CREB mediates transient and sustained genomic responses to cAMP via distinct mechanisms [ChIP-seq]

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. 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.

本研究通过对环腺苷酸反应元件结合蛋白（CREB）、CREB共激活因子、谱系决定转录因子及组蛋白乙酰化进行全基因组结合占位分析，旨在揭示环腺苷酸（cAMP）诱导胰岛组织特异性基因表达的潜在分子机制。CREB可介导cAMP对细胞基因表达的调控作用。绝大多数核心CREB靶基因在CREB及其共激活因子被招募至启动子近端结合位点后，会出现普遍性的表达诱导。我们发现，CREB可通过结合远端增强子区域内的位点，激活胰腺β细胞相关基因的表达。与核心靶基因受到的瞬时诱导效应不同，CREB以持续模式激活胰腺β细胞特异性基因的表达，这一现象反映了CBP介导的驻留核小体乙酰化水平提升，而该乙酰化可招募染色质阅读器BRD4。CREB可与谱系特异性激活因子Neurod1协同作用，在β细胞中构建cAMP响应型增强子。当Lrrc10b-Syt7超级增强子区域内CREB-Neurod1结合的增强子被敲除后，两个基因的表达均受到破坏，且葡萄糖诱导的胰岛素分泌水平下降。本研究结果证实，信号依赖型因子与谱系决定因子之间的协同作用，可响应胞外信号并调控细胞类型特异性基因程序的表达。实验整体设计：将细胞用0.75%甲醛固定10分钟，随后用125 mM甘氨酸淬灭反应5分钟。将冻存的细胞沉淀重悬于LB3缓冲液（10 mM Tris-HCl pH 8.0、100 mM NaCl、1 mM EDTA、0.5 mM EGTA、0.1% 脱氧胆酸钠、0.5% N-月桂酰肌氨酸钠、蛋白酶抑制剂混合物，Sigma P9599）中，随后使用Active Motif EpiShear探针式超声仪进行超声破碎。超声结束后，向提取物中添加Triton-X100至终浓度1%，并以17,000 g离心10分钟以澄清染色质提取物。取35 μl蛋白A琼脂糖磁珠（Thermo Scientific 20333），用PBS+0.1% BSA洗涤两次，随后与3 μg抗体在4℃下于PBS+0.1% BSA中旋转孵育4小时以偶联抗体。取500 μl提取物加入偶联抗体的磁珠中，4℃下旋转孵育过夜。用500 μl洗涤缓冲液1（20 mM Tris-HCl pH 7.4、150 mM NaCl、2 mM EDTA、0.1% SDS、1% Triton X-100）洗涤磁珠三次，再用500 μl洗涤缓冲液2（20 mM Tris-HCl pH 7.4、250 mM LiCl、1 mM EDTA、1% Triton X-100、0.7% 脱氧胆酸钠）洗涤磁珠三次。洗脱步骤如下：将磁珠置于50 μl洗脱缓冲液1（TE、1% SDS）中，50℃振荡孵育15分钟；再更换为50 μl洗脱缓冲液2（TE、1% SDS、300 mM NaCl），50℃振荡孵育15分钟。合并两次洗脱液，加入蛋白酶K和RNase A，37℃孵育2小时。将DNA-蛋白质复合物在65℃下振荡孵育过夜以解除交联。使用Agencourt AMPure XP磁珠（Beckman Coulter A63881）纯化染色质免疫沉淀（ChIP）所得DNA。[1] 多份样本生成的bed.txt文件以系列补充文件形式提供，详细说明见readme.txt，并在对应样本描述字段中予以标注。[2] bed.txt文件包含峰的基因组坐标信息，因此无法直接在基因组浏览器中展示。