Transcription profiling of mouse thymus from ZAS3-null vs wild-type

The HIVEP/ZAS/Schnurri genes encode large zinc finger proteins that regulate gene expression through DNA binding to the kappaB motif. The ZAS proteins have also been shown to associate with signaling molecules to regulate the TNFa and TGFb signaling pathways. Because ZAS3 transcript and protein expression is rapidly abrogated in primary lymphocytes upon tissue culture, we have generated a ZAS3-null mouse model to study the physiological function of ZAS3. Mice with targeted disruption of ZAS3 are viable with life span comparable to controls. Additionally, the gross anatomy and histology of the major organs, proliferation rates of splenocytes and thymocytes, and the diversity of the TCRb chains of ZAS3 mice are comparable to wild-type. However, compared to wild-type mice, there are decreased CD3+CD4+ and CD3+CD4+CD69+ thymocyte populations. Additionally, CD44hi/CD62Llo subset and CD25 and CD69 expression are increased in splenocytes of ZAS3 mice. Microarray analysis validated by real time PCR revealed changes in the expression level of specific transcripts in ZAS3-null thymus, including several proteins in the G-protein coupled olfactory receptor family. Furthermore, EMSA shows that disruption of ZAS3 results in varying changes in binding activities towards NF-kB and AP1 binding sites. Other phenotypes observed in the ZAS3 mice include generalized increased bone density in older animals and infertility in females. As with ZAS2/shn-2 mice, disruption of ZAS3 is not lethal and does not grossly affect development or homeostasis. We propose that the ZAS proteins likely have overlapping functions, which may be uncovered with deletion of multiple ZAS genes in a single animal. Experiment Overall Design: Five independent microarray hybridizations were performed with thymocytes isolated from sex- and age-matched mutant and wild-type littermates at 6-8 weeks of age. The Agilent Whole Mouse Genome Oligo Microarray (G4121A) containing over 20,000 60-mer oligonucleotide probes representing mouse genes, ESTs and EST clusters was used (Agilent, Cincinnati, OH). Probe labeling and hybridization were preformed by the Microarray Core Faculty (Columbus Children’s Research Institute) following the manufacturer's protocols. Briefly, cDNA probes were synthesized with Superscript III, oligo-dT primers and dNTPs supplemented with amino-allyl-UTP to improve hybridization characteristics and stability. After purification, cDNA samples from mutant or control mice were labeled with Cy3 or Cy5, respectively, and hybridized to the microarray for 14 hours at 48o C. Slide image was acquired using an Affymetrix 428 scanner with gain settings set so that 95% of spots were below saturation to yield the maximum dynamic range within an experiment. Images were converted into “.gpr” files using GenePix software (Axon, Union City, CA). Data were analyzed using GeneTraffic 2.6 software (Iobion, La Jolla, CA). Lowess-global normalization was applied to all experiments. Flagging parameters were set as spot intensity lower than the intensity of local spot background, spot intensity lower than average background, and raw spot intensity less than 100. Flagged spots were not included in normalization or aggregate calculations. Only genes that were induced or repressed 1.5-fold in at least four of the five independent analyses and hybridization were targeted for potential further study.HIVEP/ZAS/Schnurri基因编码大型锌指蛋白，可通过与κB基序（kappaB motif）结合DNA调控基因表达。研究表明，ZAS蛋白还可与信号分子结合，进而调控肿瘤坏死因子α（TNFα）与转化生长因子β（TGFβ）信号通路。由于在体外培养原代淋巴细胞时，ZAS3的转录本与蛋白表达会快速被抑制，我们构建了ZAS3敲除小鼠模型（ZAS3-null mouse model）以研究ZAS3的生理功能。 定向敲除ZAS3的小鼠可正常存活，寿命与野生型对照小鼠相当。此外，ZAS3敲除小鼠的主要器官大体解剖与组织学特征、脾细胞与胸腺细胞的增殖速率，以及T细胞受体β链（TCRβ chains）多样性均与野生型小鼠无显著差异。但与野生型小鼠相比，ZAS3敲除小鼠的CD3+CD4+及CD3+CD4+CD69+胸腺细胞群体数量有所减少。同时，ZAS3敲除小鼠脾细胞中CD44hi/CD62Llo亚群比例，以及CD25与CD69的表达水平均有所升高。 经实时PCR（real time PCR）验证的芯片分析结果显示，ZAS3敲除小鼠胸腺中特定转录本的表达水平发生了改变，其中包括G蛋白偶联嗅觉受体家族的多种蛋白。此外，电泳迁移率变动分析（EMSA）结果表明，ZAS3敲除会导致核因子κB（NF-κB）与活化蛋白1（AP-1）结合位点的结合活性出现不同程度的变化。ZAS3敲除小鼠还表现出其他表型：老年小鼠出现全身性骨密度升高，雌性小鼠存在不育问题。与ZAS2/shn-2小鼠一致，ZAS3敲除不会导致小鼠死亡，也不会显著影响其发育或体内稳态。我们推测ZAS蛋白可能存在功能重叠，若在单只动物中同时敲除多个ZAS基因，可能会揭示其潜在功能。 实验总体设计：选取6~8周龄、性别与年龄匹配的突变型与野生型同窝仔鼠，分离其胸腺细胞进行5次独立的芯片杂交实验。使用安捷伦全小鼠基因组寡核苷酸芯片（Agilent Whole Mouse Genome Oligo Microarray, G4121A），该芯片包含超过20000条代表小鼠基因、表达序列标签（Expressed Sequence Tags, ESTs）及EST簇的60聚体寡核苷酸探针（安捷伦公司，俄亥俄州辛辛那提）。探针标记与杂交步骤由哥伦布儿童医院研究所芯片核心实验室严格按照制造商的实验方案完成。 简要而言，使用SuperScript III反转录酶、寡聚dT引物及添加了氨基烯丙基UTP的脱氧核苷三磷酸（dNTPs）合成cDNA探针，以提升杂交特性与稳定性。纯化后，将突变型与对照组小鼠的cDNA样本分别用菁3（Cy3）与菁5（Cy5）标记，随后在48℃下与芯片杂交14小时。使用Affymetrix 428扫描仪采集芯片图像，设置增益参数使95%的斑点信号未达到饱和，以确保实验获得最大动态范围。使用GenePix软件（Axon公司，加州联合城）将图像转换为".gpr"格式文件。使用GeneTraffic 2.6软件（Iobion公司，加州拉霍亚）进行数据分析。对所有实验数据应用Lowess全局归一化处理。标记参数设置为：斑点强度低于局部背景强度、斑点强度低于平均背景强度，以及原始斑点强度低于100。被标记的斑点不纳入归一化或汇总计算环节。仅选取在5次独立分析与杂交实验中至少4次出现1.5倍上调或下调的基因，作为后续潜在研究的目标。