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Title: Epigenetic events underlying somatic cell reprogramming
Author: Ribeiro Lemos Pereira, Carlos Filipe
ISNI:       0000 0004 2686 9783
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2009
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Although differentiated cells normally retain cell-type-specific gene expressionpatterns throughout their lifetime, cell identity can sometimes be modified or reversedin vivo by transdifferentiation, or experimentally through cell fusion or by nucleartransfer. Several studies have illustrated the importance of chromatin remodelling, DNAdemethylation and dominant transcriptional factor expression for changes in lineageidentity. Here the epigenetic mechanisms required to ?reset? genome function wereinvestigated using experimental heterokaryons.To examine the epigenetic changes that are required for the dominantconversion of lymphocytes to muscle, I generated stable heterokaryons betweenhuman B-lymphocytes and mouse C2C12 myotubes. I show that lymphocyte nucleiadopt an architecture resembling that of muscle and initiate the expression of musclespecificgenes in the same temporal order as developing muscle. The establishment ofthis novel gene expression program is coordinated with the shutdown of severallymphocyte-associated genes. Interestingly, inhibition of histone deacetylase (HDAC)activity during reprogramming selectively blocks the silencing of lymphocyte-specificgenes but does not prevent the establishment of muscle-specific gene expression.In order to reprogram somatic cells to pluripotency, I fused human Blymphocytesand mouse embryonic stem (ES) cells. The conversion of human cells isinitiated rapidly, occurring in heterokaryons before nuclear fusion. Reprogramming ofhuman lymphocytes by mouse ES cells elicits the expression of a human ES-specificgene expression profile in which endogenous hSSEA4, hFgf receptors and ligands areexpressed while factors that are characteristic of mouse ES cells, such as Bmp4 andLif receptor are not. Using genetically engineered mouse ES cells I demonstrate thatsuccessful reprogramming requires the expression of Oct4, but importantly, does notrequire Sox2, a factor implicated as critical for the induction of pluripotency. Followingreprogramming, mOct4 becomes dispensable for maintaining the multi-potent state ofhybrid cells. Finally, I have examined the reprogramming potential of embryonic germ(EG), embryonic carcinoma (EC) and ES cells deficient for the Polycomb repressivecomplex 2 (PRC2) proteins Eed, Suz12 and Ezh2. While EC and EG cells share theability to reprogram human lymphocytes with ES cells, the lack of Polycomb proteinsabolishes reprogramming. Thus, the repressive chromatin mark (H3K27 methylation)catalysed by PRC2 play a crucial role in keeping ES cells with full reprogrammingcapacity. Collectively my results underscore the importance of chromatin events duringcell fate reprogramming.
Supervisor: Fisher, Amanda Sponsor: FCT Portugal ; Medical Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral