Faculty Collaboration Database

Deletion of Tet proteins results in quantitative disparities during ESC differentiation partially attributable to alterations in gene expression. BMC Dev Biol 2019 Jul 08;19(1):16

Date

07/10/2019

Pubmed ID

31286885

Pubmed Central ID

PMC6615237

DOI

10.1186/s12861-019-0196-6

Scopus ID

2-s2.0-85068878011 (requires institutional sign-in at Scopus site)   7 Citations

Abstract

BACKGROUND: The Tet protein family (Tet1, Tet2, and Tet3) regulate DNA methylation through conversion of 5-methylcytosine to 5-hydroxymethylcytosine which can ultimately result in DNA demethylation and play a critical role during early mammalian development and pluripotency. While multiple groups have generated knockouts combining loss of different Tet proteins in murine embryonic stem cells (ESCs), differences in genetic background and approaches has made it difficult to directly compare results and discern the direct mechanism by which Tet proteins regulate the transcriptome. To address this concern, we utilized genomic editing in an isogenic pluripotent background which permitted a quantitative, flow-cytometry based measurement of pluripotency in combination with genome-wide assessment of gene expression and DNA methylation changes. Our ultimate goal was to generate a resource of large-scale datasets to permit hypothesis-generating experiments.

RESULTS: We demonstrate a quantitative disparity in the differentiation ability among Tet protein deletions, with Tet2 single knockout exhibiting the most severe defect, while loss of Tet1 alone or combinations of Tet genes showed a quantitatively intermediate phenotype. Using a combination of transcriptomic and epigenomic approaches we demonstrate an increase in DNA hypermethylation and a divergence of transcriptional profiles in pluripotency among Tet deletions, with loss of Tet2 having the most profound effect in undifferentiated ESCs.

CONCLUSIONS: We conclude that loss of Tet2 has the most dramatic effect both on the phenotype of ESCs and the transcriptome compared to other genotypes. While loss of Tet proteins increased DNA hypermethylation, especially in gene promoters, these changes in DNA methylation did not correlate with gene expression changes. Thus, while loss of different Tet proteins alters DNA methylation, this change does not appear to be directly responsible for transcriptome changes. Thus, loss of Tet proteins likely regulates the transcriptome epigenetically both through altering 5mC but also through additional mechanisms. Nonetheless, the transcriptome changes in pluripotent Tet2^-/- ESCs compared to wild-type implies that the disparities in differentiation can be partially attributed to baseline alterations in gene expression.

Author List

Reimer M Jr, Pulakanti K, Shi L, Abel A, Liang M, Malarkannan S, Rao S

Authors

Subramaniam Malarkannan PhD Professor in the Medicine department at Medical College of Wisconsin
Sridhar Rao MD, PhD Associate Professor in the Pediatrics department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Animals
CRISPR-Cas Systems
Cell Differentiation
DNA Methylation
DNA-Binding Proteins
Dioxygenases
Embryonic Stem Cells
Gene Editing
Gene Expression Regulation, Developmental
Mice
Mice, Knockout
Proto-Oncogene Proteins