Utilizing oocyte biology to understand nuclear reprogramming

Stanford University, Stanford CA

Investigators

Abstract

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.