부산대학교 도서관

The epigenetic memory of a cells defines its identity. Upon reprogramming towards pluripotency a somatic cell undergoes epigenetic remodeling in order to become pluripotent. The work I am presenting here is divided into two parts. In the first part, I am using cell fusion mediated reprogramming to investigate Xi reactivation, a typical example of epigenetic remodeling, as well as how ploidy affects reprogramming efficiency. Among all the technique established for in vitro reprogramming towards pluripotency, cell fusion has the advantage of allowing us to study early events of reprogramming as well as having a higher efficiency compared to induced Pluripotent Stem cells (iPS). Despites intensive research on the molecular mechanisms and the epigenetic changes leading a somatic cell towards pluripotency, a lot remain unanswered. In this thesis, I showed that somatic cells were able to reprogram towards pluripotency and that some X-linked genes were reactivated upon reprogramming. I also demonstrated that haploid Embryonic Stem cells (ES) cells are able to reprogram somatic cells with a lower potential compared to diploid ES cells. This low reprogramming potential can be partially rescued by overexpression of Nanog. In the second part of my thesis, I focused on setting up a technique to visualize chromatin and aimed at studying the structure of mitotic chromosomes 19 using high resolution microscopy techniques such as Structured Illumination Microscopy (SIM) and cryo-Electron Microscopy (cryo-EM). Higher order chromatin structure remains unknown in mitosis. During mitosis, chromatin become highly compacted to form a mitotic chromosome. In order to isolate a specific chromosome (chromosome 19), first I reestablished flow karyotyping technique to sort the chromosomes 19. I also showed that chromosomes after sorting retained a certain degree of compaction as well as protein important for centromeric integrity such as Centromere Protein A (CENPA). To validate the isolation of mitotic chromosomes and the use of cryo-EM in chromosomes structure analysis, I assessed the compaction of mitotic chromosomes 19 upon loss of cohesin. Chromosomes 19 lacking Rad21 cohesin subunit possess a larger area and their chromatin looked more decondensed compared to mitotic chromosomes 19 with cohesin. This promising tool could be used in the future to study how epigenetic modifications affect chromatin structure.