Developmental genome-wide occupancy analysis of bZIP transcription factor NRL uncovers the role of c-Jun in early differentiation of rod photoreceptors in the mammalian retina [RNA-seq]. Developmental genome-wide occupancy analysis of bZIP transcription factor NRL uncovers the role of c-Jun in early differentiation of rod photoreceptors in the mammalian retina [RNA-seq]

The basic motif-leucine zipper (bZIP) transcription factor NRL determines rod photoreceptor cell fate during retinal development, and its loss leads to cone-only retina in mice. NRL works synergistically with homeodomain protein Cone-Rod Homeobox and other regulatory factors to control the transcription of most genes associated with rod morphogenesis and functional maturation, which span over a period of several weeks in the mammalian retina. We predicted that NRL gradually establishes rod cell identity and function by temporal and dynamic regulation of stage-specific transcriptional targets. Therefore, we mapped the genomic occupancy of NRL at four stages of mouse photoreceptor differentiation by CUT&RUN analysis. Dynamics of NRL binding revealed concordance with the corresponding changes in transcriptome of the developing rods. Notably, we identified c-Jun proto-oncogene as one of the targets of NRL, which could bind to specific cis-elements in the c-Jun promoter and modulate its activity in HEK293 cells. Coimmunoprecipitation studies showed the association of NRL with c-Jun, also a bZIP protein, in transfected cells as well as in developing mouse retina. Additionally, shRNA-mediated knockdown of c-Jun in the mouse retina in vivo resulted in altered expression of almost 1000 genes, with reduced expression of phototransduction genes and many direct targets of NRL in rod photoreceptors. We propose that c-Jun-NRL heterodimers prime the NRL-directed transcriptional program in neonatal rod photoreceptors before high NRL expression suppresses c-Jun at later stages. Our study highlights a broader cooperation among cell-type restricted and widely expressed bZIP proteins, such as c-Jun, in specific spatiotemporal contexts during cellular differentiation. Overall design: shRNA-mediated knockdown of mouse Jun was performed using electroporation with shRNA-expressing vector or control pLKO.1-Scrambled vector co-transfected with Nrlp-EGFP vector in neonatal mice at P0. EGFP positive rod photoreceptors electroporated with control or Jun shRNA constructs were purified by flow-sorting at P10. Experiments were performed in triplicate.

碱性亮氨酸拉链（basic motif-leucine zipper, bZIP）转录因子NRL在视网膜发育过程中决定视杆感光细胞的细胞命运，其缺失会导致小鼠视网膜仅存留视锥感光细胞。NRL可与同源结构域蛋白视锥-视杆同源框（Cone-Rod Homeobox）及其他调控因子协同作用，调控绝大多数与视杆细胞形态发生和功能成熟相关的基因的转录——这一过程在哺乳动物视网膜中可持续数周之久。我们推测，NRL通过对阶段特异性转录靶标进行时序性、动态性调控，逐步建立视杆细胞的身份特征与功能。因此，我们通过CUT&RUN分析，绘制了小鼠感光细胞分化四个阶段中NRL的基因组结合位点图谱。NRL结合的动态变化与发育中视杆细胞的转录组相应变化呈现一致性。值得注意的是，我们鉴定出c-Jun原癌基因是NRL的靶标之一：NRL可结合c-Jun启动子区域的特定顺式作用元件，并在HEK293细胞中调控其活性。免疫共沉淀实验证实，在转染细胞与发育中的小鼠视网膜中，NRL均可与同为bZIP蛋白的c-Jun发生结合。此外，在小鼠视网膜内通过短发夹RNA（short hairpin RNA, shRNA）介导的c-Jun敲低实验，导致近1000个基因的表达发生改变，其中光转导相关基因以及视杆感光细胞中众多NRL直接靶标的表达均出现下调。我们提出，在新生视杆感光细胞中，c-Jun-NRL异二聚体可启动NRL介导的转录程序，而在后续阶段高表达的NRL会抑制c-Jun的活性。本研究揭示了在细胞分化过程中，细胞类型限制性表达与广泛表达的bZIP蛋白（如c-Jun）在特定时空环境下存在更为广泛的协同作用。
实验整体设计：于新生小鼠P0（出生后第0天）时期，通过电转染将携带shRNA的载体或对照pLKO.1-Scrambled载体与Nrlp-EGFP载体共转染，以实现小鼠Jun基因的shRNA介导体内敲低。于P10（出生后第10天）时期，通过流式分选获取经对照或Jun shRNA构建体电转染的EGFP阳性视杆感光细胞。实验均设置三次生物学重复。