SRC-2 Metabolism Project Description:
Glucose homeostasis is a tightly regulated process that relies on dynamic rapid transcriptional responses. We define SRC-2 as an integral coactivator of opposing enzymes (i.e. glucokinase and glucose-6-phosphatase) at the opposing steps of glycolysis and gluconeogenesis. Previous studies have offered an incomplete picture of the dynamics of transcriptional machinery recruitment under varying metabolic conditions. To address this deficit we have employed a global proteomic approach to define the influence of both metabolism and SRC-2 on the recruitment and stabilization of promoter complexes.
Here we used biotinylated promoter DNA fragments that represent ~1kb of the proximal promoter regions of Gck or G6pc genes that were bound to streptavidin beads. The immobilized DNA-beads were used to recruit proteins from hepatic nuclear extract generated from SRC-2 WT or SRC-2 KO mice that were fasted for 24 hours, or fasted for 24 hours followed by a 3 hour refeed. The pull-down elutant was then used for mass spectrometry (MS) or immunoblotting to identify nuclear proteins that localized to either the Gck or G6pc promoter regions.
Results:
Table 1. Differential methods of quantity normalization of MS data, ratios comparing different methods, MS parameters, and quantile normalization iFOTs used to generate heat maps in Figure 3C. Data are organized from high iFOTs to low for either Gck SRC-2 WT Refed or G6pc SRC-2 WT Fasted.
Table 2. Quantile normalized iFOTs and functionally annotated category used to generate heatmap in Figure S3B.
Table 3. Quantile normalized iFOTs used to generate heatmaps in Figure 3E.
Table 4. Differential methods of quantity normalization of MS data, ratios comparing different methods, MS parameters, and a heatmap that represents the ratio of iFOTs (blue is <2 fold; red is >2 fold) (comparisons defined in header column) used to generate models in Figure 5A.
Raw MS Data Files:
Below are the original mass spectrometry files (Thermo Fisher instruments, ".raw" format) for the 8-experiments promoter affinity dataset:
