Research Highlights:Highlights from the latest articles in DNA methylation research

Transfection of a Methylated Promoter Drives Mesenchymal Stem Cell Differentiation

Evaluation of: Hsiao SH, Lee KD, Hsu CC et al.: DNA methylation of the Trip10 promoter accelerates mesenchymal stem cell lineage determination. Biochem. Biophys. Res. Commun. 400(3), 305–312 (2010).

Mesenchymal stem cells (MSCs) are responsible for cell regeneration in adult organisms. Under specific conditions, they are able to differentiate into many cell types, including hepatocytes, adipocytes and neural cells [1]. For this reason, MSCs are considered a very promising tool for tissue regeneration. Despite encouraging preclinical and clinical evidence, controversial results on MSC application have been reported [2]. This suggests the need for a deeper understanding of the molecular mechanisms orchestrating MSC self-renewal and differentiation.

Epigenetic gene silencing is a major mechanism of stem cell development, and plays a crucial role in both physiological and pathological conditions [3]. During MSC differentiation, lineage-specific gene silencing is orchestrated by histone post-translational modifications and DNA methylation. Predeposited DNA methylation has been detected during neural lineage differentiation, thereby determining cell fate [4]. Based on this background, Hsiao and collegues identified 204 epigenetically silenced loci, during MSC-to-hepatocyte differentiation [5]. Among these, they found the Trip10 gene, encoding for a scaffold protein that regulates cytoskeleton and membrane trafficking [6]. In committed MSCs, the Trip10 promoter is marked by the polycomb-mediated histone H3 lysine 27 trimethylation, and is then progressively methylated during liver cell differentiation. This phenomenon is coupled to efficient Trip10 mRNA silencing. To investigate the causal relationship between MSC differentiation and Trip10 methylation, the authors synthesized an in vitro methylated Trip10 promoter sequence. The denatured sequence was transfected into MSCs. This causes the formation of a hemimethylated template during S phase. This template is then bound by DNA-methyltransferase (DNMT), that in turn methylates the complimentary DNA strand. The transfection system was efficient, since it was able to induce DNA methylation and silencing of the genomic Trip10 locus. More importantly, extrinsically induced Trip10 methylaytion was able to drive neuronal and osteocytic differentiation. In addition, the authors developed an enhanced green fluorescent protein reporter gene system to monitor DNA methylation and gene silencing at a specific locus. This method could prove to be a promising tool for the real-time tracing of DNA methylation dynamics in live cells.

Despite these promising results, some questions remain open: why was Trip10 methylation able to induce neural and osteocytic differentiation while it was detected during liver cell specification? What is the relationship between polycomb- and DNMT-meditated gene silencing? In specific contexts like cancer, histone methylation may mediate gene silencing in a DNMT-independent manner [7].

Financial & competing interests disclosure

The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Biological Significance of Dna Methylation Patterns in Human Progenitor Cells

Evaluation of: S⊘rensen AL, Timoskainen S, West FD et al.: Lineage-specific promoter DNA methylation patterns segregate adult progenitor cell types. Stem Cells Dev. 19(8), 1257–1266 (2010); S⊘rensen AL, Jacobsen BM, Reiner AH, Andersen IS, Collas P: Promoter DNA methylation patterns of differentiated cells are largely programmed at the progenitor stage. Mol. Biol. Cell 21(12), 2066–2077 (2010).

Recent evidence indicates that most human organs hold populations of progenitor cells, with multilineage differentiation ability [1]. Among these, mesenchymal stem cells (MSCs) can be isolated from bone marrow, skeletal muscle and adipose tissue, and therefore differentiate into myocyes, adipocytes, osteocytes and chondrocytes. MSCs are thought to be derived from common progenitors, called pericytes, residing within the perivascular compartment of mesenchymal tissues [2]. Pericytes display differentiation capacity and surface markers common to all MSCs, as well as to hematopoietic stem cells (HSCs). The common ontological origin of MSCs and HSCs is at present an interesting hypothesis. Few data are available to corroborate this.

Epigenetic gene regulation is crucial in orchestrating tissue specification and progenitor cell development [3]. DNA methylation is an epigenetic mark that often silences gene expression during differentiation. However, promoter DNA methylation is not always followed by gene silencing, and depends on CpG density. In addition, histone post-translational modifications contribute to gene-expression control during differentiation. Among these, the ‘histone code‘ recognises both permissive (i.e., histone H3K4 trimethylation) and repressive (i.e., histone H3K27 trimethylation) chromatin marks. Thus, epigenetic control of tissue specification emerges from a complex interplay between histone and DNA modifications.

For this reason, tracing DNA methylation patterns during progenitor cell differentiation may provide insight into the ontogeny of these cells. S⊘rensen and coworkers had previously demonstrated that a subset of lineage-specific promoters in adipocyte-, bone marrow- and myocyte-MSCs demonstrated very similar patterns of DNA methylation [4]. To corroborate these findings, they performed a genome-wide screening of promoter methylation in the same cell types [5]. They found that DNA methylation patterns are highly overlapping among these cells, arguing for their common origin. In addition, 90% of commonly methylated promoters in MSCs are methylated in HSCs. This corroborates the hypothesis that both HSCs and MSCs derive from pericytes. Despite this striking finding, the authors failed to identify a clear relationship between DNA methylation and gene silencing. Gene-expression analysis of methylated genes demonstrated that 54–57% of these were expressed. Promoter methylation profiles revealed that hypermethylation at the transcriptional start site is a unique feature of silenced genes. Despite this, the authors did not compare the overlap between actually silenced genes in MSCs and HSCs. This analysis would have likely shed new light on the relationship between epigenetic silencing and differentiation programs. In addition, the authors investigated the relationship between histone modifications and DNA methylation. In embryonic stem cells, the promoters of early differentiation genes are poised in a temporarily repressed status. These promoters hold both histone H3K4 and H3K27 methylation [6]. As differentiation into progenitor cells occurs, these genes are definitively activated (maintaining H3K4 trimethylation) or silenced (maintaining H3K27 trimethylation). In MSCs, these genes are often silenced by DNA methylation and/or H3K27 trimethylation [5]. Conversely, lineage-specific promoters are mainly methylated in embryonic stem cells, and unmethylated in MSCs. When compiled, these data corroborate the hypothesis of lineage priming in human stem cells, and indicates that epigenetic regulation of organ development is a complex process, far from being completely understood.

Induced Pluripotent Stem Cells Derived From Liver Disease Patients Can Differentiate Into Functional Hepatocytes

Evaluation of: Ghodsizadeh A, Taei A, Totonchi M: Generation of liver disease-specific induced pluripotent stem cells along with efficient differentiation to functional hepatocyte-like cells. Stem Cell Rev. 6(4), 622–632 (2010).

Induced pluripotent stem cells (iPSCs) were first generated by Takahashi and Yamanaka in 2006 [1]. They demonstrated that the introduction of four embryonic stem cell (ESC)-specific transcription factors into mouse adult fibroblasts was sufficient to induce ESC-like morphology and in vivo pluripotency. This evidence paved the way for the generation of human iPSCs. These cells have since been produced from patients affected by several diseases, especially genetic disorders [2]. In this context, iPSCs can be employed to investigate embryological defects causing the disease. In addition, iPSCs are a promising therapeutic tool. Defective genes can be replaced in patient-derived iPSCs, that eventually are driven to differentiate into a specific cell type.

With this background in mind, Ghodsizadeh and coworkers generated and characterized liver disease-specific iPSCs [3]. In particular, iPSCs were generated from human dermal fibroblasts of patients affected by tyrosinemia, glycogen storage disease, progressive familiar cholestasis and Crigler–Najjar syndrome. All these disorders share two main features: they are hereditary conditions and progressively lead to liver failure. In most cases, replacement of the defective gene in patient-derived iPSCs could be curative.

Using a previously described technique [4], the authors generated iPSCs introducing Oct4, Sox2, c-Myc and Klf4 genes in differentiated skin cells. This was sufficient to generate clones that morphologically resembled human ESCs. The iPSCs expressed high levels of Oct4 and Nanog, which are silenced by DNA methylation in differentiated cells [5]. This phenomenon was coupled to gene-specific promoter demethylation. In addition, iPSCs could be directed to differentiate into hepatocyte-like cells, secreting albumin, urea and other liver-specific factors. Phenobarbital was able to induce cytochrome P450 expression in these cells. Thus, hepatocyte-like cells derived form iPSCs seem to be metabolically active.

In the future, these promising results should challenge both the biologist and the clinician. The first should test iPSCs as a disease model; the latter should identify effective therapeutic protocols.