Global analysis of the interplay between site-specific CREB O-GlcNAc glycosylation and phosphorylation

Purpose: CREB (cAMP response element binding protein) is a transcription factor that is critical for learning and memory. The activity of CREB is mediated through its post-translational modifications (PTMs); specifically, phosphorylation at serine 133 and glycosylation at serine 40. In this study, we used RNA-Seq and weighted gene network coexpression analysis (WGCNA) to determine the CREB-mediated transcriptional programmes that are regulated by phosphorylation at serine 133 and glycosylation at serine 40 through the use of various PTM-deficient CREB mutants. Methods: The mRNA profiles of E16.5 Creb1-/- mouse cortical neurons expressing GFP, WT CREB, S40A-CREB, S133A-CREB, and S40A-S133A-CREB were generated by deep sequencing, in triplicate, using Illumina HiSeq 2500. The sequence reads that passed quality filters were analyzed using Bowtie, aligned using TopHat, and quantified using Cufflinks in Galaxy. Results: Through differential expression analysis with glycosylation-deficient (S40A) and phosphorylation-deficient (S133A) CREB mutants, we show that CREB O-GlcNAcylation is important for neuronal activity and excitability, while phosphorylation at serine 133 regulates the expression of genes involved in neuronal differentiation. Furthermore, many of the S40A and S133A differentially-expressed genes were directly bound by (1) CREB and its co-activators, CREB-binding protein and p300, (2) activating histone modifications, (3) OGT and O-GlcNAc, and (4) Tet1, an critical regulator of neuronal activity and differentiation. Finally, we observed a positive correlation between S40A and activity- and excitotoxicity-related gene networks and a negative correlation between S133A and neuronal differentiation and amino and fatty acid metabolism-related gene networks. This study demonstrates that CREB O-GlcNAcylation at serine 40 and phosphorylation mediate mutually exclusive gene networks. Together, O-GlcNAc and phosphorylation impart a TF code, which CREB must integrate and decode to modulate neuronal activity, differentiation, and metabolism. The mRNA profiles from E16.5 Creb1-/- mouse cortical neurons expressing glycosylation- and/or phosphorylation-deficient and WT CREB using HSV (herpes simplex virus) were generated by deep sequencing, in duplicate or quadruplicate, using the Illumina HiSeq 2500.

研究目的：环腺苷酸应答元件结合蛋白（cAMP response element binding protein, CREB）是一类对学习与记忆至关重要的转录因子（transcription factor）。其活性通过翻译后修饰（post-translational modifications, PTMs）介导，具体为丝氨酸133位的磷酸化与丝氨酸40位的糖基化。本研究借助RNA测序（RNA-Seq）与加权基因共表达网络分析（weighted gene coexpression analysis, WGCNA），通过构建多种PTM缺陷型CREB突变体，解析受丝氨酸133磷酸化及丝氨酸40糖基化调控的CREB介导转录程序。
实验方法：将表达绿色荧光蛋白（GFP）、野生型（wild-type, WT）CREB、S40A-CREB、S133A-CREB及S40A-S133A-CREB的E16.5 Creb1基因敲除（Creb1-/-）小鼠皮层神经元的mRNA表达谱，采用Illumina HiSeq 2500平台进行三次生物学重复深度测序获取。对通过质量过滤的序列读段，在Galaxy分析平台中使用Bowtie进行序列预处理、TopHat完成基因组比对，并通过Cufflinks进行表达量定量。
实验结果：通过对糖基化缺陷型（S40A）与磷酸化缺陷型（S133A）CREB突变体开展差异表达分析，本研究证实：CREB的O-连接N-乙酰葡糖胺糖基化（O-GlcNAcylation）对神经元活性与兴奋性具有关键作用，而丝氨酸133位的磷酸化则调控神经元分化相关基因的表达。进一步分析显示，诸多在S40A与S133A突变体中呈现差异表达的基因，可被四类分子直接结合：（1）CREB及其共激活因子CREB结合蛋白（CREB-binding protein）与p300；（2）激活型组蛋白修饰复合物；（3）O-连接N-乙酰葡糖胺转移酶（O-GlcNAc transferase, OGT）与O-GlcNAc；（4）Tet1——神经元活性与分化的核心调控因子。此外，本研究观察到S40A突变体的差异基因集与神经元活性、兴奋毒性相关基因网络呈正相关，而S133A突变体的差异基因集则与神经元分化、氨基酸及脂肪酸代谢相关基因网络呈负相关。本研究证明，CREB丝氨酸40位的O-GlcNAc糖基化与磷酸化介导了两套相互排他的基因调控网络。综上，O-GlcNAc糖基化与磷酸化共同构成了一套转录因子编码（TF code），CREB需整合并解码这套编码，以精准调控神经元活性、分化与代谢过程。本研究补充数据集：采用单纯疱疹病毒（herpes simplex virus, HSV）介导表达糖基化和/或磷酸化缺陷型及野生型CREB的E16.5 Creb1-/-小鼠皮层神经元的mRNA表达谱，通过Illumina HiSeq 2500平台进行二次或四次生物学重复深度测序获取。