MicroRNAs and hematopoietic differentiation

 描述（由申请人提供）：我们鉴定了13种核定位的长非编码（lncRNA）转录物，其长度超过200个核苷酸，不具有功能性蛋白质编码能力，对于小鼠胎肝红细胞生成素依赖性CFU-E阶段的终末红细胞分化至关重要。所有这些基因在终末红细胞发育期间均高度上调，并且主要或仅在红系细胞中表达;这些基因中的大多数的启动子被关键的红系转录因子GATA 1、TAL 1和/或KLF 1靶向。这些lncRNA中的三种调节相邻基因：一种增强子转录物alncRNA-EC 7是激活编码BAND 3的基因所特别需要的，另一种elncRNA-EC 3是上调KIF 2A基因所必需的。shlncRNA-EC 6，也称为DLEU 2，仍然定位于其在细胞核中的转录位点，并且DLEU 2基因座与同一染色体上至少8个相邻基因的推定启动子相互作用。shlncRNA-EC 6的表达对于这些基因在终末红系细胞增殖和分化期间的正常下调是必不可少的。因此，我们假设并将测试alncRNA-EC 7和elncRNA-EC 3在其相邻基因周围建立激活染色质结构，而shlncRNA-EC 6建立抑制性结构。更一般地说，这项资助的重点是精确确定这13种红细胞特异性lncRNA中的每一种如何调节终末红细胞发育。为此，我们将克隆每个lncRNA的全长转录本，并确定每个lncRNA转录本是否保持在其遗传位点附近。同时，我们将鉴定与每个lncRNA相互作用的所有基因组DNA片段，并确定是否如我们预测的那样，lncRNA遗传位点与其他染色体片段的相互作用取决于lncRNA的表达。我们将鉴定与每个红系lncRNA结合的核蛋白，并确定红系重要lncRNA与红系发育期间特异性结合它们的蛋白质之间的功能关联。同时，我们将确定这13个lncRNA在红系发育中的广泛功能。通过测定RNA敲低细胞对终末分化的影响，包括细胞增殖、细胞周期， 细胞凋亡、诱导红系重要标志基因和核浓缩。同时，我们将使用深度测序来鉴定所有mRNA，其在发育过程中的上调或下调直接或间接依赖于每个lncRNA的诱导。总之，这些实验将使我们能够制定一个具体的假设，每个lncRNA如何调节红细胞生成和开始，以测试这一假设。最后，我们将通过使用Cas9系统产生敲除小鼠来确认这13种红细胞重要核lncRNA中的每一种在体内的作用。我们预计所有的都是胚胎致死性的严重贫血，我们将详细分析胎儿和成人红细胞的发育。

 DESCRIPTION (provided by applicant): We identified 13 nuclear-localized long noncoding (lncRNAs), transcripts longer than 200 nucleotides that do not have functional protein coding capacity, that are essential for terminal erythroid differentiation from the mouse fetal liver erythropoietin-dependent CFU-E stage. All are highly upregulated during terminal red cell development and are expressed mainly or exclusively in erythroid cells; the promoters of most of these genes are targeted by the key erythroid transcription factors GATA1, TAL1, and/or KLF1. Three of these lncRNAs regulate adjacent genes: One enhancer transcript, alncRNA-EC7, is specifically needed for activation of the gene encoding BAND3 and another, elncRNA-EC3, is essential for upregulation of the KIF2A gene. shlncRNA-EC6, also called DLEU2, remains localized to its site of transcription in the nucleus, and the DLEU2 gene locus interacts with the putative promoters of at least 8 neighboring genes on the same chromosome. Expression of shlncRNA-EC6 is essential for normal downregulation of these genes during terminal erythroid proliferation and differentiation. Therefore, we hypothesize and will test that alncRNA-EC7 and elncRNA-EC3 establish an activating chromatin structure around their adjacent genes, whereas shlncRNA-EC6 establishes a repressive one. More generally this grant is focused on determining precisely how each of these 13 erythroid-specific lncRNAs regulates terminal red cell development. To this end, we will clone the full-length transcript of each lncRNA and determine whether each lncRNA transcript remains near its genetic locus. In parallel, we will identify all of the genomic DNA segments with which each lncRNA interacts, and determine whether, as we predict, interaction of the lncRNA genetic locus with other chromosome segments is dependent on expression of the lncRNA. We will identify the nuclear proteins bound to each erythroid lncRNA, and determine the functional association between erythroid-important lncRNAs and the proteins that specifically bind them during erythroid development. In parallel, we will determine the broad functions of these 13 lncRNAs in erythroid development. An overview of the requirement for each lncRNA will be done by assaying RNA knockdown cells for effects on terminal differentiation including cell proliferation, cell cycling, apoptosis, induction of erythroid important marker genes, and nuclear condensation. In parallel we will use deep sequencing to identify all mRNAs whose up- or down-downregulation during development is directly or indirectly dependent on induction of each lncRNA. Taken together, these experiments will enable us to formulate a specific hypothesis as to how each lncRNA regulates erythropoiesis and begin to test this hypothesis. Finally, we will confirm the roles of each of these 13 erythroid-important nuclear lncRNAs in vivo by generating knockout mice using the Cas9 system. We expect all to be embryonic lethal with severe anemia, and we will analyze in detail fetal and if need be adult erythroid development.