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Characterize CREB target genes in different tissue types

ABSTRACT: The CREB family of transcription factors stimulates cellular gene expression following phosphorylation at a conserved serine (Ser133 in CREB1) in response to cAMP and other extracellular signals. In order to characterize CREB target genes in various tissues, we give a cAMP agonist, forskolin (FSK), to cell lines or primary cultures and monitor the gene expression. To eliminate CREB-independent effects of FSK on cellular gene expression, we employed a dominant negative form of CREB called A-CREB, which dimerizes selectively with and blocks the DNA binding activity of CREB but not other bZIP family members. Therefore, genes that are induced by cAMP and the induction was blocked by A-CREB treatment likely represents CREB target genes. Notes: 1) In HEK293T cells, besides the Control+FSK+(FSK-ACREB) experiments, a different set of experiments showing FSK effect on 1hr and 4hr is included. The two sets of data in HEK293T were generated at different times with different batch of cells, and comparison should be limited within each set. The cAMP induced genes at 1hr, however, was similar between the two sets. 2) These is no ACREB data for pancreatic islets or hepatocytes. For hepatocytes, however, we have included fasting liver and refed liver in additional to FSK treated primary hepatocytes. During fasting, glucagon induces cAMP increase in the liver and CREB is activated. Therefore, a more reliable list of CREB target genes in hepatocytes can be obtained by selecting those genes are that induced both during fasting and in FSK treated primary culture. Keywords: parallel sample

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE2060 | GEO | 2005/03/02

SECONDARY ACCESSION(S): PRJNA91305

REPOSITORIES: GEO

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Project description:During fasting, increases in circulating pancreatic glucagon maintain glucose balance by up-regulating hepatic gluconeogenesis. Triggering of the cAMP pathway stimulates the gluconeogenic program through the phosphorylation of CREB and via the de-phosphorylation of the CREB coactivator CRTC2. Hormonal and nutrient signals are also thought to modulate gluconeogenic genes by promoting epigenetic changes that facilitate assembly of the transcriptional machinery, although the nature of these modifications is unclear. Here we show that histone H3 acetylation at Lys 9 (H3K9Ac) is elevated over gluconeogenic genes during fasting and in diabetes, where it contributes to increases in hepatic glucose production. Following its dephosphorylation, CRTC2 promoted increases in H3K9Ac by mediating the recruitment of the lysine acetyltransferase 2B (KAT2B) and WD repeat-containing protein 5 (WDR5), a core subunit of histone methyltransferase (HMT) complexes. In turn, KAT2B and WDR5 stimulated the gluconeogenic program through a self-reinforcing cycle whereby increases in H3K9Ac further potentiated CRTC2 occupancy at CREB binding sites. Breaking this cycle, by depletion of KAT2B or WDR5, decreased gluconeogenic gene expression. As administration of a small molecule KAT2B antagonist lowered circulating blood glucose concentrations in insulin resistance, our results demonstrate how this enzyme may be a useful target for diabetes treatment. A subset of cAMP responsive genes depend on specific recruitment of KAT2B (pcaf), which in concert with WDR5 acetylates H3K9. By selectively depleting hepatocytes for KAT2B or WDR5 prior to glucagon stimulation we explore, which genes rely on KAT2B and WDR5 activity. mKAT2B or mWDR5 were knocked down in primary mouse hepatocytes using adenoviral transduction with appropriate shRNAs. Control cells were transduced with a non-specific (NS) shRNA. 72 hours post transduction some cells were stimulated for 90 minutes with 100nM glucagon and others with PBS. Total RNA was purified and subjected to micro-RNA analysis. All samples are pools of RNA from three sepearate dishes. One replicate is included for most samples.

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Characterize CREB target genes in different tissue types

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