Project description:By binding to specific DNA elements, known collectively as “κB sites”, contained within the promoters/enhancers of target genes, NF-κB regulates gene expression. We found that the identity of the central base pair (bp) of κB sites profoundly impacts the transcriptional activity of NF-κB dimers. RelA dimers prefer an A/T bp at this position for optimum transcriptional activation (A/T-centric) and discriminate against G/C-centric κB sites. The p52 homodimer, in contrast, activates transcription from G/C-centric κB sites in complex with Bcl3 but represses transcription from the A/T-centric sites. The p52:Bcl3 complex binds to these two classes of κB sites in distinct modes permitting recruitment of coactivator, corepressor, or both coactivator and corepressor complexes in promoters containing G/C, A/T or both G/C and A/T-centric sites. Therefore, through sensing of bp differences within κB sites, NF-κB dimers modulate biological programs by activating, repressing and altering expression of effector genes. Total RNA extracted from bone marrow derived macrophages (BMDMs) with Bcl3 siRNA knockdown or mouse scramble siRNA knockdown were subjected to LPS stimulation.
Project description:We use NGS to assess the ability of TALE-guided DNA methyltranferases to make targeted changes to DNA methylation Targeted bisulfite sequencing of cells infected with wild-type or mutant TALE-DNMT constructs directed to the CDKN2A (p16) locus
Project description:This deposition describes DNA sequences at integration sites in primary tumors and lung metastases from a sleeping-beauty transposon-mediated mutagenesis screen on Rb-deficient background in the mammary gland in MMTV-Cre:Rbf/f:T2/Onc3a:R26lsl_SB11 and MMTV-Cre:Rbf/f:T2/Onc3b:R26lsl_SB11 female mice. In these mice, Rb floxed (Rbf/f) alleles and a SB11 transposase, knocked into the ROSA26 locus (R26lsl_SB11), are mobilized via MMTV-Cre. Two different transgenic transposon concatemers, T2/Onc3a and T2/Onc3b, containing 11 and 28 copies on chromosomes 9 and 12 were used so that local hoping in the respective chromosomes can be discarded. These mice developed large primary tumors and macroscopic lung metastases; biopsies from the primary tumors and entire lung metastases were used to extract DNA. The DNA was subjected to sonication, ligation-mediated PCR with 79 bar coded primers, followed by next-generation DNA sequencing. We identified Primary-specific and lung Metastasis-specific gene-centric Common Integration Sites (gCIS) as well as shared gCIS, observed in both primary mammary tumors and lung metastases. Using sequence analysis of integration sites, we were able to demonstrate in multiple cases clonal relationship between primary lesions and metastases. The metastatic gCIS form specific hubs that may be prioritized for targeted therapy. For details, see reference below.