CHD5, A Brain-Specific Chromatin Remodeling Enzyme, Regulates Expression Of Neuronal Genes.

CHD5 is frequently deleted in neuroblastoma, and appears to be a tumor suppressor gene; however, little is known about the role of CHD5. We found CHD5 mRNA was restricted to brain; by contrast most other remodeling ATPases were broadly expressed. CHD5 protein isolated from mouse brain was associated with HDAC2, p66, MTA3 and RbAp46 in a megadalton complex. CHD5 protein was detected in several rat brain regions and appeared to be enriched in neurons. CHD5 protein was predominantly nuclear in primary rat neurons and brain sections. Microarray analysis revealed genes that were upregulated and downregulated when CHD5 was depleted from primary neurons. CHD5 depletion altered expression of neuronal genes, transcription factors, and brain-specific subunits of the SWI/SNF remodeling enzyme. Aging and Alzheimers gene sets were strongly affected by CHD5 depletion from primary neurons. Chromatin immunoprecipitation revealed CHD5 bound to these genes, suggesting the regulation was direct. Together, these results indicate that CHD5 is found in a NuRD-like multi-protein complex. CHD5 is restricted to the brain, unlike the closely related family members CHD3 and CHD4. CHD5 regulates expression of neuronal genes, cell cycle genes and remodeling genes. CHD5 is linked to regulation of aging and Alzheimer’s genes. CHD5 KD shRNA sequences were designed according to the instructions for the pLKO.1 system (Addgene). Control was as described (Sci307-1098,2005) Scramble, clone 1864 from Addgene). Virus was packaged using HEK-293T cells, pLKO.1 vector with shRNA inserts for CHD5, and the control. 48 hours after transfection of 293 cells, medium containing virus was filtered (0.45 micron), then applied for 6 hours to primary cortical neurons one day after the neurons were plated (Day 1). Medium was removed, and replaced with Neural Basal Medium, and the cells were cultured until Day 5, 9 or 12. RNA was harvested from 3 replicates of the treated primary cortical neurons at each time point. RNA was isolated using RNAeasy Kit (Qiagen), Quality and quantity of the total RNA was checked with the Agilent 2100 bioanalyzer using RNA 6000 Nano chips. RNA was labeled using the standard Illumina protocol and Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX, cat # IL1791) Biotin labeled cRNA was hybridized to Illumina's Sentrix Rat Ref-12 v1 Expression BeadChips.

CHD5是神经母细胞瘤中常见缺失的基因，被认为是一种肿瘤抑制基因（tumor suppressor gene），但目前对其功能所知甚少。我们发现CHD5的信使RNA（mRNA）仅在脑组织中表达；与之形成对比的是，大多数其他重塑ATP酶（remodeling ATPase）的表达范围较为广泛。从小鼠脑组织中分离得到的CHD5蛋白，以兆道尔顿级复合物的形式与HDAC2、p66、MTA3及RbAp46相结合。在多个大鼠脑区中均可检测到CHD5蛋白，且似乎在神经元中富集。原代大鼠神经元及脑组织切片中的CHD5蛋白主要定位于细胞核。微阵列分析（microarray analysis）揭示了原代神经元中CHD5敲低后上调和下调的基因。CHD5敲低会改变神经元基因、转录因子以及SWI/SNF重塑酶（SWI/SNF remodeling enzyme）脑特异性亚基的表达。衰老和阿尔茨海默病（Alzheimer’s）相关基因集在原代神经元CHD5敲低后受到显著影响。染色质免疫沉淀（chromatin immunoprecipitation）实验显示，CHD5可结合至这些基因，表明其调控作用是直接的。综合以上结果，表明CHD5存在于一个类似NuRD的多蛋白复合物中。与密切相关的家族成员CHD3和CHD4不同，CHD5的表达仅局限于脑组织。CHD5可调控神经元基因、细胞周期基因及重塑相关基因的表达。CHD5与衰老及阿尔茨海默病相关基因的调控存在关联。CHD5的短发夹RNA（shRNA）敲低序列是按照pLKO.1系统（Addgene）的操作指南设计的。对照组设置如前文所述（Sci307-1098,2005），即来自Addgene的1864号克隆的随机序列（Scramble）。病毒包装使用HEK-293T细胞、携带CHD5 shRNA插入片段的pLKO.1载体及对照载体。转染293细胞48小时后，收集含病毒的培养基并以0.45微米滤膜过滤，随后在原代皮层神经元接种1天后（第1天）将其施加于神经元，处理6小时。移除培养基，更换为神经基础培养基（Neural Basal Medium），将细胞培养至第5、9或12天。在每个时间点，从3份重复的处理组原代皮层神经元中提取RNA。总RNA的提取使用RNAeasy试剂盒（Qiagen），总RNA的质量与浓度通过Agilent 2100生物分析仪结合RNA 6000 Nano芯片进行检测。RNA的标记按照Illumina标准流程进行，使用Illumina TotalPrep RNA扩增试剂盒（Ambion; 美国德克萨斯州奥斯汀，货号IL1791）。将生物素标记的互补RNA（cRNA）与Illumina的Sentrix Rat Ref-12 v1表达微珠芯片进行杂交。