Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Integrative genomic analysis of CREB defines a critical role for transcription factor networks in mediating the fed/fasted switch in liver [array]

ABSTRACT: Metabolic homeostasis in mammals critically depends on the regulation of fasting-induced genes by CREB in the liver. Previous genome-wide analysis has shown that only a small percentage of CREB target genes are induced in response to fasting-associated signaling pathways. The precise molecular mechanisms by which CREB specifically targets these genes in response to alternating hormonal cues remain to be elucidated. We performed chromatin immunoprecipitation coupled to high-throughput sequencing of CREB in livers from both fasted and re-fed mice. In order to quantitatively compare the extent of CREB-DNA interactions genome-wide between these two physiological conditions we developed a novel, robust analysis method, termed the ‘single sample independence’ (SSI) test that greatly reduced the number of false positive peaks. We found that CREB remains constitutively bound to its target genes in the liver regardless of the metabolic state. Integration of the CREB cistrome with expression microarrays of fasted and re-fed mouse livers and ChIP-seq data for additional transcription factors revealed that the gene expression switches between the fasted and fed states are associated with co-localization of additional transcription factors at CREB sites. Our results support a model in which CREB is constitutively bound to thousands of potential target genes and combinatorial interactions between DNA-binding factors are necessary to achieve the specific transcriptional response of the liver to fasting. Furthermore, our genome-wide analysis identifies thousands of novel CREB target genes in liver, including a previously unknown role for CREB in regulating ER stress genes in response to nutrient influx. Individual livers were collected from 24hr fasted and re-fed mice, with 4 biological replicates per condition. Extracted RNA was hybridized to Agilent two-color arrays, using randomly paired fasted/re-fed replicates.

ORGANISM(S): Mus musculus

SUBMITTER: Logan Everett

PROVIDER: E-GEOD-45731 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Little is known about the role of the transcription factor PPARÃ?/d in liver. Here we set out to better elucidate the function of PPARÃ?/d in liver by comparing the effect of PPARa and PPARÃ?/d deletion using whole genome transcriptional profiling and analysis of plasma and liver metabolites. In fed state, the number of genes altered by PPARa and PPARÃ?/d deletion was similar, whereas in fasted state the effect of PPARa deletion was much more pronounced, consistent with the pattern of gene expression of PPARa and PPARÃ?/d. Minor overlap was found between PPARa- and PPARÃ?/d-dependent gene regulation in liver. Pathways upregulated by PPARÃ?/d deletion were connected to innate immunity. Pathways downregulated by PPARÃ?/d deletion included lipoprotein metabolism and various pathways related to glucose utilization, which correlated with elevated plasma glucose and triglycerides and reduced plasma cholesterol in PPARÃ?/d-/- mice. Downregulated genes that may underlie these metabolic alterations included Pklr, Fbp1, Apoa4, Vldlr, Lipg, and Pcsk9, which may represent novel PPARÃ?/d target genes. In contrast to PPARa-/- mice, no changes in plasma FFA, plasma Ã?-hydroxybutyrate, liver triglycerides and liver glycogen were observed in PPARÃ?/d-/- mice. Our data indicate a role for PPARÃ?/d in hepatic glucose utilization and lipoprotein metabolism but not in the adaptive response to fasting. Keywords: Analysis of target gene regulation by using microarrays Pure-bred Sv129 PPARÎ± -/- mice and corresponding wildtype mice were used. Male mice (n=4-5 per group) were either fed or fasted for 24 hours. At the end of the experiment, mice were anaesthetized with a mixture of isofluorane (1.5%), nitrous oxide (70%) and oxygen (30%). Blood was collected by orbital puncture, after which the mice were sacrificed by cervical dislocation. Livers were dissected, snap frozen in liquid nitrogen and kept at -80ÂºC until further analysis. For RNA analyses, tissue from the same part of the liver lobe was used.

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Integrative genomic analysis of CREB defines a critical role for transcription factor networks in mediating the fed/fasted switch in liver [array]

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