纯合失活致死基因ZFHX4对脑发育的影响及机制研究

8q21.11 microdeletion syndrome is the birth defect caused by the microdeletion of a segment of chromosome 8, characterized by intellectual disability, unusual behavior and some distinctive facial features. The minimum deletion region have been mapped to a DNA segment of 539.7Kb, in which ZFHX4 is the only one protein-coding gene. ZFHX4 is a transcription factor. The Zfhx4 knockout mice were generated, mice homozygous for the Zfhx4 deletion presented dyspnea, convulsions, no food intake, died within 24 hours and displayed abnormal brain morphology. Many apoptotic cells appeared and the expression of neural stem cell marker Nestin decreased in the Zfhx4-/- brain. Mice homozygous for the Zfhx4 deletion were viable, but there were also some apoptotic cells in their brains and displayed abnormality of learning and memory. So, we suspected that ZFHX4, as a transcription factor, plays important roles in the brain development by regulating gene expression. We will investigate: (1) effects of Zfhx4 homozygous deletion on the brain development; (2)effects of Zfhx4 heterozygous deletion on the ability of learning and memory; (3) confirm whether Zfhx4 directly regulates Nestin and screen other key target genes by using ChIP-seq and study the cellular and molecular mechanisms of Zfhx4 by the methods of DNA-protein/protein-protein interaction and rescue experiments. This research will provide theoretical basis and therapeutic target for the future treatment of 8q21.11 microdeletion syndrome.

8q21.11微缺失综合症主要表现为特殊面容、智障和行为异常。其最小缺失区已锁定在一539.7 kb区域，ZFHX4是该区域内唯一的编码基因，为一锌指结构蛋白。我们制备了Zfhx4敲除小鼠，纯合缺失小鼠出生后即呼吸困难和抽搐，并24h内死亡，出现脑形态异常、脑细胞凋亡增加、神经干细胞标志物Nestin表达降低。杂合子则表现出行为异常。我们推测ZFHX4作为一脑内特异高表达转录因子，通过调控关键基因的表达影响脑发育。本项目拟研究：（1）通过分子病理学手段分析其纯合缺失对小鼠脑发育的影响；（2）通过行为学检测杂合缺失小鼠的学习记忆能力变化；（3）通过ChIP、二代测序、GST-pulldown、体外脑片培养等手段，确定Zfhx4是否直接调控Nestin，筛选更多直接调控的靶基因，明确Zfhx4调控靶基因的分子机制。本项目有望为8q21.11微缺失综合症的防治提供理论依据和干预靶点。

Zfhx4是一种锌指转录因子，约有3550个氨基酸，带有4个同源结构域和22个锌指结构域，包括2个假锌指结构基序。Zfhx4在发育中的大脑和肌肉中显著表达，其胚胎期表达最高，而在成人中逐渐下降到几乎检测不到的水平，并在P19胚胎癌细胞的神经元分化过程中诱导表达。已有的研究报道表明ZFHX4是8q21.11微缺失综合症（8q21.11 Microdeletion Syndrome）中唯一蛋白编码基因，目前关于该基因的功能与机制研究甚少。我们希望通过小鼠动物模型结合分子机制研究，部分阐明Zfhx4的生物学功能。.我们采用QPCR的方法检测了P0小鼠的各个脏器中Zfhx4的表达水平，发现Zfhx4主要表达于小鼠的脑组织中，尤其是脑干部位。进一步比较不同发育时期的小鼠脑组织，结果显示Zfhx4在胚胎时期尤其是F16.5的时候高表达，随着出生表达下降。全脑免疫荧光结果显示，Zfhx4在小鼠的中后脑表达，并且在脑桥呼吸群、腹侧呼吸群以及背侧呼吸群等部位显著高表达，提示Zfhx4在小鼠的呼吸系统中发挥重要作用。PB转座子介导的Zfhx4纯合缺失小鼠(Zfhx4PB/PB)出生后即出现紫绀，抽搐，无法进食等行为，表型分析发现其呼吸节律相较于野生型小鼠明显降低，再次提示Zfhx4对呼吸系统的建立至关重要。Zfhx4PB/PB小鼠的脑干组织的表达谱芯片结果显示，Zfhx4基因的有效缺失伴随着与呼吸节律生成相关的基因（Phox2b, Math1, Lbx1）的表达的显著降低。免疫荧光染色显示，Zfhx4在脑干中与Phox2b和Math1共表达，随着Zfhx4的敲除的同时也伴随着Phox2b和Math1的表达降低，尤其是在与先天性中央性换气不足综合征(CCHS)相关的旁呼吸群/后梯形核(pFRG/RTN)区域表达的Phox2b和Math1显著降低。ChIP-Seq与mRNA表达芯片结合分析，鉴定Phox2b为Zfhx4的直接下游靶基因，并采用ChIP-qPCR验证。本研究发现Zfhx4是Phox2b表达的关键调控因子，对围产期呼吸至关重要。