The DNA dioxygenase Tet1 regulates H3K27 modification and embryonic stem cell biology independent of its catalytic activity

Tet enzymes (Tet1/2/3) oxidize 5-methylcytosine to promote DNA demethylation and partner with chromatin modifiers to regulate gene expression. Tet1 is highly expressed in embryonic stem cells (ESCs), but its enzymatic and non-enzymatic roles in gene regulation are not dissected. We have generated Tet1 catalytically inactive (Tet1^m/m) and knockout (Tet1^−/−) ESCs and mice to study these functions. Loss of Tet1, but not loss of its catalytic activity, caused aberrant upregulation of bivalent (H3K4me3⁺; H3K27me3⁺) developmental genes, leading to defects in differentiation. Wild-type and catalytic-mutant Tet1 occupied similar genomic loci which overlapped with H3K27 tri-methyltransferase PRC2 and the deacetylase complex Sin3a at promoters of bivalent genes and with the helicase Chd4 at active genes. Loss of Tet1, but not loss of its catalytic activity, impaired enrichment of PRC2 and Sin3a at bivalent promoters leading to reduced H3K27 trimethylation and deacetylation, respectively, in absence of any changes in DNA methylation. Tet1^−/−, but not Tet1^m/m, embryos expressed higher levels of Gata6 and were developmentally delayed. Thus, the critical functions of Tet1 in ESCs and early development are mediated through its non-catalytic roles in regulating H3K27 modifications to silence developmental genes, and are more important than its catalytic functions in DNA demethylation.     

MicroRNA-29b/Tet1 regulatory axis epigenetically modulates mesendoderm differentiation in mouse embryonic stem cells

Ten eleven translocation (Tet) family-mediated DNA oxidation on 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) represents a novel epigenetic modification that regulates dynamic gene expression during embryonic stem cells (ESCs) differentiation. Through the role of Tet on 5hmC regulation in stem cell development is relatively defined, how the Tet family is regulated and impacts on ESCs lineage development remains elusive. In this study, we show non-coding RNA regulation on Tet family may contribute to epigenetic regulation during ESCs differentiation, which is suggested by microRNA-29b (miR-29b) binding sites on the Tet1 3′ untranslated region (3′ UTR). We demonstrate miR-29b increases sharply after embyoid body (EB) formation, which causes Tet1 repression and reduction of cellular 5hmC level during ESCs differentiation. Importantly, we show this miR-29b/Tet1 regulatory axis promotes the mesendoderm lineage formation both in vitro and in vivo by inducing the Nodal signaling pathway and repressing the key target of the active demethylation pathway, Tdg. Taken together, our findings underscore the contribution of small non-coding RNA mediated regulation on DNA demethylation dynamics and the differential expressions of key mesendoderm regulators during ESCs lineage specification. MiR-29b could potentially be applied to enrich production of mesoderm and endoderm derivatives and be further differentiated into desired organ-specific cells.     

Tet1 is dispensable for maintaining pluripotency and its loss is compatible with embryonic and postnatal development

The Tet family of enzymes (Tet1/2/3) converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Mouse embryonic stem cells (mESCs) highly express Tet1 and have an elevated level of 5hmC. Tet1 has been implicated in ESC maintenance and lineage specification in?vitro but its precise function in development is not well defined. To establish the role of Tet1 in pluripotency and development, we have generated Tet1 mutant mESCs and mice. Tet1(-/-) ESCs have reduced levels of 5hmC and subtle changes in global gene expression, and are pluripotent and support development of live-born mice in tetraploid complementation assay, but display skewed differentiation toward trophectoderm in?vitro. Tet1 mutant mice are viable, fertile, and grossly normal, though some mutant mice have a slightly smaller body size at birth. Our data suggest that Tet1 loss leading to a partial reduction in 5hmC levels does not affect pluripotency in ESCs and is compatible with embryonic and postnatal development.     

Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells

TET family enzymes convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. Here, we show that Tet1 and Tet2 are Oct4-regulated enzymes that together sustain 5hmC in mouse embryonic stem cells (ESCs) and are induced concomitantly with 5hmC during reprogramming of fibroblasts to induced pluripotent stem cells. ESCs depleted of Tet1 by RNAi show diminished expression of the Nodal antagonist Lefty1 and display hyperactive Nodal signaling and skewed differentiation into the endoderm-mesoderm lineage in embryoid bodies in?vitro. In Fgf4- and heparin-supplemented culture conditions, Tet1-depleted ESCs activate the trophoblast stem cell lineage determinant Elf5 and can colonize the placenta in midgestation embryo chimeras. Consistent with these findings, Tet1-depleted ESCs?form aggressive hemorrhagic teratomas with increased endoderm, reduced neuroectoderm, and ectopic appearance of trophoblastic giant cells. Thus, 5hmC is an epigenetic modification associated with the pluripotent state, and Tet1 functions to regulate the lineage differentiation potential of ESCs.     

Genome-wide mapping of miRNAs expressed in embryonic stem cells and pluripotent stem cells generated by different reprogramming strategies

Background

Reprogrammed cells, including induced pluripotent stem cells (iPSCs) and nuclear transfer embryonic stem cells (NT-ESCs), are similar in many respects to natural embryonic stem cells (ESCs). However, previous studies have demonstrated that iPSCs retain a gene expression signature that is unique from that of ESCs, including differences in microRNA (miRNA) expression, while NT-ESCs are more faithfully reprogrammed cells and have better developmental potential compared with iPSCs.

Results

We focused on miRNA expression and explored the difference between ESCs and reprogrammed cells, especially ESCs and NT-ESCs. We also compared the distinct expression patterns among iPSCs, NT-ESCs and NT-iPSCs. The results demonstrated that reprogrammed cells (iPSCs and NT-ESCs) have unique miRNA expression patterns compared with ESCs. The comparison of differently reprogrammed cells (NT-ESCs, NT-iPSCs and iPSCs) suggests that several miRNAs have key roles in the distinct developmental potential of reprogrammed cells.

Conclusions

Our data suggest that miRNAs play a part in the difference between ESCs and reprogrammed cells, as well as between MEFs and pluripotent cells. The variation of miRNA expression in reprogrammed cells derived using different reprogramming strategies suggests different characteristics induced by nuclear transfer and iPSC generation, as well as different developmental potential among NT-ESCs, iPSCs and NT-iPSCs.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-488) contains supplementary material, which is available to authorized users.     

Ontogeny,distribution and potential roles of 5-hydroxymethylcytosine in human liver function

Background

Interindividual differences in liver functions such as protein synthesis, lipid and carbohydrate metabolism and drug metabolism are influenced by epigenetic factors. The role of the epigenetic machinery in such processes has, however, been barely investigated. 5-hydroxymethylcytosine (5hmC) is a recently re-discovered epigenetic DNA modification that plays an important role in the control of gene expression.

Results

In this study, we investigate 5hmC occurrence and genomic distribution in 8 fetal and 7 adult human liver samples in relation to ontogeny and function. LC-MS analysis shows that in the adult liver samples 5hmC comprises up to 1% of the total cytosine content, whereas in all fetal livers it is below 0.125%. Immunohistostaining of liver sections with a polyclonal anti-5hmC antibody shows that 5hmC is detected in most of the hepatocytes. Genome-wide mapping of the distribution of 5hmC in human liver samples by next-generation sequencing shows significant differences between fetal and adult livers. In adult livers, 5hmC occupancy is overrepresented in genes involved in active catabolic and metabolic processes, whereas 5hmC elements which are found in genes exclusively in fetal livers and disappear in the adult state, are more specific to pathways for differentiation and development.

Conclusions

Our findings suggest that 5-hydroxymethylcytosine plays an important role in the development and function of the human liver and might be an important determinant for development of liver diseases as well as of the interindividual differences in drug metabolism and toxicity.     

Identification of Five Developmental Processes during Chondrogenic Differentiation of Embryonic Stem Cells

Background

Chondrogenesis is the complex process that leads to the establishment of cartilage and bone formation. Due to their ability to differentiate in vitro and mimic development, embryonic stem cells (ESCs) show great potential for investigating developmental processes. In this study, we used chondrogenic differentiation of ESCs as a model to analyze morphogenetic events during chondrogenesis.

Methodology/Principal Findings

ESCs were differentiated into the chondrocyte lineage, forming small cartilaginous aggregates in suspension. Differentiated ESCs showed that chondrogenesis was typically characterized by five overlapping stages. During the first stage, cell condensation and aggregate formation was observed. The second stage was characterized by differentiation into chondrocytes and fibril scaffold formation within spherical aggregates. Deposition of cartilaginous extracellular matrix and cartilage formation were hallmarks of the third stage. Apoptosis of chondrocytes, hypertrophy and/or degradation of cartilage occurred during the fourth stage. Finally, during the fifth stage, bone replacement with membranous calcified tissues took place.

Conclusions/Significance

We demonstrate that ESCs show the chondrogenic differentiation pathway from the pluripotent stem cell to terminal skeletogenesis through these five stages in vitro. During each stage, morphological changes acquired in preceding stages played an important role in further development as a scaffold or template in subsequent stages. The study of chondrogenesis via ESC differentiation may be informative to our further understanding of skeletal growth and regeneration.     

KDM5B focuses H3K4 methylation near promoters and enhancers during embryonic stem cell self-renewal and differentiation

Background

Pluripotency of embryonic stem (ES) cells is controlled in part by chromatin-modifying factors that regulate histone H3 lysine 4 (H3K4) methylation. However, it remains unclear how H3K4 demethylation contributes to ES cell function.

Results

Here, we show that KDM5B, which demethylates lysine 4 of histone H3, co-localizes with H3K4me3 near promoters and enhancers of active genes in ES cells; its depletion leads to spreading of H3K4 methylation into gene bodies and enhancer shores, indicating that KDM5B functions to focus H3K4 methylation at promoters and enhancers. Spreading of H3K4 methylation to gene bodies and enhancer shores is linked to defects in gene expression programs and enhancer activity, respectively, during self-renewal and differentiation of KDM5B-depleted ES cells. KDM5B critically regulates H3K4 methylation at bivalent genes during differentiation in the absence of LIF or Oct4. We also show that KDM5B and LSD1, another H3K4 demethylase, co-regulate H3K4 methylation at active promoters but they retain distinct roles in demethylating gene body regions and bivalent genes.

Conclusions

Our results provide global and functional insight into the role of KDM5B in regulating H3K4 methylation marks near promoters, gene bodies, and enhancers in ES cells and during differentiation.     

A primary role of TET proteins in establishment and maintenance of De Novo bivalency at CpG islands

Ten Eleven Translocation (TET) protein-catalyzed 5mC oxidation not only creates novel DNA modifications, such as 5hmC, but also initiates active or passive DNA demethylation. TETs’ role in the crosstalk with specific histone modifications, however, is largely elusive. Here, we show that TET2-mediated DNA demethylation plays a primary role in the de novo establishment and maintenance of H3K4me3/H3K27me3 bivalent domains underlying methylated DNA CpG islands (CGIs). Overexpression of wild type (WT), but not catalytic inactive mutant (Mut), TET2 in low-TET-expressing cells results in an increase in the level of 5hmC with accompanying DNA demethylation at a subset of CGIs. Most importantly, this alteration is sufficient in making de novo bivalent domains at these loci. Genome-wide analysis reveals that these de novo synthesized bivalent domains are largely associated with a subset of essential developmental gene promoters, which are located within CGIs and are previously silenced due to DNA methylation. On the other hand, deletion of Tet1 and Tet2 in mouse embryonic stem (ES) cells results in an apparent loss of H3K27me3 at bivalent domains, which are associated with a particular set of key developmental gene promoters. Collectively, this study demonstrates the critical role of TET proteins in regulating the crosstalk between two key epigenetic mechanisms, DNA methylation and histone methylation (H3K4me3 and H3K27me3), particularly at CGIs associated with developmental genes.     

Microenvironment Modulates Osteogenic Cell Lineage Commitment in Differentiated Embryonic Stem Cells

Background

Due to their self-renewal, embryonic stem cells (ESCs) are attractive cells for applications in regenerative medicine and tissue engineering. Although ESC differentiation has been used as a platform for generating bone in vitro and in vivo, the results have been unsatisfactory at best. It is possible that the traditional culture methods, which have been used, are not optimal and that other approaches must be explored.

Methodology/Principal Findings

ESCs were differentiated into osteoblast lineage using a micro-mass approach. In response to osteogenic differentiation medium, many cells underwent apoptosis, while others left the micro-mass, forming small aggregates in suspension. These aggregates were cultured in three different culture conditions (adhesion, static suspension, and stirred suspension), then examined for osteogenic potential in vitro and in vivo. In adhesion culture, ESCs primed to become osteoblasts recommitted to the adipocyte lineage in vitro. In a static suspension culture, resulting porous aggregates expressed osteoblasts markers and formed bone in vivo via intermembranous ossification. In a stirred suspension culture, resulting non-porous aggregates suppressed osteoblast differentiation in favor of expanding progenitor cells.

Conclusions/Significance

We demonstrate that microenvironment modulates cell fate and subsequent tissue formation during ESC differentiation. For effective tissue engineering using ESCs, it is important to develop optimized cell culture/differentiation conditions based upon the influence of microenvironment.     

Defining Differentially Methylated Regions Specific for the Acquisition of Pluripotency and Maintenance in Human Pluripotent Stem Cells via Microarray

Background

Epigenetic regulation is critical for the maintenance of human pluripotent stem cells. It has been shown that pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells, appear to have a hypermethylated status compared with differentiated cells. However, the epigenetic differences in genes that maintain stemness and regulate reprogramming between embryonic stem cells and induced pluripotent stem cells remain unclear. Additionally, differential methylation patterns of induced pluripotent stem cells generated using diverse methods require further study.

Methodology

Here, we determined the DNA methylation profiles of 10 human cell lines, including 2 ESC lines, 4 virally derived iPSC lines, 2 episomally derived iPSC lines, and the 2 parental cell lines from which the iPSCs were derived using Illumina''s Infinium HumanMethylation450 BeadChip. The iPSCs exhibited a hypermethylation status similar to that of ESCs but with distinct differences from the parental cells. Genes with a common methylation pattern between iPSCs and ESCs were classified as critical factors for stemness, whereas differences between iPSCs and ESCs suggested that iPSCs partly retained the parental characteristics and gained de novo methylation aberrances during cellular reprogramming. No significant differences were identified between virally and episomally derived iPSCs. This study determined in detail the de novo differential methylation signatures of particular stem cell lines.

Conclusions

This study describes the DNA methylation profiles of human iPSCs generated using both viral and episomal methods, the corresponding somatic cells, and hESCs. Series of ss-DMRs and ES-iPS-DMRs were defined with high resolution. Knowledge of this type of epigenetic information could be used as a signature for stemness and self-renewal and provides a potential method for selecting optimal pluripotent stem cells for human regenerative medicine.