expression of E2F1 or E2F3 induces JNK phosphorylation and activation. The mechanism by which E2F modulates JNK phosphorylation involves transcriptional induction of the kinase GCK, a MAP4K that can activate JNK indirectly. Hence, inhibition of GCK expression impairs E2F1-induced JNK phosphorylation. The JNK pathway is an important mediator of stress-induced apoptosis and we show here that inhibition of JNK expression or activity significantly hinders E2F1-induced apoptosis. Overall, our data identify the kinase GCK as a novel E2F-regulated gene and reveal a functional link between a central signaling pathway, namely the JNK pathway, and the transcription factor E2F.
    Cellular Signalling 01/2011; 23(1):65-70. · 4.47 Impact Factor