CREB binds to multiple loci on human chromosome 22

Abstract

The cyclic AMP-responsive element-binding protein (CREB) is an important transcription factor that can be activated by hormonal stimulation and regulates neuronal function and development. An unbiased, global analysis of where CREB binds has not been performed. We have mapped for the first time the binding distribution of CREB along an entire human chromosome. Chromatin immunoprecipitation of CREB-associated DNA and subsequent hybridization of the associated DNA to a genomic DNA microarray containing all of the nonrepetitive DNA of human chromosome 22 revealed 215 binding sites corresponding to 192 different loci and 100 annotated potential gene targets. We found binding near or within many genes involved in signal transduction and neuronal function. We also found that only a small fraction of CREB binding sites lay near well-defined 5' ends of genes; the majority of sites were found elsewhere, including introns and unannotated regions. Several of the latter lay near novel unannotated transcriptionally active regions. Few CREB targets were found near full-length cyclic AMP response element sites; the majority contained shorter versions or close matches to this sequence. Several of the CREB targets were altered in their expression by treatment with forskolin; interestingly, both induced and repressed genes were found. Our results provide novel molecular insights into how CREB mediates its functions in humans.

FIG. 1.

Immunoprecipitation of CREB1. (A) CREB1 immunoprecipitated as a single 43-kDa band with the CREB NT antibody in JEG-3 cells. The CREB NT antibody was used for both the immunoprecipitation (lanes 1 and 2) and the immunoblot (lane 3) analysis. (B) PCR analysis of a control CREB1 target region after chromatin immunoprecipitation with the CREB NT antibody. The region around the tandem CREs in the hCGα promoter was selectively amplified in DNA purified from CREB1 immunoprecipitations in JEG-3 cells, whereas no enrichment was seen in a genomic region not known to bind CREB.

FIG. 2.

Map of CREB1 binding regions on chromosome 22q. (A) The sequence is ordered from centromere to telomere on the long arm of chromosome 22. The dark gray center band represents all of the nonrepetitive genomic sequence included on the DNA microarray. Sanger annotated genes are oriented 5′ to 3′ above the center band and 3′ to 5′ below the center band. Colored bars depict the differential expression analysis results in response to forskolin: upregulated genes are red, downregulated genes are green, and nondifferentially regulated genes are yellow. Blue triangles indicate the positions of CREB1-bound fragments. (B and C) Higher-resolution examples of genes with CREB1-bound fragments. (B) A total of 14 CREB1 binding regions occur within or the near the genes SERPINDI and SNAP29 (top strand) and PIK4CA (lower strand). (C) A total of six CREB1 binding regions occur within or near the genes for MAPK1, PPM1F, and TOP3b. All three genes are on the lower strand.

FIG. 3.

Real-time PCR analysis of CREB1 chromatin immunoprecipitations. DNA from forskolin-treated and untreated JEG-3 cells was immunopurified with anti-CREB1 antibody and amplified (blue circles) and compared to control experiments lacking antibody (pink squares) and control reactions lacking template (yellow triangles). Graphs show averages of triplicate PCRs. Units are ΔRn (fluorescence) on the y axis and PCR cycle number on the x axis. One ΔC_t (where C_t denotes threshold cycle) corresponds to approximately twofold enrichment. Enrichment of CREB1-bound regions is observed as an increase in fluorescence at earlier PCR cycle numbers than the no-antiserum controls. Examples shown are the positive control, hCGα promoter (A); the negative control, chromosome 22 nonspecific region (B); CACNA1I, 74.5-kbp fragment internal to the downregulated Ca²⁺ channel voltage-dependent α1I subunit (C); and dJ930L11.1, 69.7-kbp internal fragment (D). This gene is similar to KIAA0397, which has RabGAP domains and may be involved in membrane traffic. (E) Fragment >10 kb from the annotated region within chromosomal coordinates 23262970 to 23263520.

FIG. 4.

Position of CREB1 binding regions relative to chromosome 22 annotations. CREB1 binding sites were mapped relative to 5′ ends, introns, exons, internal introns and exons, and transcriptionally active regions (TARs).

FIG. 5.

Model of CREB pathway and possible positions of proteins whose genes contain CREB1 binding sites. Proteins whose mRNAs are upregulated, downregulated, or not affected by forskolin are indicated in the red, green, and yellow circles, respectively. Components of signaling pathways (light blue shapes) that converge on CREB are adapted from reference . MIF, macrophage migration inhibitory factor; CELSR1, cadherin epidermal growth factor LAG seven-pass G-type receptor; ARHGAP8, rho GTPase-activating protein 8; dJ930L11.1, similar to the RabGAP domain-containing gene KIAA0397; Em:AP000347.C22.3, similar to mouse guanine nucleotide dissociation stimulator RALGDS; Cabin 1, calcineurin binding protein; PACSIN2, protein kinase C and casein kinase substrate in neurons 2; ERK2/MAPK1, mitogen-activated protein kinase 1; PPM1F, protein phosphatase 1F; ADRBK2, adrenergic beta receptor kinase 2.