Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Tunable regulation of CREB DNA binding activity couples genotoxic stress response and metabolism

ABSTRACT: In this study we show that, in embryonic fibroblasts from mice on a high fat diet and treated with Forskolin, ionizing radiation exposure or both, phosphorylation of CREB-binding protein (CREB) by ATM (ataxia-telangiectasia-mutated) and casein kinases 1 and 2 (CK1 and CK2) on a cluster of five phosphorylation sites (the ATM/CK cluster) within the unstructured kinase-inducible domain (KID) provides an additional level of regulation through dynamic modulation of CREB DNA binding activity. Stoichiometric phosphorylation of the ATM/CK cluster in response to DNA damage inhibited cAMP-induced CREB target gene expression, CREB DNA binding activity, and CREB-CRTC2-DNA ternary complex formation proportional to the number of phosphate residues modified. Substoichiometric phosphorylation of the ATM/CK cluster promoted cAMP/Ca2+-regulated transcriptional coactivators (CRTCs) recruitment and CREB activation via an ATM-independent, PKA-dependent pathway. Mice expressing a non-phosphorylatable CREBS111A allele exhibited phenotypes consistent with CREB deregulation, including fasting hyperglycemia, susceptibility to diet-induced obesity, and reduced expression of gluconeogenic genes. Two genotypes: CREB+/+ (wild type) and CREBS111A (non-phosphorylatable CREB KID S111A mutant allele) each control treated, exposed to forskolin, ionizing radiation or both in triplicate and in two batches toataling 48 arrays

ORGANISM(S): Mus musculus

SUBMITTER: Pierre Bushel

PROVIDER: E-GEOD-83891 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The LH surge triggers dramatic transcriptional changes in genes associated with ovulation and luteinization. The present study investigated the spatiotemporal expression of nuclear factor interleukin-3 (NFIL3), a transcriptional regulator of the bZIP transcription factor superfamily, and its potential role in the ovary during the periovulatory period. NFIL3, also known as E4-binding protein 4 or NFIL3/E4BP4, was originally identified as a transcriptional repressor based on its DNA-binding activity at the promoter of the gene encoding the adenovirus E4 protein. Immature female rats were injected with PMSG, treated with hCG, and ovaries or granulosa cells were collected at various times after hCG. Nfil3 mRNA was highly induced both in intact ovaries and granulosa cells after hCG treatment. In situ hybridization demonstrated that Nfil3 mRNA was highly induced in theca-interstitial cells at 4-8 h after hCG, localized to granulosa cells at 12 h, and decreased at 24 h. Over-expression of NFIL3 in granulosa cells inhibited the induction of prostaglandin-endoperoxide synthase 2 (Ptgs2), progesterone receptor (Pgr), epiregulin (Ereg), and amphiregulin (Areg) and down regulated levels of prostaglandin E2. The inhibitory effect on Ptgs2 induction was reversed by NFIL3 siRNA treatment. In theca-interstitial cells the expression of hydroxyprostaglandin dehydrogenase 15-(NAD) (Hpgd) was also inhibited by NFIL3 over-expression. Data from luciferase assays demonstrated that NFIL3 over-expression decreased the induction of the Ptgs2 and Areg promoter activity. EMSA and ChIP analyses indicated that NFIL3 binds to the promoter region containing the DNA binding sites of CREB and C/EBP?. In summary, hCG induction of NFIL3 expression may modulate the process of ovulation and theca-interstitial and granulosa cell differentiation by regulating expression of PTGS2, PGR, AREG, EREG, and HPGD, potentially through interactions with CREB and C/EBP? on their target gene promoters. The granulosa cells were exposed to Ad- NFIL3 or Ad-GFP at a multiplicity of infection (MOI) of 50 pfu/cell. Two hours later, Medium was replaced with fresh Opti-MEM medium. At 24 h after adenovirus exposure, granulosa cells were treated with FSK + PMA for 6 h before collection for RNA isolation using a RNeasy kit according to the manufacturer’s instructions (Qiagen Inc., Valencia, CA). Five micrograms of total RNA were used as a template for cDNA synthesis by the University of Kentucky Microarray Core facility as described previously (47). The Affymetrix Rat 230-2.0 oligonucleotide array sets were hybridized, washed, and scanned using Affymetrix equipment and protocols (Affymetrix, Santa Clara, CA). The DNA microarray assays were performed on total RNA pooled from granulosa cells obtained from 3 separate experiments. The changes observed by DNA microarray analysis were confirmed by real-time PCR for a select subset of genes. Affymetrix GCS 3000 7G scanner was used.

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Tunable regulation of CREB DNA binding activity couples genotoxic stress response and metabolism

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