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Epigenetic events underlying somatic cell reprogramming

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Title: Epigenetic events underlying somatic cell reprogramming
Authors: Ribeiro Lemos Pereira, Carlos Filipe
Item Type: Thesis or dissertation
Abstract: Although differentiated cells normally retain cell-type-specific gene expression patterns throughout their lifetime, cell identity can sometimes be modified or reversed in vivo by transdifferentiation, or experimentally through cell fusion or by nuclear transfer. Several studies have illustrated the importance of chromatin remodelling, DNA demethylation and dominant transcriptional factor expression for changes in lineage identity. Here the epigenetic mechanisms required to “reset” genome function were investigated using experimental heterokaryons. To examine the epigenetic changes that are required for the dominant conversion of lymphocytes to muscle, I generated stable heterokaryons between human B-lymphocytes and mouse C2C12 myotubes. I show that lymphocyte nuclei adopt an architecture resembling that of muscle and initiate the expression of musclespecific genes in the same temporal order as developing muscle. The establishment of this novel gene expression program is coordinated with the shutdown of several lymphocyte-associated genes. Interestingly, inhibition of histone deacetylase (HDAC) activity during reprogramming selectively blocks the silencing of lymphocyte-specific genes but does not prevent the establishment of muscle-specific gene expression. In order to reprogram somatic cells to pluripotency, I fused human Blymphocytes and mouse embryonic stem (ES) cells. The conversion of human cells is initiated rapidly, occurring in heterokaryons before nuclear fusion. Reprogramming of human lymphocytes by mouse ES cells elicits the expression of a human ES-specific gene expression profile in which endogenous hSSEA4, hFgf receptors and ligands are expressed while factors that are characteristic of mouse ES cells, such as Bmp4 and Lif receptor are not. Using genetically engineered mouse ES cells I demonstrate that successful reprogramming requires the expression of Oct4, but importantly, does not require Sox2, a factor implicated as critical for the induction of pluripotency. Following reprogramming, mOct4 becomes dispensable for maintaining the multi-potent state of hybrid cells. Finally, I have examined the reprogramming potential of embryonic germ (EG), embryonic carcinoma (EC) and ES cells deficient for the Polycomb repressive complex 2 (PRC2) proteins Eed, Suz12 and Ezh2. While EC and EG cells share the ability to reprogram human lymphocytes with ES cells, the lack of Polycomb proteins abolishes reprogramming. Thus, the repressive chromatin mark (H3K27 methylation) catalysed by PRC2 play a crucial role in keeping ES cells with full reprogramming capacity. Collectively my results underscore the importance of chromatin events during cell fate reprogramming.
Issue Date: Sep-2008
Date Awarded: Feb-2009
URI: http://hdl.handle.net/10044/1/4439
DOI: https://doi.org/10.25560/4439
Supervisor: Fisher, Amanda
Sponsor/Funder: FCT Portugal and the Medical Research Council UK
Author: Ribeiro Lemos Pereira, Carlos Filipe
Department: MRC Clinical Sciences Centre
Publisher: Imperial College London
Qualification Level: Doctoral
Qualification Name: Doctor of Philosophy (PhD)
Appears in Collections:Medicine PhD theses

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