Transcriptional and epigenetic mechanisms of cellular reprogramming to induced pluripotency

Abstract

Enforced ectopic expression of a cocktail of pluripotency-associated genes such as Oct4, Sox2, Klf4 and c-Myc can reprogram somatic cells into induced pluripotent stem cells (iPSCs). The remarkable proliferation ability of iPSCs and their aptitude to redifferentiate into any cell lineage makes these cells a promising tool for generating a variety of human tissue in vitro. Yet, pluripotency induction is an inefficient process, as cells undergoing reprogramming need to overcome developmentally imposed epigenetic barriers. Recent work has shed new light on the molecular mechanisms that drive the reprogramming of somatic cells to iPSCs. Here, we present current knowledge on the transcriptional and epigenetic regulation of pluripotency induction and discuss how variability in epigenetic states impacts iPSCs' inherent biological properties.

Keywords: DNA methylation; chromatin; epigenetics; histone modifications; iPSC; induced pluripotent stem cell; pluripotency; reprogramming.

Figure 1.. Cellular, transcriptional and epigenetic changes during mouse somatic cell reprogramming to induced pluripotency.

Markers upregulated before and during the different phases of reprogramming are shown at the top. The two major waves of gene activity that occur during pluripotency induction are depicted in pink. Gray intensities of the bars denote the approximate magnitude of the indicated process over the time course of reprogramming. AP: Alkaline phosphatase; iPSC: Induced pluripotent stem cell; MET: Mesenchymal-to-epithelial transition; OSKM: Oct4, Sox2, Klf4, c-Myc.

Figure 2.. Roles of histone-modifying proteins and DNA methylation in pluripotency and reprogramming.

(A) In somatic cells, pluripotency gene promoters are methylated and enriched for H3K9me3, a heterochromatin-associated histone mark that blocks access of the OSKM factors to the DNA, hindering reprogramming. Reprogramming of somatic cells to iPSCs is associated with DNA demethylation and acquisition of H3K4me3 at promoters of pluripotency genes. The establishment of this permissive H3K4me3 chromatin state is brought about by Trithorax group (trxG) proteins, whose core component Wdr5 was found in ESCs to be directed to pluripotency loci by Oct4. The occupancy of Oct4 (and other pluripotency factors) with trxG drives strong activation of the pluripotency transcriptional network. In apparent contrast, somatic genes become silenced during reprogramming due to de novo promoter DNA methylation and the action of PRC2 that imposes a transcriptionally silent state of chromatin characterized by H3K27me3 modification. (B) Bivalent H3K4me3/H3K27me3 chromatin domains at promoter regions of developmental genes constitute a hallmark of ESC and iPSC pluripotency. The bivalent chromatin-associated protein Utf1, a target of Oct4 and Sox2, enforces a ‘poised’ state of gene expression in ESCs by preventing excessive PRC2 binding and H3K27me3, and facilitating the loading of Dcp1a to mRNAs generated from leaky transcription for cytoplasmic degradation. These contrasting functions of Utf1 prevent both the oversilencing and insufficient repression of bivalent genes, thereby properly coordinating developmental processes. 5mC: 5-methylcytosine; C: (Nonmethylated) Cytosine; ESC: Embryonic stem cell; iPSC: Induced pluripotent stem cell.

Figure 3.. Roles of TET enzymes and hydroxymethylation in the control of pluripotency.

(A) In embryonic stem cells, TET enzymes induce a poised state of chromatin at promoters of lineage-specific genes by recruiting PRC2, whose Ezh2 subunit catalyzes the methylation of nearby nucleosomes at H3K27. Mbd3, a transcriptional repressor of the NuRD complex that specifically binds to hydroxymethylated cytosines (5hmC), reinforces the repressed chromatin state via associated HDACs and chromatin remodelers. (B) At pluripotency gene promoters, 5hmC and possibly TET itself may hinder Uhrf1-mediated recruitment of the maintenance DNA methyltransferase Dnmt1 and associated transcriptional repressors, thereby keeping inhibitory DNA methylation (5mC) at a low level and safeguarding a transcriptionally permissive state of chromatin. Furthermore, MeCP2, a transcriptional repressor associated with Sin3a and HDAC-containing corepressor complexes, fails to recognize 5hmC. (C) The unhindered binding of Dnmt1 and MeCP2 at heavily methylated promoter regions of somatic genes contributes to gene repression by recruitment of chromatin-modifying transcriptional repressors. 5hmC: 5-hydroxymethylcytosine; 5mC: 5-methylcytosine.