Role of RFX4 in Brain Development and Function

该项目的启动是由于观察到在六个心脏特异性过表达人CYP 2 J2的转基因品系中的一个中，大部分小鼠表现出头部肿胀，随后迅速神经功能恶化并在成年早期死亡。我们假设转基因中断了一个重要基因的编码或调控区。我们鉴定了与单个转基因插入位点相邻的5 ′和3 ′基因组序列，发现它们与含有RFX 4基因座的人类12号染色体序列高度相关。将转基因插入RFX 4基因的内含子中，这种插入阻止了导致先天性脑积水发展的新变体转录本（称为RFX 4v 3）的表达。我们发现，野生型（）和转基因中断等位基因（-为一个中断等位基因和-为两个中断等位基因）可以很容易地区分Southern印迹和PCR。来自半合子（-）小鼠的脑表达约50%的正常水平的RFX 4v 3转录物，并表现出与连合下器官（SCO）形成失败相关的侧脑室和第三脑室的严重先天性脑积水，导致Sylvius导水管狭窄。与此相反，纯合子无效（--）小鼠端脑形成有严重缺陷，导致产前脑畸形和围产期死亡。事实上，对E12.5小鼠的研究表明，它们失去了关键的中线结构，包括半球间裂，导致形成单个中央脑室，而不是两个侧脑室。虽然脊髓也畸形，面部结构，视网膜，嗅上皮和所有其他非脑组织检查不受影响的-小鼠。克隆了人和小鼠RFX 4v 3 cDNA，发现96%相同，表明该变体转录本在这两个物种之间高度保守。基因组序列分析表明，小鼠RFX 4v 3转录本由独特的和共享的外显子与其他RFX 4转录本。RFX 4v 3转录物仅在脑中表达，最初出现在E7.5和E9.5之间。原位杂交结果显示，RFX 4v 3的表达是高度动态的大脑发育过程中。重要的是，在E14.5时，在尾侧间脑中发育中的SCO区域发现RFX 4v 3的丰富表达。总之，这些数据表明RFX 4v 3对正常的大脑发育至关重要。此外，SCO在小鼠中形成的失败表明，这种不寻常的脑器官对RFX 4v 3的正常发育表达极其敏感。 为了鉴定RFX 4v 3野生型和突变型小鼠脑中差异表达的基因，从胚胎第10.5天（E10.5）的头部分离RNA，并用Agilent小鼠寡核苷酸微阵列进行杂交。在本研究中使用四对单独的RNA和一对合并的RNA，并且每对样品与两个寡核苷酸微阵列杂交，每个RNA样品用每个荧光团标记以解释荧光团掺入偏倚。 在5对样本中至少有4对样本中，34个基因表达上调，75个基因表达下调。基于i）通过微阵列分析的相对大的倍数变化，和ii）在脑发育期间的潜在重要功能，选择离群值列表中的24个基因用于通过实时PCR进行验证。实时PCR结果表明，24个基因中的22个被证实差异表达，与微阵列数据相比，具有相似或更大的倍数变化。 微阵列数据和实时PCR结果之间的高度相关性证明了使用微阵列在脑形态发生过程中识别转录因子的潜在下游靶基因的实用性。一些已验证的基因是众所周知的神经元发育调节因子。例如，Cx 3cl 1通过其启动子中的特异性X-box（X-box 1）是RFX 4v 3的直接转录靶标，所述启动子是RFX蛋白的响应元件。为了鉴定RFX 4v 3的潜在相互作用伴侣，我们进行了酵母双杂交筛选。9个候选的相互作用被确定，包括G蛋白途径抑制因子2（GPS 2）。GPS 2和RFX 4v 3主要（如果不是排他的话）在细胞核中表达。 GPS 2和RFX 4v 3信息都存在于成年小鼠大脑的大部分区域，这表明这两种蛋白质可以相互结合。免疫共沉淀试验表明，GPS 2和RFX 4v 3之间的物理相互作用确实发生在体外。GPS 2还可以通过X-box 1增强Cx 3cl 1启动子上的RFX 4v 3激活，这表明这种相互作用对于RFX 4v 3在脑发育中的活性也具有功能意义。基于这些数据，我们得出结论，GPS 2与RFX 4v 3相互作用，以调节包括Cx 3Cl 1在内的多种参与脑形态发生的基因的反式激活。其他潜在的下游基因的检查可能会导致更好地了解RFX 4v 3在大脑发育中的功能的分子机制。目前，我们正在与Xenogen的研究人员合作开发RFX 4的条件性敲除，以便我们可以检查该基因在成人大脑中的功能。 我们也在研究RFX 4启动子，以确定调节其表达的相关转录因子结合位点。

This project was initiated by the observation that a large percentage of mice in one of six transgenic lines with cardiac-specific overexpression of human CYP2J2 exhibited head swelling followed by rapid neurological deterioration and death in young adulthood. We hypothesized that the transgene had interrupted the coding or regulatory region of an important gene. We identified the 5 prime and 3 prime genomic sequences adjacent to the single transgene insertional site and found them to be highly related to a human chromosome 12 sequence that contained the RFX4 locus. The transgene was inserted into an intron of the RFX4 gene and this insertion prevented expression of a novel variant transcript (termed RFX4v3) which led to the development of congenital hydrocephalus. We found that wild type () and transgene-interrupted alleles (- for one interrupted allele and -- for two interrupted alleles) could be readily distinguished by Southern blotting and PCR. Brains from hemizygous (-) mice expressed approximately 50% of normal levels of the RFX4v3 transcript and exhibited severe congenital hydrocephalus of the lateral and third ventricles associated with failure of formation of the subcommissural organ (SCO), leading to stenosis of the aqueduct of Sylvius. In contrast, the homozygous null (--) mice had a severe defect in telencephalon formation that led to gross prenatal brain malformations and death in the perinatal period. Indeed, investigation of -- mice at E12.5 showed that they had lost critical midline structures including the interhemispheric fissure resulting in the formation of a single central ventricle instead of two lateral ventricles. Although the spinal cords were also malformed, facial structures, retinas, olfactory epithelium and all other non-brain tissues examined were unaffected in the -- mice. The human and mouse RFX4v3 cDNAs were cloned and found to be 96% identical, indicating that this variant transcript was highly conserved between these two species. Analysis of genomic sequences revealed that the mouse RFX4v3 transcript was composed of both unique and shared exons with other RFX4 transcripts. The RFX4v3 transcript was expressed only in brain and initially appeared between E7.5 and E9.5. In situ hybrization revealed that RFX4v3 expression was highly dynamic during brain development. Importantly, abundant expression of RFX4v3 was found in the region of the developing SCO in the caudal diencephalon at E14.5. Together, these data indicate that RFX4v3 is critical for normal brain development. Moreover, the failure of formation of the SCO in - mice suggests that this unusual brain organ is extremely sensitive to normal developmental expression of RFX4v3. To identify the differentially expressed genes in the brains of RFX4v3 wild-type and mutant mice, RNAs were isolated from the embryonic day 10.5 (E10.5) heads and hybridizations were performed with the Agilent mouse oligo microarrays. Four pairs of individual RNAs and one pair of pooled RNAs were used in the present studies and each pair of samples was hybridized to two oligonucleotide microarrays with each RNA sample labeled with each fluorophore to account for fluorophore incorporation bias. Thirty-four genes were up-regulated and 75 genes were down-regulated in at least 4 out of 5 pairs of samples. Twenty-four genes in the outlier lists were chosen for validation by real-time PCR based on i) relatively large fold changes by microarray analysis, and ii) potentially important functions during brain development. Real-time PCR results indicated that 22 out of 24 genes were confirmed to express differentially with similar or larger fold changes compared to microarray data. The high correlation between microarray data and real-time PCR results demonstrates the utility of using microarrays in identifying potential downstream target genes of transcription factors during brain morphogenesis. Some of the validated genes are well-known regulators for neuronal development. For example, Cx3cl1 is the direct transcriptional target of RFX4v3 through a specific X-box (X-box1) in its promoter, the responsive element for RFX proteins. To identify the potential interacting partners for RFX4v3, we performed yeast two-hybrid screening. Nine candidate interactors were identified, including G-protein pathway suppressor 2 (GPS2). GPS2 and RFX4v3 are predominantly, if not exclusively, expressed in the nuclei. Both GPS2 and RFX4v3 messages are present in most portions of the adult mouse brain, suggesting that the two proteins could bind to each other. Co-immunoprecipitation assays indicate that physical interactions between GPS2 and RFX4v3 do indeed occur in vitro. GPS2 could also potentiate the RFX4v3 activation on the Cx3cl1 promoter through X-box 1, suggesting the interaction is also functionally significant for RFX4v3 activity in brain development. Based on these data, we conclude that GPS2 interacts with RFX4v3 to modulate transactivation of a variety of genes involved in brain morphogenesis including Cx3Cl1. Examination of other potential downstream genes may lead to better understanding of the molecular mechanisms underlying the function of RFX4v3 in brain development. Currently, we are working with investigators at Xenogen to develop a conditional knockout for RFX4 so that we can examine the function of this gene in the adult brain. We are also studying the RFX4 promoter to determine relevant transcription factor binding sites that regulate its expression.