ABSTRACT
The synthesis of fatty acids and cholesterol, the building blocks of membranes, is regulated by three membrane-bound transcription factors: sterol regulatory element-binding proteins (SREBP)-1a, -1c, and -2. Their function in liver has been characterized in transgenic mice that overexpress each SREBP isoform and in mice that lack all three nuclear SREBPs as a result of gene knockout of SREBP cleavage-activating protein (SCAP), a protein required for nuclear localization of SREBPs. Here, we use oligonucleotide arrays hybridized with RNA from livers of three lines of mice (transgenic for SREBP-1a, transgenic for SREBP-2, and knockout for SCAP) to identify genes that are likely to be direct targets of SREBPs in liver. A total of 1,003 genes showed statistically significant increased expression in livers of transgenic SREBP-1a mice, 505 increased in livers of transgenic SREBP-2 mice, and 343 showed decreased expression in Scap-/- livers. A subset of 33 genes met the stringent combinatorial criteria of induction in both SREBP transgenics and decreased expression in SCAP-deficient mice. Of these 33 genes, 13 were previously identified as direct targets of SREBP action. Of the remaining 20 genes, 13 encode enzymes or carrier proteins involved in cholesterol metabolism, 3 participate in fatty acid metabolism, and 4 have no known connection to lipid metabolism. Through application of stringent combinatorial criteria, the transgenic/knockout approach allows identification of genes whose activities are likely to be controlled directly by one family of transcription factors, in this case the SREBPs.
Subject(s)
CCAAT-Enhancer-Binding Proteins/genetics , DNA-Binding Proteins/genetics , Membrane Proteins/deficiency , Membrane Proteins/genetics , Animals , Cholesterol/biosynthesis , Fatty Acids/biosynthesis , Gene Expression Profiling , Intracellular Signaling Peptides and Proteins , Liver/metabolism , Male , Mice , Mice, Knockout , Mice, Transgenic , Oligonucleotide Array Sequence Analysis , Sterol Regulatory Element Binding Protein 1 , Sterol Regulatory Element Binding Protein 2 , Transcription Factors/geneticsABSTRACT
We developed a high-throughput method for resequencing for single nucleotide polymorphism (SNP) discovery using high-density microarrays. Over the two-year course of this study a number of improvements in sample preparation methods, hybridization assay, array handling, and analysis method were developed and implemented. DNA from 40 unrelated individuals of three different ethnic origins was amplified, labeled, and hybridized to arrays designed with probes representing genomic, coding, and regulatory regions. Protocol improvements including the use of long PCR and semi-automation reduced labeling and fragmentation costs by 33%. Automation improvements include the development of a scanner autoloader for arrays, a faster array wash station, and a linked laboratory tracking and data management system. Validation of a smaller feature size, 20 x 24 microns, allowed the simultaneous screening of 30-kb sense and 30-kb antisense DNA on each microarray, increasing throughput to 1.4 Mb per day per two laboratory personnel. More than 15,000 SNPs were identified in 8.3 Mb of the human genome using high-density resequencing and variation detection arrays (microarrays).
