LncRNA Haunt在发育和干细胞分化中的功能和机制研究

长链非编码RNA（lncRNA）在细胞命运决定过程中扮演重要角色。我们之前报导了lncRNA Haunt的转录本和DNA序列能拮抗性地参与调控HOXA基因簇的表达，并影响干细胞分化。但是，Haunt行使功能的分子机制及其在动物体内的生理功能还不清楚。本申请以Haunt为例，从分析lncRNA的结合蛋白入手，阐述lncRNA-蛋白结合的基本规律、作用和意义。我们将通过分析Haunt RNA-蛋白芯片杂交数据，建立优化染色质RNA富集（ChIRP-MS）和Csy4 RNA标签并结合质谱检测等手段，筛选与Haunt结合的功能性蛋白。并结合生物化学、结构解析和构建Haunt条件性敲除小鼠，揭示Haunt的生物学功能和具体机制。预期研究成果将为lncRNA的机制研究提供新思路和新手段，弥补lncRNA机制和生理功能研究的不足，有助于更好认识lncRNA在细胞命运决定、谱系分化等相关发育过程中的作用。

Long noncoding RNAs (lncRNAs) have been hypothesized to be involved in diverse biological processes. However, their functions and biological significance are largely unknown. We have previously reported an opposing role for the DNA sequence and RNA transcripts of the lncRNA Haunt in regulating HOXA genes and embryonic stem cell differentiation. While the DNA sequence of Haunt functions as HOXA enhancers, Haunt RNA transcripts serve as a ‘brake’ to modulate its DNA enhancer activity, resulting in fine-tuned expression of HOXA genes during ESC differentiation (Yin et al., 2015. Cell Stem Cell). However, the detailed mechanisms by which Haunt RNA regulates HOXA gene expression and the physiological functions of Haunt are still unclear. We hypothesize that Haunt may exhibit its function by interacting with key regulatory proteins. To identify its interacting proteins in a systematic manner, we propose to use novel complementary biochemical approaches, including protein array hybridization, ChIRP and Csy4 RNA tagging following by mass spec sequencing. In addition, we will perform biochemical and structural study of the Haunt-protein complex to reveal the mechanisms of Haunt function. Meanwhile, we will study the physiological functions of Haunt by establishing a conditional knockout mouse. The results from the proposed work will provide an important framework for us to elaborate how Haunt functions to regulate local and distal chromatin structure and epigenetic landscapes of HOXA region through key RNA-protein interactions. Moreover, this work will establish novel experimental platforms and provide a new angle for mechanistic investigation of lncRNA functions, facilitating our overall understanding of the roles of noncoding RNAs in mediating transcriptional and epigenetic regulation in development and disease.