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基因作为酵母MOT1的直系同源基因，编码一种高度保守的通用转录因子。这类类似SNF-2的ATP酶的功能主要在酵母与人类细胞中得到研究，研究显示其可直接结合TATA盒结合蛋白（TATA-binding protein, TBP），并形成B-TFIID复合物。该复合物可结合RNA聚合酶II（RNA polymerase 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。
本研究报道了一株携带Btaf1等位基因的小鼠品系，该等位基因含有由N-乙基-N-亚硝基脲（ENU）诱导的点突变，该突变会导致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，该突变后的蛋白稳定性下降，且独立于此之外，其在将TBP从染色质上释放的功能上存在缺陷，但与TBP结合的功能并未受影响。
本研究利用全胚胎RNA，针对Btaf1 V1330M突变型胚胎相较于野生型（wild type, WT）胚胎中表达存在差异的基因进行了微阵列筛选。我们对Btaf1-/-与Btaf1+/+胚胎混合样本中的mRNA转录本进行了比较分析。在条件允许的情况下，我们选取了3-5体节期的胚胎——该阶段被我们认为是可观测表型出现前的临界时期。本实验采用重复设计，即开展两组比对实验：一组以胚胎发育阶段匹配（即体节计数一致）为首要原则；另一组则在允许最多5个体节差异的前提下，尽可能选取同窝胚胎进行混合样本比对。
我们通过使用Agilent Technologies（美国加利福尼亚州圣克拉拉市）的Low RNA Input Fluorescent Linear Amplification Kit试剂盒，合成了cDNA并制备了经Cy-3/Cy-5标记的cRNA。将标记好的cRNA与4×44K Agilent Whole Mouse Genome Dual Color Microarrays（货号G4122F）进行杂交。开展了两次染料互换实验，最终得到四张独立的芯片杂交数据。使用Feature Extraction软件（版本V9.5.3，Agilent Technologies）获取微阵列的信号与背景信息。所有数据分析均通过ArrayAssist软件（版本5.5.1，Stratagene Inc，美国加利福尼亚州拉霍亚市）与Microsoft Excel（微软公司，美国华盛顿州雷德蒙德市）完成。
遗传背景：研究人员对C57BL/6J小鼠进行诱变，并将其与FVB/NJ小鼠杂交以进行定位作图。本实验所用小鼠的遗传背景为C57BL/6J与FVB/NJ的杂交品系，FVB基因组占比介于50%至90%之间。