Utilizing oocyte biology to understand nuclear reprogramming

描述（由申请人提供）：迄今为止，卵母细胞仍然是唯一一种以有效和刻板的方式内化、重塑和重编程外源细胞核的细胞，但对这种重编程能力的机制知之甚少。因此，虽然很多人关注的是通过转录因子过表达在哺乳动物细胞类型中诱导多能性，但卵母细胞含有重塑和重编程终分化细胞核所需的所有信息和组织。本研究利用卵母细胞独特的生物学特性，对卵母细胞的组织、重编程和体细胞对重编程的反应等方面提出了具体的问题。为了确定卵母细胞如何在结构上组织以驱动受精和重编程，我将使用测序和质谱法的最新进展来确定卵母细胞内RNA转录物和蛋白质的定位（目的1）。为了确定进行重编程的因素，我将使用质谱法鉴定在重编程的特定时间点进入卵母细胞生发囊泡（GV）的蛋白质（目的2）。与此同时，我还将通过鉴定重编程过程中体细胞核的转录组和蛋白质组来研究体细胞核对重编程的反应（目的2）。我发现卵母细胞GV中一些最丰富的蛋白质是染色质重塑因子NuRD（核小体重塑和组蛋白去乙酰化酶）复合体的成员。最近的工作已经确定了NuRD复合体的一个成员对诱导多能细胞的产生至关重要。我将研究NuRD复合体对于驱动卵母细胞内的重编程是否必要和充分（目的3）。很明显，卵母细胞的组织结构是为了一个任务：重新编程，并利用一个外源核来驱动发育。通过利用反向遗传学来识别卵母细胞内进行重编程的因素，我们可以开始识别在没有卵母细胞的情况下重编程过程所需的蛋白质或rna。同样，细胞核被重新编程的刻板和确定的过程使我们能够独特地一瞥体细胞核必须经历的基本步骤，以成为多能性。最后，卵母细胞提供了一个很好的模型来测试染色质重塑复合物在重编程过程中的作用。这项工作将有助于深入了解分化的可塑性、有助于重编程和受精的内源性因素以及分化和多能细胞的基因组组织。

DESCRIPTION (provided by applicant): To date, the oocyte remains the only cell that internalizes, remodels and reprograms a foreign nucleus in an efficient and stereotypic manner, but little is known about the mechanisms underlying this reprogramming ability. Thus, while much focus has been on inducing pluripotency via transcription factor overexpression in mammalian cell types, the oocyte harbors all the information and organization necessary to remodel and reprogram a terminally differentiated nucleus. This proposal utilizes the unique biology of the oocyte to drive specific questions on oocyte organization, reprogramming, and the somatic response to reprogramming. To determine how the oocyte is structurally organized to drive fertilization and reprogramming, I will use recent advancements in sequencing and mass spectrometry to identify the localization of RNA transcripts and proteins within the oocyte (Aim 1). To determine the factors that carry out reprogramming, I will use mass spectrometry to identify the proteins entering oocyte germinal vesicle (GV) during specific time points in reprogramming (Aim 2). In concert, I will also investigate how somatic nuclei respond to reprogramming by identifying the transcriptome and proteome of somatic nuclei during reprogramming (Aim 2). I have found that some of the most abundant proteins within the oocyte GV are members of the NuRD (Nucleosome Remodeling and histone Deacetylase) complex of chromatin remodeling factors. Recent work has identified one member of the NuRD complex as critical for induced pluripotent cell generation. I will investigate whether the NuRD complex is necessary and sufficient to drive reprogramming within the oocyte (Aim 3). The oocyte is very clearly organized for one task: to reprogram and employ a foreign nucleus to drive development. By utilizing reverse genetics to identify factors within the oocyte that carry out reprogramming, we can begin to identify proteins or RNAs required for the reprogramming process in the absence of the oocyte. Similarly, the stereotypic and determined process in which nuclei are reprogrammed allows us a unique glimpse into the essential steps a somatic nucleus must undergo to become pluripotent. And lastly, the oocyte provides an excellent model in which to test the roles of chromatin remodeling complexes in the process of reprogramming. This work will contribute significantly to insights into the plasticity of differentiation, the endogenous fators that contribute to reprogramming and fertilization and the genomic organization of differentiated and pluripotent cells.