RNA-seq expression profiling of cerebral cortices from Taf8 deleted and Taf8 intact mouse embryos

Mutations in genes encoding general transcription factors cause neurological disorders. Despite clinical prominence, the consequences of defects in the basal transcription machinery during brain development are unclear. We found that loss of the TATA-box binding protein associated factor TAF8, a component of the general transcription factor TFIID, in the developing central nervous system affected the expression of many, but notably not all genes. Taf8 deletion caused apoptosis, unexpectedly restricted to forebrain regions. Nuclear levels of the transcription factor p53 were elevated in the absence of TAF8, as were the mRNAs of the pro-apoptotic p53 target genes Noxa, Puma and Bax. The cell death in Taf8-/- forebrain regions was completely rescued by additional loss of p53, but Taf8-/-p53-/- brains failed to initiate a neuronal expression program. Taf8 deletion caused aberrant transcription of promoter regions and splicing anomalies. We propose that TAF8 supports the directionality of transcription and co-transcriptional splicing, and that failure of these processes causes p53- induced apoptosis of neuronal cells in the developing mouse embryo. Taf8 was deleted in the developing mouse central nervous system (CNS) using our Taf8 conditional allele. In the absence of global effects of loss of TAF8 on RNA levels, we determined effects on individual gene loci by RNA sequencing of cortices from Taf8 deleted (E14.5 Taf8lox/-NesCreT/+p53-/-) and control (E14.5 Taf8+/+NesCreT/+p53-/-) cortices. The p53-deficient background was used to avoid complications due to apoptosis, but prevented us from examining changes in p53-induced gene transcription. Four Taf8 deleted and four control cortical samples were prepared, each from a separate mouse. Stranded and unstranded RNA-seq libraries were prepared from each RNA sample.编码通用转录因子（general transcription factors）的基因突变可引发神经系统疾病。尽管该类疾病在临床上备受关注，但脑发育过程中基础转录装置（basal transcription machinery）缺陷所导致的具体效应仍未明确。本研究发现，在发育中的中枢神经系统内，通用转录因子TFIID（transcription factor TFIID）的组成组分TATA盒结合蛋白相关因子8（TATA-box binding protein associated factor 8, TAF8）的缺失，会影响大量而非全部基因的表达。TAF8缺失可引发细胞凋亡（apoptosis），且该细胞死亡意外地仅局限于前脑（forebrain）区域。在TAF8缺失的情况下，转录因子p53的核内水平显著升高，促凋亡p53靶基因Noxa、Puma与Bax的mRNA表达量亦同步上调。TAF8单敲除小鼠前脑区域的细胞死亡可通过额外敲除p53完全挽救，但TAF8与p53双敲除（Taf8-/-p53-/-）小鼠的大脑无法启动神经元表达程序。TAF8缺失会导致启动子区域转录异常与剪接异常。我们提出如下假说：TAF8可维持转录的方向性与共转录剪接过程，上述过程的缺陷会引发p53介导的发育小鼠胚胎神经元细胞凋亡。本研究利用TAF8条件性等位基因（conditional allele）在发育中的小鼠中枢神经系统中特异性敲除Taf8。鉴于TAF8缺失未对RNA整体水平产生全局性影响，我们通过对Taf8敲除组（E14.5 Taf8lox/-NesCreT/+p53-/-）与对照组（E14.5 Taf8+/+NesCreT/+p53-/-）的小鼠皮层进行RNA测序（RNA sequencing, RNA-seq），分析其对单个基因位点的调控效应。本研究采用p53缺陷背景以规避细胞凋亡带来的实验干扰，但这也使得我们无法检测p53诱导的基因转录变化。本研究共制备4份Taf8敲除皮层样本与4份对照皮层样本，每份样本均取自单只独立小鼠。每份RNA样品均被用于构建链特异性与非链特异性RNA测序文库（RNA-seq libraries）。