MRC Clinical Sciences Centre, Imperial College London
Doi
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