Integrative genomic analysis of CREB defines a critical role for transcription factor networks in mediating the fed/fasted switch in liver [ChIP-seq]

Metabolic homeostasis in mammals critically depends on the regulation of fasting-induced genes by CREB in the liver. Previous genome-wide analysis has shown that only a small percentage of CREB target genes are induced in response to fasting-associated signaling pathways. The precise molecular mechanisms by which CREB specifically targets these genes in response to alternating hormonal cues remain to be elucidated. We performed chromatin immunoprecipitation coupled to high-throughput sequencing of CREB in livers from both fasted and re-fed mice. In order to quantitatively compare the extent of CREB-DNA interactions genome-wide between these two physiological conditions we developed a novel, robust analysis method, termed the 'single sample independence' (SSI) test that greatly reduced the number of false positive peaks. We found that CREB remains constitutively bound to its target genes in the liver regardless of the metabolic state. Integration of the CREB cistrome with expression microarrays of fasted and re-fed mouse livers and ChIP-seq data for additional transcription factors revealed that the gene expression switches between the fasted and fed states are associated with co-localization of additional transcription factors at CREB sites. Our results support a model in which CREB is constitutively bound to thousands of potential target genes and combinatorial interactions between DNA-binding factors are necessary to achieve the specific transcriptional response of the liver to fasting. Furthermore, our genome-wide analysis identifies thousands of novel CREB target genes in liver, including a previously unknown role for CREB in regulating ER stress genes in response to nutrient influx. Overall design: CREB ChIP-seq was performed on mouse liver from fasted and re-fed mice, using 5 separate biological replicates for each condition. GR and C/EBP(beta) ChIP-seq were performed as single replicates on ad-lib fed mice.

哺乳动物的代谢稳态核心依赖于肝脏中CREB对禁食诱导基因的调控。此前的全基因组分析显示，仅少数CREB靶基因会响应禁食相关信号通路而被诱导表达。CREB如何响应周期性激素信号而特异性靶向这些基因的精确分子机制，仍有待阐明。
我们对禁食及复喂养小鼠的肝脏组织开展了CREB的染色质免疫共沉淀结合高通量测序（chromatin immunoprecipitation coupled to high-throughput sequencing, ChIP-seq）实验。为了在全基因组层面定量比较这两种生理状态下CREB与DNA的结合程度，我们开发了一种名为"单样本独立性检验（single sample independence, SSI）"的新型稳健分析方法，该方法可大幅降低假阳性峰的数量。
我们发现，无论代谢状态如何，CREB都会在肝脏中持续性结合其靶基因。
将CREB顺反组（cistrome）与禁食及复喂养小鼠肝脏的表达微阵列（expression microarrays，基因芯片）数据，以及其他转录因子的ChIP-seq数据进行整合分析后，我们发现禁食与进食状态间的基因表达转换，与其他转录因子在CREB结合位点处的共定位存在关联。
我们的研究结果支持如下模型：CREB会持续性结合数千个潜在靶基因，而DNA结合因子间的协同互作，是实现肝脏对禁食产生特异性转录应答的必要条件。
此外，我们的全基因组分析在肝脏中鉴定出数千个全新的CREB靶基因，其中包括CREB在响应营养物质流入时调控内质网应激（ER stress）基因的此前未知的功能。
整体实验设计：针对禁食及复喂养小鼠的肝脏组织开展CREB ChIP-seq实验，每个生理条件设置5个独立生物学重复；糖皮质激素受体（glucocorticoid receptor, GR）及CCAAT增强子结合蛋白β（C/EBPβ）的ChIP-seq实验则以自由摄食小鼠为对象，仅设置1个生物学重复。