Functional characterization of the long non-coding RNA Scirocco in development an

描述（由申请人提供）：长链非编码rna （lncrna）已成为染色质结构和功能的重要调节因子。脊椎动物lncrna通过向靶基因募集表观遗传抑制因子而与表观遗传沉默密切相关。提出的研究背后的具体假设是，新型lncRNA Scirocco （SCIR）在发育和癌症中控制细胞分化和增殖。具体目的是为了全面评估SCIR在发育和疾病中细胞分化和增殖中的作用。目的1。为了分析SCIR与EZH2的相互作用：在体外，SCIR与PRC2的EZH2亚基相互作用，SCIR与EZH2的相互作用将PRC2从染色质中驱逐出去。SCIR如何与EZH2相互作用尚不清楚。参与SCIR与EZH2关联的RNA和蛋白质基序将通过体外RNA-蛋白结合试验揭示，该试验将研究EZH2与突变SCIR转录本的相互作用，反之亦然。为了评估鉴定的RNA和蛋白基序是否支持体内EZH2-SCIR相互作用，我们将在表达突变SCIR转录物或突变EZH2蛋白的细胞中研究EZH2与SCIR的关联。目标2。分析SCIR在发育中的作用：我们的数据表明，全反式维甲酸介导的SCIR激活导致HOXA1和HOXA2的激活，并最终导致细胞分化和增殖。RA如何激活SCIR转录以及scr介导的HOXA1和HOXA2的激活如何控制细胞增殖和分化仍然未知。我们将结合RNA和ChIP测序测定与功能测定来鉴定HOXA1和HOXA2的靶基因。为了揭示SCIR在发育中的作用，我们将研究scr缺陷细胞和小鼠的发育。我们将研究RA信号转导通路成分的失活是否会减弱SCIR转录。目的3：解剖SCIR在癌症中的作用：我们在原发性乳腺癌和肺癌中检测到SCIR的异常转录，并证明SCIR控制肺癌和乳腺癌细胞的增殖、迁移和侵袭性。我们的研究结果表明，异常的SCIR转录参与了癌症的发生和发展。我们将研究SCIR的异位表达是否会导致小鼠癌症。通过提供lncRNA SCIR直接与表观遗传抑制因子EZH2相互作用的证据（Aim 1），阐明SCIR在细胞分化和增殖中的作用（Aim 2），揭示SCIR在癌症的发生和进展中的作用（Aim 3），实现本提案的三个目标将支持SCIR在发展和癌症中的作用。

DESCRIPTION (provided by applicant): Long, non-coding RNAs (lncRNAs) have emerged as important regulators of chromatin structure and function. Vertebrate lncRNAs have been closely associated with epigenetic silencing by recruiting epigenetic repressors to target genes. The specific hypothesis behind the proposed research is that the novel lncRNA Scirocco (SCIR) controls cell differentiation and proliferation in development and cancer. The specific aims are designed to provide a comprehensive assessment of the role of SCIR in cell differentiation and proliferation in development and disease. Aim 1. To dissect the interaction of SCIR with EZH2: SCIR interacts with the EZH2 subunit of PRC2 in vitro and the interaction of SCIR with EZH2 evicts PRC2 from chromatin. How SCIR interacts with EZH2 remains unclear. The RNA and protein motifs involved in the association of SCIR with EZH2 will be uncovered by in vitro RNA-protein binding assays, which investigate the interaction of EZH2 with mutant SCIR transcripts and vice versa. To assess whether the identified RNA and protein motifs support the EZH2-SCIR interaction in vivo, we shall investigate the association of EZH2 with SCIR in cells, which express mutant SCIR transcripts or mutant EZH2 proteins. Aim 2. To dissect the role of SCIR in development: Our data suggest that all-trans retinoic acid mediated activation of SCIR results in activation of HOXA1 and HOXA2 and culminates in cell differentiation and proliferation. How RA activates SCIR transcription and how the SCIR-mediated activation of HOXA1 and HOXA2 controls cell proliferation and differentiation remains unknown. We shall combine RNA- and ChIP- sequencing assays with functional assays to identify target genes for HOXA1 and HOXA2. To uncover the role of SCIR in development, we shall investigate the development SCIR-deficient cells and mice. We shall investigate whether the inactivation of components of the RA signal transduction pathway attenuates SCIR transcription. Aim 3: To dissect the role of SCIR in cancer: We have detected aberrant transcription of SCIR in primary breast and lung cancer and demonstrated that SCIR controls the proliferation, migration and invasiveness of lung and breast cancer cells. Our results suggest that anomalous SCIR transcription is involved in initiation and progression of cancer. We shall investigate whether the ectopic expression of SCIR causes cancer in mice. Accomplishing the three aims of this proposal will support he role of SCIR in development and cancer by providing evidence that the lncRNA SCIR directly interacts with epigenetic repressor EZH2 (Aim 1), elucidating the role of SCIR in cell differentiation and proliferation (Aim 2), and uncovering the role of SCIR in initiation and progression of cancer (Aim 3). PUBLIC HEALTH RELEVANCE: Epigenetic mechanisms establish and maintain the identity of cells during development. Errors in epigenetic mechanisms have been correlated with various human diseases such as cancer. Long, non-coding RNAs (ncRNAs) have emerged as important regulators of epigenetic phenomena. The proposed project will assess the role and function of the ncRNA Scirocco in epigenetic gene expression in development and cancer. Accomplishing the described project will provide novel insights into epigenetic mechanisms and open novel avenues for the development of therapeutic assays that attenuate cancer.