Transcriptomic analysis of wild type and Del(Hotair)-/- mouse tissues

Despite decades of interest, the mechanisms that control Hox gene expression are not yet fully understood. It was recently proposed that Hotair, a lncRNA transcribed from the HoxC cluster, regulates HoxD gene expression via Polycomb targeting and thus is important for correct skeletal development. However, genetic manipulations of the locus led to conflicting results regarding the roles of Hotair. Here, we analyze the molecular and phenotypic consequences of deleting the Hotair locus in vivo. In contradiction with previous findings, we show that deleting Hotair has no detectable effect on HoxD gene expression in vivo. We could not observe any morphological alteration in mice lacking the Hotair locus. However, we find a significant impact of deleting Hotair on the expression of neighboring genes Hoxc11 and Hoxc12. Our results do not support an RNA-dependent role for Hotair in vivo, but argue in favor of a DNA-dependent effect of Hotair deletion on the transcriptional landscape in cis. We micro-dissected wild type and Del(Hotair)-/- E12.5 embryos into 6 segments: forelimbs (FL), hindlimbs (HL), genital tubercle (GT), trunk section corresponding to the lumbar/sacral region (T1); trunk section corresponding to the sacral/caudal region (T2) and trunk section corresponding to the caudal region (T3). We generated strand-specific RNA-seq data for each segment, in two biological replicates and we performed differential expression analyses for each tissue. Furthermore, we analyzed the impact of deleting the Hotair locus on the local transcriptional landscape, in the HoxC cluster.尽管学界对同源盒（Hox）基因的表达调控机制已有数十年的研究兴趣，但相关机制至今尚未完全阐明。此前有研究提出，源自同源盒C（HoxC）基因簇转录的长链非编码RNA（long non-coding RNA, lncRNA）Hotair，可通过靶向多梳蛋白复合体（Polycomb）调控同源盒D（HoxD）基因的表达，进而对正常骨骼发育发挥重要作用。然而，针对该基因座的遗传操作实验，却得到了关于Hotair功能相互矛盾的研究结果。本研究通过体内敲除Hotair基因座，分析其带来的分子与表型层面的后果。与此前的研究结论相悖，我们发现体内敲除Hotair后，并未检测到同源盒D（HoxD）基因的表达出现显著变化；在缺失Hotair基因座的小鼠体内，亦未观察到任何形态学异常。但我们发现，敲除Hotair会对相邻基因Hoxc11与Hoxc12的表达产生显著影响。本研究结果并不支持Hotair在体内以RNA依赖的方式发挥功能，反而表明Hotair敲除通过顺式（cis）作用，对转录组景观产生了DNA依赖的影响。我们将胚胎发育第12.5天（E12.5）的野生型及Del(Hotair)-/-纯合突变小鼠胚胎，显微切割为6个组织片段：前肢（forelimbs, FL）、后肢（hindlimbs, HL）、生殖结节（genital tubercle, GT）、对应腰/骶区域的躯干段（T1）、对应骶/尾区域的躯干段（T2）以及对应尾区域的躯干段（T3）。我们为每个组织片段设置2次生物学重复，生成了链特异性RNA测序（RNA-seq）数据，并针对每个组织开展了差异表达分析。此外，我们还分析了敲除Hotair基因座对同源盒C（HoxC）基因簇局部转录组景观的影响。