Project description:The Nuclear Factor I (NFI) family of DNA binding proteins (also called CAAT box transcription factors or CTF) is involved both in replication of adenoviral DNA and in regulation of gene expression. Using chromatin immuno-precipitation and high throughput sequencing (method termed ChIP-Seq) we performed genome-wide mapping of Nuclear Factor I DNA binding sites in mouse embryonic fibroblasts. We found that in vivo and in vitro NFI binding specificities are identical, since previously established position weight matrix was found to accurately predict binding sites for NFI group of proteins. Positional correlation between + and - strand ChIP-Seq tags revealed that NFI is a nucleosome-binding protein, unlike some other transcription factors. We further found that NFI binding correlates with the specific histone modification H3K4me3, the marker of transcribed promoters. Combining ChIP-Seq with the microarray expression data, we found that NFI associates with promoters with higher transcription level. We estimate that transcribed promoters may be more accessible to transcription factor binding than their nearby regions since NFI preferentially bind their DNA target sites located around transcription start sites. Knocking-out one of the NFI proteins (NFI-C), reduced the occupancy of predicted sites, however at the same time indicating that cells could compensate the missing protein with the other members of the family. Keywords: ChIP-Seq (chromatin immunoprecipitation and high throughput sequencing) ChIP-Seq (chromatin immunoprecipitation and high throughput sequencing) using antibody against NFI family of transcription factors in two cell types (wild type and NFI-C knock-out mouse embryonic fibroblasts MEFs)
Project description:The aim of the study was to investigate whether the trefoil peptide genes, in concerted action with a miRNA regulatory network, were contributing to nutritional maintrenance. Using a Tff2 knock-out mouse model, 48 specific miRNAs were noted to be significantly deregulated when compared to the wild type strain.
Project description:The aim of the study was to investigate whether the trefoil peptide genes, in concerted action with a miRNA regulatory network, were contributing to nutritional maintrenance. Using a Tff3 knock-out mouse model, 21 specific miRNAs were noted to be significantly deregulated when compared to the wild type strain.
Project description:The Nuclear Factor I C (NFI-C) transcription factor has been implicated in TGF-β signaling, extracellular matrix deposition and skin appendage pathologies. We performed a global gene expression analysis in NFI-C-/- and wild-type embryonic primary murine fibroblasts. This indicated that NFI-C acts mostly to repress gene expression in response to TGF-β1. Misregulated genes featured a prominent over-representation of regulators of connective tissue inflammation and repair. Experiment Overall Design: mRNAs were isolated from embryonic fibroblasts obtained from 3 wild-type and 3 NFI-C knock-out mice, and their levels were probed using microarrays. Prior to RNA extraction, fibroblast cultures were treated or not with TGF-β1 for 1 hour to examine the immediate response to the growth factor, or treated for 10 hours to assess the delayed response.
Project description:MBD-affinity purification (MAP) was employed to investigate the DNA methylation status at promoters of mouse embryonic fibroblasts (MEFs), both wild type and Lsh knock-out cells.<br>MAP is conceptually identical to ChIP, using an affinity column rather than an antibody.