Expression data from NFI-C knock out embryonic fibroblasts
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE15871 | GEO | 2009/11/05
SECONDARY ACCESSION(S): PRJNA117021
REPOSITORIES: GEO
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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.
2009-11-15 | E-GEOD-15871 | biostudies-arrayexpress
Project description:The conserved Nuclear Factor I (NFI) family of transcription factors is unique to animals and essential for mammalian development. The C. elegans genome encodes a single NFI family member, whereas vertebrate genomes encode four distinct NFI protein subtypes (A, B, C, and X). NFI-1 deficient worms exhibit abnormalities including reduced lifespan, defects in movement and pharyngeal pumping, and delayed egg-laying. To explore the functional basis of these phenotypes, we sought to comprehensively identify NFI-1 binding targets in C. elegans. We first established NFI-1 DNA-binding specificity using an in vitro DNA-selection strategy. Analysis yielded a consensus motif of TTGGCA(N3)TGCCAA, which occurs 586 times in the genome, a 100-fold higher frequency than expected. We next asked which sites were occupied by NFI-1 in vivo by performing chromatin immunoprecipitation of NFI-1 followed by microarray hybridization (ChIP-chip). Only 55 genomic locations were identified, an unexpectedly small target set. In vivo NFI-1 binding sites tend to be upstream of genes involved in core cellular processes such as chromatin remodeling, mRNA splicing, and translation. Remarkably, 67/80 (84%) of the C. briggsae homologs of the identified targets contain conserved NFI binding sites in their promoters. These experiments provide a foundation for understanding how NFI-1 is recruited to unexpectedly few in vivo sites to perform its developmental functions, despite a vast over-representation of its binding site. To our knowledge, this represents the first genome-wide location study of any transcription factor in C. elegans, and the first genome-wide analysis in any species of an NFI transcription factor. 4 biological replicates of NFI-1 ChIP-chip (including 1 dye-swap) were performed with 2 pre-immune Mock ChIP-chip controls from corresponding extracts.
2009-01-30 | E-GEOD-13918 | biostudies-arrayexpress
Project description:The conserved Nuclear Factor I (NFI) family of transcription factors is unique to animals and essential for mammalian development. The C. elegans genome encodes a single NFI family member, whereas vertebrate genomes encode four distinct NFI protein subtypes (A, B, C, and X). NFI-1 deficient worms exhibit abnormalities including reduced lifespan, defects in movement and pharyngeal pumping, and delayed egg-laying. To explore the functional basis of these phenotypes, we sought to comprehensively identify NFI-1 binding targets in C. elegans. We first established NFI-1 DNA-binding specificity using an in vitro DNA-selection strategy. Analysis yielded a consensus motif of TTGGCA(N3)TGCCAA, which occurs 586 times in the genome, a 100-fold higher frequency than expected. We next asked which sites were occupied by NFI-1 in vivo by performing chromatin immunoprecipitation of NFI-1 followed by microarray hybridization (ChIP-chip). Only 55 genomic locations were identified, an unexpectedly small target set. In vivo NFI-1 binding sites tend to be upstream of genes involved in core cellular processes such as chromatin remodeling, mRNA splicing, and translation. Remarkably, 67/80 (84%) of the C. briggsae homologs of the identified targets contain conserved NFI binding sites in their promoters. These experiments provide a foundation for understanding how NFI-1 is recruited to unexpectedly few in vivo sites to perform its developmental functions, despite a vast over-representation of its binding site. To our knowledge, this represents the first genome-wide location study of any transcription factor in C. elegans, and the first genome-wide analysis in any species of an NFI transcription factor.
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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)
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Project description:Dendrite and synapse development are critical for establishing appropriate neuronal circuits, and disrupted timing of these events can alter connectivity leading to disordered neural function. In early postnatal development of cerebellar granule neurons (CGNs), the expression of many genes is temporally regulated. Further, NFI (Nuclear Factor I) proteins have been shown to play important roles in the regulation of gene expression in developing CGNs. To identify NFI-regulated genome-wide targets involved in late maturation of mouse CGNs, we performed two groups of microarray expression analysis: (1) temporal expression arrays using 1.5 and 6 day cultures of mouse CGNs representing immature and more mature CGNs, respectively, which identified 844 Temporal-Up or Down genes; and (2) NFI-regulated genes in 6 day CGN cultures that were infected at the time of plating with lentiviral vectors expressing either NFI dominant repressor (NFI-EnR) or EnR control protein. This identified 686 NFI-Up (NFI-EnR down-regulated) or NFI-Down (NFI-EnR up-regulated) genes. Overlap analysis identified 212 temporal genes that were regulated by NFI. These results indicated that NFI plays a pivotal role in the regulation of late CGN maturation. For temporal arrays, mouse CGN progenitors were purified from P6 mouse cerebellum and cultured for either 1.5 or 6 days. Three biological replicates were analyzed for each time point. For NFI arrays, CGN progenitors from P6 mice were transduced upon plating with either NFI-EnR or EnR control lentivirus and cultured 6 days. Four pairs of biological replicates were performed.
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