Epigenetic modification by active DNA demethylation

Epigenetic modification by active DNA demethylation or histone demethylation has been shown to facilitate iPSC induction in human and mouse (Huangfu et al., 2008a, 2008b). Tet1, a DNA methylcytosine dioxygenase, can facilitate iPSC induction by promoting Oct4 demethylation and re-activation, and even can replace Oct4 to initiate somatic cell reprogramming (Gao et al., 2013). H3K9me3 acts as a block to pluripotency, and Kdm3a/Jmjd1a as a histone H3K9 demethylase, or vitamin C that also can demethylate the histones, enhances reprogramming (Chen et al., 2013; Ma et al., 2008). Tet3 is another dioxygenase of Tet enzymes, and Tet3-mediated DNA hydroxylation is involved in epigenetic reprogramming of the zygotic paternal DNA (Gu et al., 2011); however, it has not been determined whether Tet3 can facilitate iPSC generation. Recently, a new pluripotent state, called “F-class iPSCs” was found (Hussein et al., 2014; Tonge et al., 2014). The F-class iPSCs is at a Nanog-positive cell state that is stable, occurs frequently, and is dependent on high expression of reprogramming factors, and these shk do not form typical embryonic stem cell (ESC)-like colonies. The F-class cells express significantly reduced levels of many PluriNet genes (Muller et al., 2008), including Dnmt3b, Rex1 (Zfp42), and Tdgf1 (Cripto). Nevertheless, they also express many genes at ESC levels such as Sall4, endogenous Oct4, and Nanog. After treatment with histone deacetylase inhibitors (HDACi), sodium butyrate (NaB), or trichostatin A (TSA), these cells could be converted to transgene-independent ESC-like cells capable of contributing to chimeras and the germline (Tonge et al., 2014). We speculated that piPSCs might resemble F-class iPSCs to some extent given that the exogenous genes of piPSCs are not silenced, the edges of early piPSCs appear fuzzy, and piPSCs express relatively high levels of endogenous Oct4 and Nanog, but low level of Rex1 as one of important naive state marker genes (as seen below).
Results
Discussion We show that epigenetic regulatory factors can enhance reprogramming of piPSCs and addition of HDACi can further downregulate exogenous genes. Consistent with the notion that Rex1 is an indicator of the naive pluripotent state (Nichols and Smith, 2009), piPSCs induced by epigenetic factors, especially Tet1, show significantly activated Rex1 and other genes important for naive state, including Utf1, Esrrb, and Dppa2. piPSCs are maintained dependent on exogenous genes, more like the “F-class” iPSCs (Hussein et al., 2014; Tonge et al., 2014). By exposure to HDACi, the two selected piPSC lines (4F + Tet1 and 4F + Kdm3a) show reduced expression of exogenous genes, especially in 4F + Kdm3a, and increased expression of endogenous genes associated with pluripotency. It is possible to convert F-class to naive-like state in some piPSCs. piPSCs in the presence of HDACi, including NaB, VPA, and suberoylanilide hydroxamic acid, express higher levels of Oct4, Nanog, Utf1, Rex1, Epcam, and Esrrb, compared with control (Petkov et al., 2016), confirmed in our study. VPA has been used for conversion of piPSCs from primed to naive-like state (Telugu et al., 2010). Moreover, we find that HDACi can moderately reduce expression of exogenous genes in piPSCs and increase expression of genes associated with pluripotency. Although exogenous genes are not effectively silenced, these approaches provide the basis for optimizing derivation and culture of piPSCs.
Experimental Procedures
Author Contributions
Acknowledgments This work was supported by the China MOST National Major Basic Research Program (2011CBA01002, 2012CB911202), the National Natural Science Foundation of China (31271587), and PCSIRT (no. IRT13023). We thank Zhongcheng Zhou, Yudong Fu, and Yu Yin for assisting the experiments, and John Tsibris for critical reading of the manuscript.
Introduction Adipose tissue is formed at specific locations as a major energy storage compartment and is an important source of signaling activity. The distribution of adipose reservoirs within the body undergoes major changes during normal aging (Caso et al., 2013), while excess or dysfunctional fat tissue leads to reduced lifespan and accelerates the onset of age-related diseases (Ahima, 2009; Muzumdar et al., 2008). Moreover, loss of subcutaneous fat and increased visceral adiposity is observed in patients with segmental progeroid syndromes such as Werner syndrome (Mori et al., 2001), Cockayne syndrome, or trichothiodystrophy. These diseases, mirroring certain aspects of accelerated aging, are characterized by mutations in DNA damage repair (DDR) factors, leading to accumulation of DNA damage over time and hence potentially to reduced proliferation and differentiation or to senescence of pre-adipocytes (Tchkonia et al., 2010). Mouse models deficient in DNA repair also show adipose tissue degeneration (Karakasilioti et al., 2013). However, it remains unclear whether DNA repair factors themselves have an impact on adipogenic differentiation of human adipose stromal cells (hASCs).