FOXE3 Contributes to Peters Anomaly through Transcriptional Regulation of an Autophagy Associated Protein termed DNAJB1

FOXE3 is a lens specific transcription factor that has been associated with anterior segment ocular dysgenesis. To determine the transcriptional target(s) of FOXE3 that are indispensable for the anterior segment development, we examined the transcriptome and the proteome of cells expressing truncated FOXE3 responsible for Peters anomaly identified through linkage-coupled next-generation whole exome sequencing. We found that DNAJB1, an autophagy-associated protein, was the only candidate exhibiting differential expression in both screens. We confirmed the candidacy of DNAJB1 through chromatin immunoprecipitation and luciferase assays while knockdown of DNAJB1 in human lens epithelial cells resulted in mitotic arrest. Subsequently, we targeted dnajb1a in zebrafish through injection of a splice-blocking morpholino. The dnajb1a morphants exhibited underdeveloped cataractous lenses with persistent apoptotic nuclei. In conclusion, we have identified DNAJB1 as a transcriptional target of FOXE3 in a novel pathway that is crucial for development of the anterior segment of the eye. Human Embryonic Kidney (HEK293FT) cells were transfected with the expression vector (pT-RexTM-DEST30) harboring either the wild type or the mutant (C240*) FOXE3 ORF (open reading frame). The experimental design included a total of eight biological replicates of cells expressing the wild type and eight replicates of mutant FOXE3 along with eight non-transfected controls. Cells were harvested 24-hour post-transfection and subjected to total RNA isolation for the preparation of whole transcriptome next-generation sequencing libraries. Initially, we examined the quality of transcriptome libraries on a MiSeq genome analyzer. Subsequent to confirmation of the quality, all libraries were paired-end sequenced (2 x 100 bp) using Illumina TruSeq Cluster V3 flow cell at a concentration of 13.0 pM in two separate lanes (12 bar-coded mRNA pooled libraries in each lane) on a HiSeq 2000 genome analyzer.

FOXE3是一种晶状体特异性转录因子（transcription factor），与眼前段发育异常（anterior segment ocular dysgenesis）密切相关。为明确FOXE3的转录靶标（transcriptional target）——该靶标对眼前段发育不可或缺——我们通过连锁耦合式新一代全外显子测序（linkage-coupled next-generation whole exome sequencing）鉴定出了可导致Peters异常（Peters anomaly）的截短型FOXE3，并对表达该截短型FOXE3的细胞开展转录组（transcriptome）与蛋白质组（proteome）分析。结果显示，自噬相关蛋白DNAJB1（autophagy-associated protein）是唯一在两组筛选中均呈现差异表达的候选靶标。我们通过染色质免疫沉淀（chromatin immunoprecipitation）与荧光素酶报告基因实验（luciferase assays）验证了DNAJB1作为FOXE3转录靶标的候选身份；在人晶状体上皮细胞中敲低DNAJB1可引发有丝分裂阻滞（mitotic arrest）。随后，我们通过注射剪接阻断型吗啉代寡核苷酸（splice-blocking morpholino）靶向斑马鱼中的dnajb1a基因，dnajb1a基因敲低型斑马鱼（morphants）表现出晶状体发育不全并伴随白内障样病变，且存在持续的凋亡细胞核。综上，我们鉴定出DNAJB1是FOXE3的转录靶标，这一全新通路对眼前段发育至关重要。

我们将携带有野生型或突变型（C240*）FOXE3开放阅读框（open reading frame, ORF）的表达载体pT-Rex™-DEST30转染至人胚肾293FT细胞（Human Embryonic Kidney, HEK293FT）。本实验设置共包含8个生物学重复（biological replicates）的野生型FOXE3表达细胞、8个生物学重复的突变型FOXE3表达细胞，以及8个未转染的阴性对照细胞。转染24小时后收集细胞，提取总RNA以构建全转录组新一代测序文库。我们首先在MiSeq基因组分析仪（MiSeq genome analyzer）上对转录组文库的质量进行质控。确认文库质量合格后，使用Illumina TruSeq Cluster V3流动池（Illumina TruSeq Cluster V3 flow cell），以13.0 pM的浓度在两个独立泳道（每个泳道包含12个带条形码的mRNA混合文库）中，通过HiSeq 2000基因组分析仪（HiSeq 2000 genome analyzer）进行双端测序（paired-end sequencing，2×100 bp）。