ID1基因m6A修饰调控鸡卵泡选择的分子机理研究

Follicle selection is the key point throughout follicle development and largely determines the egg production in chicken. The elevated expression of follicle stimulating hormone receptor (FSHR) in the granulosa layer of one particular pre-hierarchical follicle will make this follicle respond to circulating FSH and being selected into pre-ovulatory hierarchy. Thus, the rising FSHR is unequivocally accepted as the hallmark of granulosa cell differentiation and follicle selection. But the upstream factors directly responsible for this selective FSHR up-regulation remain unknown. We found that the dominant negative protein of bHLH transcription factors, inhibition of DNA binding (ID1), could inhibit FSHR expression in granulosa cells. During follicle selection, the inhibition was lifted by ID1 down-regulation which was coincident with massive N6-Methyladenosine (m6A) modification of its own mRNA. Therefore, we propose that ID1 RNA m6A modification results in ID1 degradation and thus promote follicle selection. In this project, we will (1) clarify how the ID1 mRNA were modified by m6A writers or erasers (2) determine the bHLH transcription factor(s) that interacting with ID1 and FSHR promoter. (3) establish the “secreted factor-m6A enzymes-ID1m6A-bHLH-FSHR” signal cascade that control follicle selection. This study will not only identify the molecular mechanisms regulating follicle selection in chicken, but also provide new clues for the understanding the physiology of follicle development and improving egg production in chicken.

卵泡选择是卵泡发育过程的关键环节，也是影响鸡产蛋性状的重要因素。促卵泡素受体（FSHR）在选择前卵泡颗粒细胞中升高，使卵泡对FSH产生应答而被选择进入排卵队列。但是FSHR只在一个特定卵泡中上调的机制尚不明确。申请人发现bHLH转录因子的显性抑制蛋白ID1(DNA结合抑制因子1)，抑制颗粒细胞FSHR表达，而ID1的RNA甲基化（m6A）修饰致使ID1自身在卵泡选择过程中下调。据此我们假设：鸡ID1基因RNA的m6A修饰降低了ID1的表达量，从而促进颗粒细胞FSHR表达和卵泡选择。本项目拟开展（1）揭示鸡颗粒细胞中ID1发生m6A修饰的机理；（2）鉴定与ID1蛋白和FSHR启动子互作的bHLH因子；（3）阐明“胞外信号-m6A修饰酶-ID1-bHLH-FSHR”调控卵泡选择的完整信号通路。本研究将系统揭示ID1调控鸡卵泡选择的分子机理，为理解动物卵泡发育过程和改良鸡产蛋性状提供依据。

蛋鸡的卵巢发育和卵泡选择直接影响产蛋的数量和质量，因此研究鸡卵巢发育的分子调控机制，在畜牧业生产中具有重要意义。卵泡选择是整个产蛋过程中的关键环节，在被选择卵泡颗粒细胞中FSHR急剧升高，本研究主要揭示了M6A修饰、FSHR和ID1蛋白在卵泡发育中的作用，取得的主要结果如下：1）构建了鸡卵泡选择前后m6A修饰差异图谱，发现等级卵泡的m6A峰在起始密码子区和终止密码子区比编码区更丰富，11.59%的m6A修饰的转录本在样品间甲基化水平存在差异。2）ID1基因在选择后卵泡颗粒细胞中m6A修饰水平显著升高，存在4个m6A修饰保守位点，通过CO-IP技术鉴定分析了ID1蛋白与FSHR的互作。3）分析了等级前后卵泡，AMH、BMP15和FSH等蛋白和激素对m6A修饰酶的调控作用以及ID1蛋白与Mettl13之间的关系。4）通过荧光素酶报告基因载体构建，研究了ID1基因启动子区域活性，发现TSS上游1002bp具有调控基因转录重要的元件。5）研究了鸡卵母细胞发生过程中原始生殖细胞的迁移定植性腺规律，分离鉴定了成年鸡卵巢中存在着的生殖干细胞，研究发现了ID1基因在原始生殖细胞中表达显著高于卵原干细胞，推测可能与ID1蛋白与保持细胞干性相关。总之，本研究系统揭示了鸡卵巢发育及卵泡选择中激素-M6A修饰-ID1蛋白之间表达调控的分子机理，从表观组、转录组和细胞功能验证等方面研究了鸡卵巢中卵子发生过程及IDI蛋白的差异，期望以上研究为揭示禽类卵巢发育分子机制提供重要证据，为今后提高鸡的繁殖和产蛋性能等提供服务。