Role of RFX4 in Brain Development and Function

该项目的启动是因为观察到，在六个心脏特异性过表达人CYP2J2的转基因品系之一的小鼠中，有很大比例的小鼠出现了头部肿胀，随后神经迅速恶化，并在成年后死亡。我们假设转基因已经中断了一个重要基因的编码或调节区。我们鉴定了与单个转基因插入位点相邻的5个启动子和3个启动子的基因组序列，发现它们与包含RFX4基因座的人类12号染色体序列高度相关。转基因被插入到RFX4基因的一个内含子中，该插入阻止了一种新的变异型转录本(称为RFX4v3)的表达，该转录本导致先天性脑积水的发生。我们发现，通过Southern blotting和PCR可以很容易地区分野生型()和转基因中断的等位基因(一个中断的等位基因-和两个中断的等位基因)。来自半合子(-)小鼠的大脑表达约50%的正常水平的RFX4v3转录本，并表现出严重的先天性侧脑室和第三脑室积水，并伴随着连合下器官(SCO)的形成失败，导致Sylvius导水管狭窄。相比之下，纯合子空(--)小鼠在端脑形成方面存在严重缺陷，导致出生前大脑严重畸形，并在围产期死亡。事实上，对E12.5的小鼠的研究表明，它们失去了关键的中线结构，包括大脑半球间裂隙，导致形成一个中央脑室，而不是两个侧脑室。尽管脊髓也是畸形的，但面部结构、视网膜、嗅觉上皮和所有其他被检查的非脑组织在小鼠中没有受到影响。克隆了人和小鼠的RFX4v3cDNAs，同源性为96%，表明这一变异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RFX4v3转录本既有独特的外显子，又有与其他转录本相同的外显子。RFX4v3转录本只在脑内表达，最初出现在E7.5到E9.5之间。原位杂交结果显示，RFX4v3在脑发育过程中呈高度动态表达。重要的是，在胚胎14.5天的尾侧间脑，RFX4v3在发育中的SCO区域有丰富的表达。综上所述，这些数据表明，RFX4v3对正常的大脑发育至关重要。此外，在小鼠体内形成SCO的失败表明，这个不寻常的脑器官对RFX4v3的正常发育表达极其敏感。为了鉴定RFX4v3野生型和突变型小鼠的脑内差异表达基因，从胚胎第10.5天(E10.5)的小鼠头部提取RNA，与Agilent小鼠寡核苷酸芯片进行杂交。在本研究中使用了四对单独的RNA和一对汇集的RNA，每对样品被杂交到两个寡核苷酸微阵列中，每个RNA样品被每个荧光团标记以解释荧光团的掺入偏差。在5对样本中，至少有4对中有34个基因上调，75个基因下调。在异常值列表中选择了24个基因进行实时PCR验证，基于i)微阵列分析的相对较大的折叠变化，以及ii)在大脑发育过程中潜在的重要功能。实时荧光定量聚合酶链式反应结果显示，24个基因中有22个基因被证实表达差异，与微阵列数据相比有相似或更大的倍数变化。微阵列数据和实时聚合酶链式反应结果之间的高度相关性证明了微阵列在脑形态发生过程中识别潜在的转录因子下游靶基因的有效性。其中一些经过验证的基因是众所周知的神经元发育调节基因。例如，Cx3cl1是RFX4v3通过其启动子中特定的X-box(X-box1)直接转录的靶标，X-box1是RFX蛋白的反应元件。为了确定RFX4v3的潜在相互作用伙伴，我们进行了酵母双杂交筛选。确定了9个候选相互作用因子，包括G蛋白途径抑制因子2(GPS2)。GPS2和RFX4v3主要(如果不是排他性的)在细胞核中表达。GPS2和RFX4v3信息都存在于成年小鼠大脑的大部分区域，这表明这两种蛋白可能相互结合。免疫共沉淀分析表明，GPS2和RFX4v3之间确实在体外发生了物理作用。GPS2还可以通过X-box 1增强Cx3cl1启动子上的RFX4v3的激活，表明这种相互作用在脑发育中对RFX4v3的活性也具有重要的功能意义。基于这些数据，我们得出结论，GPS2与RFX4v3相互作用，调节包括Cx3Cl1在内的多种参与脑形态发生的基因的反式激活。对其他潜在下游基因的检测可能有助于更好地理解RFX4v3在脑发育中潜在的分子机制。目前，我们正在与Xenogen的研究人员合作开发RFX4的条件敲除，以便我们可以检查该基因在成人大脑中的功能。我们还在研究RFX4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.