An ENU-induced point mutation in the mouse Btaf1 gene causes post-gastrulation embryonic lethality and protein instability

The mouse Btaf1 gene, an ortholog of yeast MOT1, encodes a highly conserved general transcription factor. The function of this SNF-2 like ATPase has been studied mainly in yeast and human cells, which has revealed that it binds directly to TBP, forming the B-TFIID complex. This complex binds to core promoters of RNA polymerase II- transcribed genes and, of crucial importance, BTAF1-TBP interactions have been shown to affect the kinetics of TBP-promoter interactions. Here we report the isolation of a mouse line carrying a Btaf1 allele containing an ENU-induced point mutation that causes a substitution mutation in the BTAF1 ATPase domain. Embryos homozygous for this loss-of-function mutation appear to be morphologically normal until early somite stages, but die between embryonic day 9 and 10.5 displaying growth arrest and edema. Analyses in vitro suggest that the altered protein is less stable and, independent from this, functionally impaired in releasing of TBP from chromatin, but not in binding to TBP. A microarray screen for genes with altered expression in the Btaf V1330M mutant as compared to WT embryos was performed with RNA from whole embryos. We compared mRNA transcripts from pools of Btaf1-/- and Btaf1+/+ embryos. Where possible, we used 3-5-somite embryos, a stage we considered to be immediately prior to the emergence of a perceptible phenotype. The experiments were done in duplo, i.e. two sets of comparisons were made: in one case stage matching (i.e. number of somites counted) was the highest priority, in the other case the pools were designed such that a maximum number of litter mates were compared while a difference of up to five somites was allowed. cDNAs were synthesized and Cy-3/Cy-5 labeled cRNAs were generated by using a Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies, Santa Clara, CA). The labeled cRNAs were hybridized on 4X44K Agilent Whole Mouse Genome Dual Color Microarrays (G4122F). Two dye-swap experiments were performed, resulting in four individual arrays. Microarray signal and background information were retrieved with Feature Extraction (V9.5.3, Agilent technologies). All data analyses were performed by using ArrayAssist (5.5.1, Stratagene Inc, La Jolla, CA) and Microsoft Excel (Microsoft Corporation, Redmont, WA). Genetic background: C57BL/6J were mutagenized, and crossed against FVB/NJ mice for positional mapping. The background of the mice used in the experiment is hybrid C57BL/6J *FVB/NJ with a percentage FVB between 50 and 90%.小鼠Btaf1基因是酵母MOT1的同源基因，编码一种高度保守的通用转录因子。这类属于SNF-2样ATP酶的蛋白的功能主要在酵母和人类细胞中得到研究，结果显示其可直接结合TBP（TATA盒结合蛋白），形成B-TFIID复合物。该复合物可结合RNA聚合酶II转录基因的核心启动子，尤为关键的是，已有研究证实BTAF1与TBP的相互作用会影响TBP-启动子相互作用的动力学特性。 本研究报道了一种携带Btaf1等位基因的小鼠品系的分离构建：该等位基因含有ENU（N-乙基-N-亚硝基脲）诱导的点突变，会导致BTAF1的ATP酶结构域发生氨基酸替换突变。携带该功能丧失型突变的纯合子胚胎在早期体节阶段之前形态学上均表现正常，但会在胚胎发育第9天至10.5天之间死亡，表现为生长停滞与水肿。 体外实验分析表明，该突变后的蛋白质稳定性下降，且独立于稳定性降低之外，其在从染色质上释放TBP的功能上存在缺陷，但结合TBP的功能并未受影响。本研究利用全胚胎RNA开展了芯片筛选，以比对Btaf1 V1330M突变体与WT（野生型）胚胎中表达异常的基因。我们对Btaf1-/-和Btaf1+/+胚胎的mRNA转录本进行了比对，在条件允许的情况下，选用了3-5体节期的胚胎——我们认为该阶段恰好是可观测表型出现之前的时期。 实验采用平行重复设计，共开展两组比对：第一组以发育阶段匹配（即体节计数一致）为最高优先级；第二组的样本池设计则优先保证尽可能多的同窝胚胎进行比对，同时允许体节数最多相差5个。使用Low RNA Input Fluorescent Linear Amplification Kit（Agilent Technologies，加利福尼亚州圣克拉拉）合成cDNA，并通过该试剂盒制备Cy-3/Cy-5标记的cRNA。将标记好的cRNA与4X44K Agilent Whole Mouse Genome Dual Color Microarrays（G4122F）进行杂交。本研究开展了两次染料互换实验，最终得到四张独立的芯片。 使用Feature Extraction（V9.5.3，Agilent Technologies）获取芯片信号与背景信息。所有数据分析均通过ArrayAssist（5.5.1，Stratagene Inc，加利福尼亚州拉霍亚）与Microsoft Excel（Microsoft Corporation，华盛顿州雷德蒙德）完成。 遗传背景：研究人员对C57BL/6J品系小鼠进行了ENU诱变，并将其与FVB/NJ小鼠杂交以进行图位克隆。本实验所用小鼠的遗传背景为杂交型C57BL/6J * FVB/NJ，其FVB基因组占比为50%至90%。