[Show abstract][Hide abstract] 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
[Show abstract][Hide abstract] 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.