MicroRNAs and hematopoietic differentiation

 描述(申请人提供)：我们鉴定了13个核定位的长非编码(LncRNAs)，转录本长度超过200个核苷酸，不具有功能性蛋白质编码能力，对于红系终末与小鼠胎肝促红细胞生成素依赖的CFU-E阶段的分化是必不可少的。这些基因的启动子主要或仅在红系细胞中表达，这些基因的启动子被关键的红系转录因子GATA1、TAL1和/或KLF1靶向。其中三个lncRNAs调节相邻的基因：一个增强子转录本，alncRNA-EC7，是编码BAND3的基因激活所特需的，另一个，elncRNA-EC3，是上调KIF2A基因所必需的。ShlncRNA-EC6，也称为DLEU2，定位于其在细胞核中的转录位置，DLEU2基因与同一染色体上至少8个相邻基因的启动子相互作用。在终末红系增殖和分化过程中，shlncRNA-EC6的表达对于这些基因的正常下调至关重要。因此，我们假设并将检验alncRNA-EC7和elncRNA-EC3在其邻近基因周围建立一个激活的染色质结构，而shlncRNA-EC6建立一个抑制结构。更广泛地说，这项资助的重点是准确地确定这13个红系特异的lncRNAs中的每一个是如何调控终末红细胞发育的。为此，我们将克隆每个lncRNA的全长转录本，并确定每个lncRNA转录本是否保留在其遗传位点附近。同时，我们将确定每个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，并确定红系重要的lncRNA与红系发育过程中特异结合它们的蛋白质之间的功能联系。同时，我们将确定这13个lncRNAs在红系发育中的广泛功能。通过分析RNA敲除细胞对末端分化的影响，包括细胞增殖，细胞周期， 细胞凋亡，红系重要标记基因的诱导，以及核固缩。同时，我们将使用深度测序来识别其在发育过程中上调或下调的所有mRNAs，这些mRNAs的上调或下调直接或间接依赖于每个lncRNA的诱导。综上所述，这些实验将使我们能够就每个lncRNA如何调节红细胞生成形成一个特定的假说，并开始检验这个假说。最后，我们将通过使用Cas9系统产生敲除小鼠来确认这13个红系重要核incRNAs中的每一个在体内的作用。我们预计ALL是胚胎致死的，并伴有严重的贫血，我们将详细分析胎儿和成人的红系发育情况。

 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.