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| Status |
Public on Jun 16, 2019 |
| Title |
CREB mediates transient and sustained genomic responses to cAMP via distinct mechanisms [ChIP-seq] |
| Organisms |
Mus musculus; Rattus norvegicus |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
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| Summary |
We investigated genome-wide occupancy of CREB, CREB coactivators, lineage determining transcription factors and histone acetylation to uncover mechanisms behind tissue-specific gene induction by cAMP in pancreatic islets. CREB mediates effects of cAMP on cellular gene expression. Most core CREB target genes are ubiquitously induced following recruitment of CREB and its coactivators to promoter proximal binding sites. We found that CREB stimulates the expression of pancreatic beta cell genes by binding to sites within distal enhancers. By contrast with its transient effects on core target genes, CREB stimulates pancreatic beta cell specific gene expression in a sustained manner, reflecting increases in the CBP-mediated acetylation of resident nucleosomes that recruit the chromatin reader BRD4. CREB cooperates with the lineage specific activator Neurod1 in establishing cAMP-responsive enhancers in beta cells. As deletion of a CREB-Neurod1 bound enhancer within the Lrrc10b-Syt7 super-enhancer locus disrupted the expression of both genes and decreased glucose-induced insulin secretion, our results demonstrate how cooperativity between signal dependent and lineage determining factors promotes the expression of cell type-specific gene programs in response to extracellular cues.
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| Overall design |
Cells were fixed in 0.75% formaldehyde for 10 min and quenched with 125 mM glycine for 5 min. Frozen cell pellets were resuspended in buffer LB3 (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-deoxycholate, 0.5% N-laurylsarcosine, protease inhibitor cocktail, Sigma P9599) and sonicated (Active Motif EpiShear Probe Sonicator). After sonication, Triton-X100 was added to the extract (1% final) and chromatin was cleared by centrifugation at 17,000 g for 10 min. 35 μl protein A agarose beads (Thermo Scientific 20333) were washed twice with PBS + 0.1% BSA and coupled with 3 ug antibody for 4 hours in PBS + 0.1% BSA at 4°C with rotation. 500 μl extract was added to antibody coupled beads and incubated overnight at 4°C with rotation. Beads were washed three times in 500 μl wash buffer 1 (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100) and three times in wash buffer 2 (20 mM Tris-HCl pH 7.4, 250 mM LiCl, 1 mM EDTA, 1% Triton X-100, 0.7% Na-deoxycholate). Elution was then performed by incubating beads in 50 μl elution buffer 1 (TE, 1% SDS) for 15 min at 50°C and 50 μl elution buffer 2 (TE, 1% SDS, 300 mM NaCl) for 15 min at 50°C while shaking. Both elutions were combined and incubated with proteinase K and RNase A for 2 hours at 37°C. DNA-protein complexes were de-crosslinked at 65°C while shaking overnight. ChIP DNA was purified using Agencourt AMPure XP beads (Beckman Coulter A63881).
[1] The bed.txt files generated from multiple samples are linked as Series supplementary files, described in the readme.txt in detail, and indicated in the corresponding sample description field.
[2] The bed.txt contains genomic coordinates of peaks and thus, can not be displayed in the genome browser.
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| Contributor(s) |
Van de Velde S |
| Citation(s) |
31182641 |
| Submission date |
Feb 14, 2019 |
| Last update date |
Jun 16, 2019 |
| Contact name |
Sam Van de Velde |
| Organization name |
Salk Institute
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| Department |
PBL
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| Street address |
10010 North Torrey Pines Road
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platforms (2) |
| GPL17021 |
Illumina HiSeq 2500 (Mus musculus) |
| GPL18694 |
Illumina HiSeq 2500 (Rattus norvegicus) |
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| Samples (107)
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| This SubSeries is part of SuperSeries: |
| GSE126558 |
CREB mediates transient and sustained genomic responses to cAMP via distinct mechanisms |
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| Relations |
| BioProject |
PRJNA522364 |
| SRA |
SRP185889 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE126556_BRD4inducedRNA.bed.txt.gz |
3.4 Kb |
(ftp)(http) |
TXT |
| GSE126556_BRD4repressedRNA.bed.txt.gz |
19.8 Kb |
(ftp)(http) |
TXT |
| GSE126556_CREBNeuroD1PDX1cobound.bed.txt.gz |
6.9 Kb |
(ftp)(http) |
TXT |
| GSE126556_CREBNeuroD1cobound.bed.txt.gz |
1.6 Kb |
(ftp)(http) |
TXT |
| GSE126556_CREBPDX1cobound.bed.txt.gz |
53.8 Kb |
(ftp)(http) |
TXT |
| GSE126556_CREBnoNeuroD1PDX1.bed.txt.gz |
97.8 Kb |
(ftp)(http) |
TXT |
| GSE126556_CREBtargetgenesinsuperenhancersRNA.bed.txt.gz |
2.1 Kb |
(ftp)(http) |
TXT |
| GSE126556_CREBtargetspromoterdrivenRNA.bed.txt.gz |
698 b |
(ftp)(http) |
TXT |
| GSE126556_FSKupgenesRNA.bed.txt.gz |
2.8 Kb |
(ftp)(http) |
TXT |
| GSE126556_HAcKoverCREB4000differential.xlsx |
3.6 Mb |
(ftp)(http) |
XLSX |
| GSE126556_INS1inducibleenhancers.bed.txt.gz |
10.1 Kb |
(ftp)(http) |
TXT |
| GSE126556_INS1noninducibleenhancers.bed.txt.gz |
45.1 Kb |
(ftp)(http) |
TXT |
| GSE126556_NeuroD1noCREB.bed.txt.gz |
8.1 Kb |
(ftp)(http) |
TXT |
| GSE126556_PDX1NeuroD1noCREB.bed.txt.gz |
11.2 Kb |
(ftp)(http) |
TXT |
| GSE126556_PDX1noCREB.bed.txt.gz |
141.5 Kb |
(ftp)(http) |
TXT |
| GSE126556_RAW.tar |
20.5 Mb |
(http)(custom) |
TAR (of TXT) |
| GSE126556_readme.txt |
2.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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