Programming the genome in embryonic and somatic stem cells

Abstract

In opposition to terminally differentiated cells, stem cells can self-renew and give rise to multiple cell types. Embryonic stem cells retain the ability of the inner cell mass of blastocysts to differentiate into all cell types of the body and have acquired in culture unlimited self-renewal capacity. Somatic stem cells are found in many adult tissues, have an extensive but finite lifespan and can differentiate into a more restricted array of cell types. A growing body of evidence indicates that multi-lineage differentiation ability of stem cells can be defined by the potential for expression of lineage-specification genes. Gene expression, or as emphasized here, potential for gene expression, is largely controlled by epigenetic modifications of DNA and chromatin on genomic regulatory and coding regions. These modifications modulate chromatin organization not only on specific genes but also at the level of the whole nucleus; they can also affect timing of DNA replication. This review highlights how mechanisms by which genes are poised for transcription in undifferentiated stem cells are being uncovered through primarily the mapping of DNA methylation, histone modifications and transcription factor binding throughout the genome. The combinatorial association of epigenetic marks on developmentally regulated and lineage-specifying genes in undifferentiated cells seems to define a pluripotent state.

1

CpG methylation. (A) Mechanism of DNA methylation. (B) CpG methylation is symmetrical and occurs on both DNA strands. (C) Simplified textbook view of the relationship between DNA methylation and gene expression.

2

Post-translational histone modifications. (A) Core histones can be methylated, acetylated, phosphorylated, ubiquitinated or SUMOylated, to modulate gene expression. (B) Known modifications on the amino-terminal tails of core histones H3 and H4.

3

Pluripotent ESCs display greater histone mobility than differentiated cells. (A) Relative mobility of H2B-green fluorescent protein (GFP), H3-yellow fluorescent protein (YFP) and of the histone variant H3.3-YFP was determined by fluorescent recovery after photobleaching in undifferentiated mouse ESCs, in ESCs cultured for 24 hrs without leukaemia inhibitory factor (Diff. ESCs) and in neuronal progenitor cells (NPC). Relative protein mobility (or protein recovery rate after photobleaching) is indicated by the width of the coloured bar. Mobility of HP1-GFP is also reduced upon differentiation of ESCs. (B) Proportion of the mobile fraction of H2B-GFP, H3-YFP and H3.3-YFP in ESCs, differentiated ESCs and neuronal progenitor cells (NPC). Figure was drawn from data presented in [74].

4

Regulation of lineage-specific gene expression by histone H3K27 methylation and PcGs. (A) In undifferentiated cells, repressed lineage-specific genes are marked by trimethylation of K4 and K27 (the bivalent marks) and acetylation of H3K9. These marks are believed to prime genes for activation. Upon differentiation, demethylation of H3K27 results in transcriptional activation of the gene. (B) In undifferentiated cells, repressed lineage-specific genes can be primed for activation by occupancy of PcGs on the promoter; differentiation coincides with removal of the PcG complex and activation of the gene. However, genes expressed in undifferentiated cells can also be primed for transcriptional repression by PcG complexes on the promoter. PcG complexes (PRCs) are therefore suggested to form a ‘pre-programmed memory system’ established during embryogenesis [87].

5

CpG methylation profile in the promoter region of lineage-specific and housekeeping genes in undifferentiated human ASCs. Genes indicative of the adipogenic lineage (LEP, PPARG2, FABP4, LPL), endothelial cell lineage (CD331, CD144) and myogenic lineage (MYOG) are represented. Lamin B1 (LMMB1) is a constitutively expressed gene. (A) The graph shows the average percentage of methylation of each cytosine in CpG dinucleotides in these promoter regions as determined by bisulphite genomic sequencing [9, 10]. (B) Mean percentage of methylation across the promoter regions examined. Note the greater percentage of methylation in CD31 and MYOG relative to adipogenic promoters (P < 0.001; t-tests). The CD144 promoter appears relatively hypomethylated due to unmethylation of the 5′ half of the region examined, while the 3′ half is fully methylated in undifferentiated ASCs [9]. The LMNB1 promoter is unmethylated.

6

Epigenetic landscape of genes associated with lineage specification as function of differentiation. ESCs, undifferentiated embryonic stem cells; MSCs, undifferentiated mesenchymal stem cells; DIFF, differentiated somatic cells. Two scenarios are presented for lineage-specific genes in differentiated cells, depending on whether the gene is activated or up-regulated (ON), or turned off (OFF). Note that relationship between promoter DNA methylation and promoter activity depends on CpG content of the promoter [37]; thus, an expressed gene in differentiated cells is not necessarily unmethylated (see text for details).

7

Ablation of DNA methylation in mouse ESCs results in reorganization of the nuclear space. (A) ESCs depleted of DNMT3a and 3b (obtained from Dnmt3a^−/− Dnmt3b^−/− embryos), compared to wild type (WT) ESCs, display a clustering of chromocentres, CpG demethylation associated with enhanced H3K9ac and reduced H3K9m2 (while H3K9m3 remains unaltered), and increased mobility of the linker histones H1 and H5. However nucleosome spacing is not altered, indicative of absence of marked chromatin compaction. (B) In ESCs depleted of DNMT1 (obtained from Dnmt1^−/− embryos), histone modification changes differ on promoters activated by loss of CpG methylation (top panels) and on those not activated by loss of CpG methylation (bottom panels).