Resetting Cell Fate by Epigenetic Reprogramming

Epigenetic modifications and their regulations govern the identity of every cell type in an organism. Cell differentiation involves a switch in gene expression profile that is accompanied by heritable changes of epigenetic signatures in the differentiated cell type. Differentiation is generally not reversible, thereby conferring cell fate decisions once an altered epigenetic pattern is set. Nevertheless, attempts have been made to reverse a differentiation cell fate to a pluripotent state by various experimental approaches, such as somatic cell nuclear transfer, cell fusion and ectopic expression of defined transcription factors. The fundamental basis of all these strategies is to mediate epigenetic reprogramming, which allows a permanent and completed conversion of cell fate. A comprehensive understanding of the dynamic of epigenetic changes during cell differentiation would provide a more precise and efficient way of reprogramming cell fate. Here we summarize the epigenetic aspects of different reprogramming strategies and discuss the possible mechanisms underlying these epigenetic reprogramming events

Global Mapping of DNA Methylation in Mouse Promoters Reveals Epigenetic Reprogramming of Pluripotency Genes

DNA methylation patterns are reprogrammed in primordial germ cells and in preimplantation embryos by demethylation and subsequent de novo methylation. It has been suggested that epigenetic reprogramming may be necessary for the embryonic genome to return to a pluripotent state. We have carried out a genome-wide promoter analysis of DNA methylation in mouse embryonic stem (ES) cells, embryonic germ (EG) cells, sperm, trophoblast stem (TS) cells, and primary embryonic fibroblasts (pMEFs). Global clustering analysis shows that methylation patterns of ES cells, EG cells, and sperm are surprisingly similar, suggesting that while the sperm is a highly specialized cell type, its promoter epigenome is already largely reprogrammed and resembles a pluripotent state. Comparisons between pluripotent tissues and pMEFs reveal that a number of pluripotency related genes, including Nanog, Lefty1 and Tdgf1, as well as the nucleosome remodeller Smarcd1, are hypomethylated in stem cells and hypermethylated in differentiated cells. Differences in promoter methylation are associated with significant differences in transcription levels in more than 60% of genes analysed. Our comparative approach to promoter methylation thus identifies gene candidates for the regulation of pluripotency and epigenetic reprogramming. While the sperm genome is, overall, similarly methylated to that of ES and EG cells, there are some key exceptions, including Nanog and Lefty1, that are highly methylated in sperm. Nanog promoter methylation is erased by active and passive demethylation after fertilisation before expression commences in the morula. In ES cells the normally active Nanog promoter is silenced when targeted by de novo methylation. Our study suggests that reprogramming of promoter methylation is one of the key determinants of the epigenetic regulation of pluripotency genes. Epigenetic reprogramming in the germline prior to fertilisation and the reprogramming of key pluripotency genes in the early embryo is thus crucial for transmission of pluripotency

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Histone demethylase JMJD2B/KDM4B regulates transcriptional program via distinctive epigenetic targets and protein interactors for the maintenance of trophoblast stem cells

Abstract Trophoblast stem cell (TSC) is crucial to the formation of placenta in mammals. Histone demethylase JMJD2 (also known as KDM4) family proteins have been previously shown to support self-renewal and differentiation of stem cells. However, their roles in the context of the trophoblast lineage remain unclear. Here, we find that knockdown of Jmjd2b resulted in differentiation of TSCs, suggesting an indispensable role of JMJD2B/KDM4B in maintaining the stemness. Through the integration of transcriptome and ChIP-seq profiling data, we show that JMJD2B is associated with a loss of H3K36me3 in a subset of embryonic lineage genes which are marked by H3K9me3 for stable repression. By characterizing the JMJD2B binding motifs and other transcription factor binding datasets, we discover that JMJD2B forms a protein complex with AP-2 family transcription factor TFAP2C and histone demethylase LSD1. The JMJD2B–TFAP2C–LSD1 complex predominantly occupies active gene promoters, whereas the TFAP2C–LSD1 complex is located at putative enhancers, suggesting that these proteins mediate enhancer–promoter interaction for gene regulation. We conclude that JMJD2B is vital to the TSC transcriptional program and safeguards the trophoblast cell fate via distinctive protein interactors and epigenetic targets

Epigenetic Regulation of Pluripotent Genes Mediates Stem Cell Features in Human Hepatocellular Carcinoma and Cancer Cell Lines

<div><p>Activation of the stem cell transcriptional circuitry is an important event in cancer development. Although cancer cells demonstrate a stem cell-like gene expression signature, the epigenetic regulation of pluripotency-associated genes in cancers remains poorly understood. In this study, we characterized the epigenetic regulation of the pluripotency-associated genes <i>NANOG</i>, <i>OCT4</i>, <i>c-MYC</i>, <i>KLF4</i>, and <i>SOX2</i> in a variety of cancer cell lines and in primary tumor samples, and investigated the re-activation of pluripotency regulatory circuits in cancer progression. Differential patterns of DNA methylation, histone modifications, and gene expression of pluripotent genes were demonstrated in different types of cancers, which may reflect their tissue origins. <i>NANOG</i> promoter hypomethylation and gene upregulation were found in metastatic human liver cancer cells and human hepatocellular carcinoma (HCC) primary tumor tissues. The upregulation of <i>NANOG</i>, together with p53 depletion, was significantly associated with clinical late stage of HCC. A pro-metastatic role of NANOG in colon cancer cells was also demonstrated, using a <i>NANOG</i>-overexpressing orthotopic tumor implantation mouse model. Demethylation of <i>NANOG</i> promoter was observed in CD133+^high cancer cells. In accordance, overexpression of <i>NANOG</i> resulted in an increase in the population of CD133+^high cells. In addition, we demonstrated a cross-regulation between <i>OCT4</i> and <i>NANOG</i> in cancer cells via reprogramming of promoter methylation. Taken together, epigenetic reprogramming of <i>NANOG</i> can lead to the acquisition of stem cell-like properties. These results underscore the restoration of pluripotency circuits in cancer cells as a potential mechanism for cancer progression.</p></div

Correlation between metastatic clinicopathological parameters and mRNA expression of <i>NANOG</i> and <i>p53</i> in HCC.

*<p><i>p</i><0.05 was considered as statistically significant.</p

Deregulation of <i>NANOG</i> in HCC primary tumor tissue.

<p>(A) H&E staining of human normal liver (left), and HCC non-tumor (middle) and tumor tissue (right). (B) Methylation status of the <i>NANOG</i> promoter (−1449 to −952) in fifteen paired HCC non-tumor and tumor tissues. DNA methylation frequency was classified into 50–69% (black), 30–49% (grey), and 19–29% (white) in HCC tumor and adjacent non-tumor tissues. (C) <i>NANOG</i> promoter methylation pattern is represented with HCC case-297 tumor and adjacent non-tumor tissue. (D) Statistical comparison of <i>NANOG</i> promoter methylation in normal liver (n = 3), HCC adjacent non-tumor (n = 15) and tumor (n = 15) tissues; data are the mean ± SD. (E) Statistical comparison of <i>NANOG</i> gene expression in paired HCC non-tumor and tumor tissue (n = 15). Each tumor tissue was normalized with its corresponding non-tumor tissue.</p

Differential methylation of pluripotency-associated genes <i>NANOG</i>, <i>OCT4</i>, and <i>c</i>-<i>MYC</i> in cancer cells.

<p>(A) Schematic diagram of gene regulatory regions of <i>NANOG</i>, <i>OCT4</i>, and <i>c-MYC</i> that were examined by bisulfite sequencing (BiS) (red bars) and ChIP (green bars) experiments. (i) The <i>NANOG</i> proximal promoter region covers 10 CpG sites from −1449 to −952. (ii) The <i>OCT4</i> promoter region is covered by 8 primer pairs for 50 CpG sites from −2973 to +320. (iii) The <i>c-MYC</i> gene region is covered by BiS primers for CpG islands before TSS1 and TSS2, and CpG sites within Exon 2 and 3; and ChIP primer for Exon 3. (B) Bisulfite sequencing analysis of the <i>NANOG</i> promoter in cancer cell lines. DNA methylation frequency is presented as percentages in: normal liver (L02) and cancer liver (PLC, 97L, and 97H) cells (blue); normal PBMC and leukemic K-562 cells (orange); and in HeLa, MCF7, HCT116, and AGS cancer cells (green). (C) Clonal <i>NANOG</i> promoter methylation patterns in 97L, HeLa and K-562 cells. Open circles represent unmethylated CpGs; closed circles represent methylated CpGs. (D) Methylation frequency of the <i>OCT4</i> proximal promoter (−530 to +7) in: normal and cancer liver cells (blue); and in HeLa and HCT116 cells (green). (E) DNA methylation frequency of upstream and downstream regions of <i>OCT4</i> in normal and cancer liver cells. “Overall” covers 50 CpG sites from −2973 to +320; “5′ TSS” covers 10 CpG sites from −530 to +7; and “3′ TSS” covers 12 CpG sites from +61 to +320. (F) Methylation frequency of exon 3 of <i>c-MYC</i> (10 CpG sites) in: normal and cancer liver cells (blue); PBMC and leukemic K-562 cells (orange); and in HeLa, MCF7, HCT116, and AGS cancer cells (green).</p

A correlation between pluripotency-associated gene expression and histone modification patterns in normal and cancer cells.

<p>Expression levels of (A) <i>NANOG</i>, (B) <i>OCT4</i>, (C) <i>c</i>-<i>MYC</i>, and (D) <i>KLF4</i> genes were determined in L02, PLC, 97L, and HCT116 cells by qRT-PCR analysis, normalized with the reference gene <i>18S</i>. The gene expression was normalized to L02 sample, which was defined as 1. Data are the mean ± SD obtained from 2 to 3 experiments with duplicates. Enrichment of histone modification H3K4me3 and H3K27me3 at the promoter regions of pluripotency-associated genes (E) <i>NANOG</i>, (F) <i>OCT4</i>, (G) <i>c</i>-<i>MYC</i>, and (H) <i>KLF4</i> were measured in L02, 97L, and HCT116 by ChIP analysis. Data are represented as fold enrichment and normalized with input and mock IgG controls. The fold enrichment was relative to L02 sample, which was defined as 1. Data are represented with mean value obtained from two ChIP experiments with error bars of standard derivation.</p