下丘脑Sonic hedgehog的转录调控网络研究

Sonic hedgehog(Shh), as an important morphogen, is involved in a large number of central nervous system (CNS) disorders. However, its transcriptional regulation mechanism within the hypothalamus is still not clear. We recently found SoxB1 factors regulate Shh expression within hypothalamus through directly binding to the specific enhancer SBE2, and the dysfunction of SoxB1 so as to down-regulation of Shh may be the pathogenesis mechanism of Septo-optic dysplasia. Tbx2/3, the downstream transcriptional repressor of BMP pathway, may repress Shh expression in the ventral midline of hypothalamus through antagonizing SoxB1 function. Based on these findings, we propose to further systematically study the transcriptional regulation of Shh in the hypothalamus. 1) Repressive regulation: the molecular mechanism employed by Tbx2/3 to antagonize SoxB1 function so as to repress hypothalamic Shh expression will be explored from different angles and levels, including generating Tbx2/3 ectopically expressed transgenic mice model; 2)Active regulation: SoxB1 interacting factors will be identified, and their potential regulation functions on Shh expression and pathogenicity roles in Septo-optic dysplasia will be evaluated; 3)Long-distance regulation: the interaction mechanism between long-distance enhancer SBE2 (400kb) and Shh transcription iniation site will be investigated from 3-dimension level. Overall, the project will present a comprehensive transcriptional regulation network of hypothalamic Shh, and help us to understand the pathagenesis mechanism of Shh-related developmental diseases.

Sonic hedgehog(Shh)作为重要的成形素，与众多中枢神经发育疾病相关，而其下丘脑转录调控机制尚不清楚。我们近期发现SoxB1直接结合特异性增强子SBE2指导Shh在下丘脑的表达，此事件失调可能是鼻眼发育畸形的致病机制，而BMP下游因子Tbx2/3可能通过拮抗SoxB1功能抑制下丘脑腹部中轴区域Shh的表达。在此基础上，本项目拟进一步系统研究Shh在下丘脑的转录调控：1）负向调控：从不同角度和层次，包括建立Tbx2/3异位表达转基因小鼠，探讨Tbx2/3抑制SoxB1功能，负向调节Shh表达的机制；2）正向调控：筛选SoxB1互作因子，研究其对下丘脑Shh潜在的调节功能及与鼻眼发育畸形的致病相关性；3）远程调控：解析远程增强子(400kb)SBE2与Shh启动子相互作用的机制。总之，该项目有望勾画一幅较完整的Shh下丘脑转录调控网络图，将有助于揭示Shh相关发育疾病分子机制。

Sonic hedgehog (Shh)在胚胎发育早期，特异性表达于整个神经管腹部，指导多种神经前体细胞的形成，因而其Shh剂量不足导致前脑无裂畸形，Shh基因缺陷小鼠出现相似表型，导致胚胎致死。可见Shh在准确的发育阶段以及部位得以表达对于其功能至关重要，因此我们多方研究Shh在中枢神经系统发育过程中的转录调控机制，以此推动其功能研究，促进对相关发育疾病致病机制的了解。.首先，我们发现间脑腹部中轴特异表达的转录抑制因子Tbx2/3缺失，伴随Shh表达增加，前体细胞过度增殖，导致垂体发育异常。机制研究发现，Tbx2/3通过物理结合，阻遏SoxB1蛋白对Shh下丘脑增强子SBE2的结合和转录激活。接着，我们构建SBE2驱动的Cre转基因小鼠，通过与Rosa26-mTmG，Rosa26-LacZ杂交，确认阳性细胞的特异性，在此基础上，与Tbx3-tg小鼠杂交，深入研究Tbx3过量表达对Shh表达及神经管发育的影响。进而，通过临床样本分析，我们发现Tbx3在甲状腺乳头状癌中过量表达，且与癌症恶性程度及淋巴转移正相关。筛选发现，周期蛋白依赖性激酶抑制剂(Cyclin-dependent kinases inhibitors, CKIs)，p57KIP2的表达在Tbx3敲低的细胞中显著上调，伴随细胞阻滞在G1-S期，增殖减慢，体内体外肿瘤形成能力下降。进一步研究发现，Tbx3通过招募HDAC1/2和PRC2复合物，发挥对p57KIP2的转录抑制功能。最后，我们结合DNA-pull down和质谱分析探求和筛选在神经管发育过程中与SoxB1的交互作用因子，并深入挖掘相关分子在神经管发育方面的表型及分子机制。.综上所述，下丘脑Shh受到精细的负向及正向调节，参与调节的分子，以Tbx3为例，参与哺乳动物重要的发育调节事件，亦在癌症等疾病发生中至关重要。本项目的研究结果为系统建立下丘脑Shh的转录调控网络，阐明相关的发育及疾病机制奠定了坚实的基础。