Loss of Tet enzymes compromises proper differentiation of embryonic stem cells - PubMed (original) (raw)

Loss of Tet enzymes compromises proper differentiation of embryonic stem cells

Meelad M Dawlaty et al. Dev Cell. 2014.

Abstract

Tet enzymes (Tet1/2/3) convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and are dynamically expressed during development. Whereas loss of individual Tet enzymes or combined deficiency of Tet1/2 allows for embryogenesis, the effect of complete loss of Tet activity and 5hmC marks in development is not established. We have generated Tet1/2/3 triple-knockout (TKO) mouse embryonic stem cells (ESCs) and examined their developmental potential. Combined deficiency of all three Tets depleted 5hmC and impaired ESC differentiation, as seen in poorly differentiated TKO embryoid bodies (EBs) and teratomas. Consistent with impaired differentiation, TKO ESCs contributed poorly to chimeric embryos, a defect rescued by Tet1 reexpression, and could not support embryonic development. Global gene-expression and methylome analyses of TKO EBs revealed promoter hypermethylation and deregulation of genes implicated in embryonic development and differentiation. These findings suggest a requirement for Tet- and 5hmC-mediated DNA demethylation in proper regulation of gene expression during ESC differentiation and development.

Copyright © 2014 Elsevier Inc. All rights reserved.

PubMed Disclaimer

Figures

Figure 1

Figure 1. Loss of 5hmC and restricted differentiation potential of Tet TKO ES cells in embryoid body and teratoma formation assays

(A) Confirmation of loss of Tet enzymes in Tet TKO ESCs and differentiated cells by Western blot. (B) Quantification of 5hmC and 5mC in ESCs and EBs of the indicated genotypes by mass spectrometry. (C) Sections of paraffin-embedded EBs derived from ES cell lines of indicated genotypes stained with Hematoxylin and Eosin (H&E). (D &E) RT-qPCR for markers of embryonic germ layers in day 10 EBs (D) and day 15 EBs (E) of the indicated genotypes. Data are normalized to Gapdh. (F) Representative images of each embryonic germ layer from hematoxylin and eosin (H&E)-stained sections of teratomas derived from ESCs of the indicated genotypes. N.D.= none detected. (G) Summary table of the various germ layer tissues detected in teratomas of indicated genotypes. For all panels, error bars indicate standard deviation. (See also Figure S1)

Figure 2

Figure 2. Limited contribution of Tet TKO ES cells to developing embryos in a chimera assay

A&B) Bright field and fluorescence images of E13.5 chimeric embryos generated by injecting Rosa26-EGFP targeted ESCs of the indicated genotypes into WT blastocysts. The very poor and weaker GFP signal in TKO ESC chimeras are highlighted in the images in B. C) Summary table for all cell lines tested in the chimera assay. D) Top: Bright field and fluorescence images of E9.5 chimeric embryos generated by injecting Rosa26-EGFP targeted ESCs of the indicated genotypes into WT blastocysts. Bottom: Summary table for all cell lines tested in this chimera assay. Asterisk indicates growth retarded and defective embryos. E) Southern blot confirming the negligible to no detection of the TKO-Rosa26-EGFP allele in DNA extracted from E13.5 TKO chimeric embryos. F) E14.5 chimeric embryos generated by injecting Rosa26-EGFP targeted ESCs of indicated genotypes into WT blastocysts. G) Left: Bright field and fluorescence images of E9.5 4N embryos generated by injecting Rosa26-EGFP targeted ESCs of the indicated genotypes into 4N WT blastocysts. Right: Summary table for all cell lines tested in the tetraploid complementation assay. For all panels, arrowheads point to poor GFP signal. (See also Figure S2)

Figure 3

Figure 3. Aberrant promoter hypermethylation and deregulation of developmental genes in Tet TKO embryoid bodies

A) Schematic of the MeDIP and gene expression analyses to identify hypermethylated and down regulated genes during differentiation of TKO ES cells. B) Scatter plots showing differentially expressed genes in red (up regulated) or in green (down regulated) across a panel of TKO EBs when compared to WT EBs. C) Gene ontology analysis of differentially expressed genes in TKO EBs. D) MeDIP-seq profile of a 3 Mb region from mouse chromosome 6 with the Hoxa cluster in the center in two independent TKO and WT EB clones as an example for the general hypermethylation observed in TKO EBs. Enrichments are indicated as normalized read counts. E) Analysis of deregulated genes in TKO EBs correlating their expression to the methylation status of their promoters or gene bodies. The pie charts show the percentage of deregulated genes in TKO EBs that show hypermethylation compared to WT (green), hypomethylation compared to WT (yellow) or do not change (grey), in the gene body or the promoter (+/− 1000 bp from the TSS). F) Gene ontology analysis of all genes with hypermethylated promoter regions (normalized average read counts > 4 in the region +/− 1000 bp from the TSS) in TKO EBs. G) Gene ontology analysis of all genes down regulated in TKO EBs that also have differentially hypermethylated promoters (TKO vs. WT). (See also Table S1)

Figure 4

Figure 4. Gene specific bisulfite sequencing for validation of hypermethylation of deregulated developmental genes in Tet TKO embryoid bodies

A) MeDIP-seq profiles of representative developmental genes in TKO EBs. Enrichments are indicated as normalized read counts. Red boxes indicate the genomic region analyzed by gene specific 454 bisulfite sequencing in B and C. B) Validation of MeDIP-data by gene specific 454 bisulfite sequencing. Results for a 200–300 bp region (see red box in A) at the promoters of indicated genes are shown as heatmaps in which each row represents one sequence read and each column an individual CpG site within the analyzed region. Individual blue boxes indicate methylated and yellow boxes indicate unmethylated CpG dinucleotides. Panels below heatmaps show the average methylation of each interrogated CpG for the analyzed DNA fragment. For color bar see C. Sequencing coverage (reads) are indicated. C) Heat maps for four additional promoter regions of the genes analyzed. Panels show the average methylation of each interrogated CpG according to the color bar shown on the right. Numbers indicate the sequencing coverage. (See also Figure S3)

Similar articles

Cited by

References

    1. Bocker MT, Tuorto F, Raddatz GUN, Musch T, Yang FC, Xu M, Lyko F, Breiling A. Hydroxylation of 5-methylcytosine by TET2 maintains the active state of the mammalian HOXA cluster. Nature Communications. 2012;3:818–12. - PMC - PubMed
    1. Dawlaty MM, Breiling A, Le T, Raddatz G, Barrasa MI, Cheng AW, Gao Q, Powell BE, Li Z, Xu M, et al. Combined deficiency of Tet1 and Tet2 causes epigenetic abnormalities but is compatible with postnatal development. Developmental Cell. 2013;24:310–323. - PMC - PubMed
    1. Dawlaty MM, Ganz K, Powell BE, Hu YC, Markoulaki S, Cheng AW, Gao Q, Kim J, Choi SW, Page DC, et al. Tet1 Is Dispensable for Maintaining Pluripotency and Its Loss Is Compatible with Embryonic and Postnatal Development. Stem Cell. 2011;9:166–175. - PMC - PubMed
    1. Gifford CA, Ziller MJ, Gu H, Trapnell C, Donaghey J, Tsankov A, Shalek AK, Kelley DR, Shishkin AA, Issner R, et al. Transcriptional and Epigenetic Dynamics during Specification of Human Embryonic Stem Cells. Cell. 2013;153:1149–1163. - PMC - PubMed
    1. Grönniger E, Weber B, Heil O, Peters N, Stäb F, Wenck H, Korn B, Winnefeld M, Lyko F. Aging and Chronic Sun Exposure Cause Distinct Epigenetic Changes in Human Skin. PLoS Genet. 2010;6:e1000971. - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources