NF-kB activation and function in B lymphocytes

National Institute On Aging

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Abstract

During FY14 we accomplished the following: 1. Carried out further studies to characterize signal-independent proliferation of naive splenic B cells. Previously described P3 (non-dividing) and P4 (dividing) cell populations were characterized by Amnis Imagestream to detect status of cell-cycle regulatory proteins, by Seahorse to characterize metabolic status and by Cytometry by time of flight (Cytof) for cell cycle analysis (in collaboration with Dr. Garry Nolan, Stanford University). The first phase of these studies is almost complete. 2. We developed chromatin immunoprecipitation to detect RelA (p65) and Rel binding in B cells. ChIP-Seq analysis was carried out in BJAB human B lymphoma cells activated by phorbol ester and calcium ionophore, and in primary murine B cells activated via the B cell antigen receptor (BCR). Cumulatively this has yielded substantial novel data which is in the process of being rigorously computationally analyzed. 3. We carried out time-dependent RNA-Seq using total RNA and chromatin-associated RNA isolated from murine B cells activated by the BCR. These data will give an accurate measure of transcription initiation rates in activated B cells. We are particularly interested in the response of NF-κB-dependent gene transcription. The results from the RNA studies will be analyzed in the context of the dynamic ChIP-Seq studies described in #2 (above). 4. We carried out genome-wide DNAase I hypersensitivity studies with activated murine spleen B cells. The goal is to determine chromatin structural changes at NF-κB-dependent promoters and enhancers as a function of time. 5. We developed ChIA-PET procedure to identify intra- and inter-chromosomal interaction of NF-κB-bound promoters and enhancers in activated B cells. The first set of experiments has been sent for sequencing, and we are actively seeking to establish collaborations to analyze the huge amount of data that will be generated.