Kolla Venkatadri, Naraparaju Koumudi, Zhuang Tiangang, Higashi Mayumi, Kolla Sriharsha, Blobel Gerd A, Brodeur Garrett M
*Division of Oncology, Department of Pediatrics, The Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia 19104, PA, U.S.A.
†Division of Hematology, Department of Pediatrics, The Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia 19104, PA, U.S.A.
Biochem J. 2015 Jun 1;468(2):345-52. doi: 10.1042/BJ20150030.
Eukaryotic gene expression is developmentally regulated, in part by chromatin remodelling, and its dysregulation has been linked to cancer. CHD5 (chromodomain helicase DNA-binding protein 5) is a tumour suppressor gene (TSG) that maps to a region of consistent deletion on 1p36.31 in neuroblastomas (NBs) and other tumour types. CHD5 encodes a protein with chromatin remodelling, helicase and DNA-binding motifs that is preferentially expressed in neural and testicular tissues. CHD5 is highly homologous to CHD3 and CHD4, which are the core subunits of nucleosome remodelling and deacetylation (NuRD) complexes. To determine if CHD5 forms a similar complex, we performed studies on nuclear extracts from NBLS, SY5Y (both with endogenous CHD5 expression), NLF (CHD5 null) and NLF cells stably transfected with CHD5 cDNA (wild-type and V5-histidine-tagged). Immunoprecipitation (IP) was performed with either CHD5 antibody or antibody to V5/histidine-tagged protein. We identified NuRD components both by GST-FOG1 (Friend Of GATA1) pull-down and by IP. We also performed MS/MS analysis to confirm the presence of CHD5 or other protein components of the NuRD complex, as well as to identify other novel proteins. CHD5 was clearly associated with all canonical NuRD components, including metastasis-associated protein (MTA)1/2, GATA zinc finger domain containing 2A (GATAD2A), histone deacetylase (HDAC)1/2, retinoblastoma-binding protein (RBBP)4/7 and methyl DNA-binding domain protein (MBD)2/3, as determined by Western blotting and MS/MS. Our data suggest CHD5 forms a NuRD complex similar to CHD4. However, CHD5-NuRD may also have unique protein associations that confer functional specificity and may contribute to normal development and to tumour suppression in NB and other cancers.
真核基因表达受发育调控，部分通过染色质重塑实现，其失调与癌症相关。CHD5（染色质结构域解旋酶DNA结合蛋白5）是一种肿瘤抑制基因（TSG），定位于神经母细胞瘤（NBs）和其他肿瘤类型中1p36.31区域的一致性缺失区域。CHD5编码一种具有染色质重塑、解旋酶和DNA结合基序的蛋白质，该蛋白质在神经组织和睾丸组织中优先表达。CHD5与CHD3和CHD4高度同源，CHD3和CHD4是核小体重塑和去乙酰化（NuRD）复合物的核心亚基。为了确定CHD5是否形成类似的复合物，我们对NBLS、SY5Y（均有内源性CHD５表达）、NLF（CHD5缺失）以及稳定转染CHD5 cDNA（野生型和V5-组氨酸标签型）的NLF细胞的核提取物进行了研究。使用CHD5抗体或V5/组氨酸标签蛋白抗体进行免疫沉淀（IP）。我们通过GST-FOG1（GATA1的朋友）下拉和IP鉴定NuRD成分。我们还进行了串联质谱（MS/MS）分析，以确认CHD5或NuRD复合物的其他蛋白质成分的存在，并鉴定其他新蛋白质。通过蛋白质免疫印迹法和MS/MS测定，CHD5显然与所有经典的NuRD成分相关，包括转移相关蛋白（MTA）1/2、含GATA锌指结构域2A（GATAD2A）、组蛋白脱乙酰基酶（HDAC）1/２、视网膜母细胞瘤结合蛋白（RBBP）4/7和甲基DNA结合结构域蛋白（MBD）2/3。我们的数据表明CHD5形成了一个类似于CHD4的NuRD复合物。然而，CHD5-NuRD也可能具有独特的蛋白质关联，赋予功能特异性，并可能有助于NB和其他癌症的正常发育和肿瘤抑制。
