NF-kB activation and function in B lymphocytes

National Institute On Aging

Investigators

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Abstract

During FY17 we accomplished the following: 1) Attempted to confirm the model that survivin/Cdk4 interaction provides the basis for G1 progression during signal-independent B cell proliferation. Co-immunoprecipitation studies from G1 phase of the cell-cycle proved more complex than anticipated and are being re-designed. We also attempted to determine the molecular mechanism underlying cell survival mediated by very low (non-mitogenic) doses of anti-CD40. A manuscript describing the basic property of signal-independent proliferation is being written. 2) Used dominant negative IB (dn-IB) expressing cell lines to identify true targets of NF-B during cell activation. We conclusively demonstrated that NF-B binding and inducible transcription were insufficient criteria to identify NF-B target genes. Using more stringent criteria we identified 130 direct NF-B target genes in activated B cells. The majority of these genes have not been linked to NF-B in earlier studies, indicating that they are novel targets of this transcription factor. We also determined that NF-B activation leads to gene repression and thereby provided evidence that NF-B affects responses in complex and unanticipated ways. Finally, we identified indirect targets of NF-B that were suppressed by dn-IB, but did not bind NF-B. We provided evidence that these genes were regulated by NF-B-dependent transcription factors, and represent the second wave of a cascade of gene expression changes initiated by NF-B. A manuscript is being written 3) GtfIIif/f mice were bred to CD19-cre strain to obtain B cells deficient in this transcription factor. GtfIIi-deficient B cells were activated with anti-Ig or LPS and RNA extracted at various times post-activation. RNA-Seq was carried out and the data are being computationally interrogated. To extend these studies, we initiated breeding of GtfIIif/f mice to CD4-cre to obtain T cells that lack this transcription factor. This strain will be ready for RNA analysis in FY18. A manuscript describing B cells studies is being written.