Genome-wide analysis of cAMP-response element binding protein occupancy, phosphorylation, and target gene activation in human tissues - PubMed (original) (raw)

. 2005 Mar 22;102(12):4459-64.

doi: 10.1073/pnas.0501076102. Epub 2005 Mar 7.

Duncan T Odom, Seung-Hoi Koo, Michael D Conkright, Gianluca Canettieri, Jennifer Best, Huaming Chen, Richard Jenner, Elizabeth Herbolsheimer, Elizabeth Jacobsen, Shilpa Kadam, Joseph R Ecker, Beverly Emerson, John B Hogenesch, Terry Unterman, Richard A Young, Marc Montminy

Affiliations

Genome-wide analysis of cAMP-response element binding protein occupancy, phosphorylation, and target gene activation in human tissues

Xinmin Zhang et al. Proc Natl Acad Sci U S A. 2005.

Abstract

Hormones and nutrients often induce genetic programs via signaling pathways that interface with gene-specific activators. Activation of the cAMP pathway, for example, stimulates cellular gene expression by means of the PKA-mediated phosphorylation of cAMP-response element binding protein (CREB) at Ser-133. Here, we use genome-wide approaches to characterize target genes that are regulated by CREB in different cellular contexts. CREB was found to occupy approximately 4,000 promoter sites in vivo, depending on the presence and methylation state of consensus cAMP response elements near the promoter. The profiles for CREB occupancy were very similar in different human tissues, and exposure to a cAMP agonist stimulated CREB phosphorylation over a majority of these sites. Only a small proportion of CREB target genes was induced by cAMP in any cell type, however, due in part to the preferential recruitment of the coactivator CREB-binding protein to those promoters. These results indicate that CREB phosphorylation alone is not a reliable predictor of target gene activation and that additional CREB regulatory partners are required for recruitment of the transcriptional apparatus to the promoter.

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Figures

Fig. 1.

Fig. 1.

Identification of CREB target genes through bioinformatic analysis. (a) Frequency of CREs in promoter, intergenic, intronic, and exonic regions of the human genome, expressed as number of sites per megabase of DNA. Relative occurrence of full-site (TGACGTCA) and half-site (TGACG/CGTCA) CREs in each category is shown. (b) Percent conservation of full and half CRE sites in orthologous sequences from human, rat, and mouse genomes. (c) Relative distribution of promoter-associated CRE sites that are conserved or not conserved between species as a function of distance from the transcription start site. (d) Identification of CRE-containing genes in the human genome by using three independent methods (conserved CRE, CRE model + position, and CRE cluster). Number of genes identified by each method and overlap between the three methods is shown. Selected Gene Ontology categories enriched in predicted CREB target genes are shown.

Fig. 2.

Fig. 2.

CRE methylation blocks CREB binding to nonfunctional sites. (a) CRE methylation frequency on promoters and exonic, intronic, or intergenic regions, as determined by endonuclease assay with methylation-sensitive restriction enzyme Aat II. (b) Effect of CRE methylation on CREB occupancy in HEK293T cells by ChIP assay. (Upper) Relative binding of CREB to unmethylated or methylated CREs in five different promoters for each group by using anti-CREB antiserum. Input levels of DNA (1%) for each gene are indicated; nonspecific IgG control is shown. (Lower) PCR analysis of genomic fragments showing that methylated CREs are resistant to Aat II digestion, whereas unmethylated CREs are completely digested. (c) Summary of CRE methylation and CREB-binding patterns in promoters, intergenic, or intronic/exonic regions of the genome. * marks one CREB-occupied CRE (LRRTM2 gene) that was partially methylated in HEK293T cells. (d) Relative methylation state (color-coded) of 34 genes across seven cell contexts. Genes are clustered based on methylation profiles.

Fig. 3.

Fig. 3.

cAMP stimulates Ser-133 phosphorylation uniformly over CREB-occupied genes. (a) Comparison of CREB occupancy and presence of CREs in human promoters. For each promoter category (those with predicted CRE, those without predicted CRE, and all genes), the distribution of confidence levels (P values) from ChIP-chip results is shown. Graph shows percentage of promoters within each category at specific _P_-value range. P values were computed from three independent CREB ChIP experiments with HEK293T cells. (b) Analysis of P-CREB levels over human promoters by ChIP-chip assay of HEK293T cells at 0, 1, and 4 h after exposure to FSK. Randomly selected subsets of ChIP-positive CREB target genes from cAMP responsive and nonresponsive genes were used to compare P-CREB profiles. (c) Western blot assay showing levels of total CREB and P-CREB in HEK293T cells after FSK treatment for times indicated.

Fig. 4.

Fig. 4.

cAMP stimulates distinct profiles of CREB target genes in different tissues. (a) Venn diagram showing overlap between CREB target genes in human islets, hepatocytes, and HEK293T cells. (b) ChIP-chip assay of promoters occupied by CREB and P-CREB in human hepatocytes compared with HEK293T cells. Top 1,000 and bottom 1,000 scoring genes from hepatocyte P-CREB ChIP-chip assays were plotted. Relative binding ratio observed for each gene is color-coded. (c) Comparison of P-CREB binding profiles from ChIP-chip assays of HEK293T cells, human pancreatic islets, and human hepatocytes. Top 200 and bottom 200 scoring genes from islet P-CREB ChIP-chip assays were plotted. (d) ChIP assay of HEK293T cells showing effects of FSK on recruitment of CBP to cAMP inducible (NR4A2) vs. noninducible (CDC37) genes. Levels of P-CREB on each promoter are indicated. (e) Quantitative PCR assay showing relative levels of CBP over cAMP inducible and noninducible promoters after 0, 1, or4hofFSK treatment by ChIP.

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