Overlapping Requirements for Tet2 and Tet3 in Normal Development and Hematopoietic Stem Cell Emergence.

Cheng Li,Evgenia Korol,Todd Evans,Mamta Tahiliani,Yahui Lan,Lianna Schwartz-Orbach,Mary G Goll

doi:10.1016/j.celrep.2015.07.025

Cheng Li, Evgenia Korol + Show 5 more

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https://doi.org/10.1016/j.celrep.2015.07.025

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The Tet family of methylcytosine dioxygenases (Tet1, Tet2, and Tet3) convert 5-methylcytosine to 5-hydroxymethylcytosine. To date, functional overlap among Tet family members has not been examined systematically in the context of embryonic development. To clarify the potential for overlap among Tet enzymes during development, we mutated the zebrafish orthologs of Tet1, Tet2, and Tet3 and examined single-, double-, and triple-mutant genotypes. Here, we identify Tet2 and Tet3 as the major 5-methylcytosine dioxygenases in the zebrafish embryo and uncover a combined requirement for Tet2 and Tet3 in hematopoietic stem cell (HSC) emergence. We demonstrate that Notch signaling in the hemogenic endothelium is regulated by Tet2/3 prior to HSC emergence and show that restoring expression of the downstream gata2b/scl/runx1 transcriptional network can rescue HSCs in tet2/3 double mutant larvae. Our results reveal essential, overlapping functions for tet genes during embryonic development and uncover a requirement for 5hmC in regulating HSC production.

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In vertebrate species, the epigenetically modified base 5-methylcytosine (5mC) is associated with transcriptional repression and is essential for normal development (Goll and Bestor, 2005)
In tet2/3DM larvae, we found that expression of the hematopoietic stem cell (HSC)-associated genes runx1 and c-myb was reduced in the dorsal aorta (DA) at 36 hpf, whereas runx1 expression in tet2 and tet3 single homozygous mutant larvae was indistinguishable from wild-type (Figures 3E–3M)
While exposure to the nitric oxide (NO) agonist S-nitroso-N-acetyl-penicillamine (SNAP) was sufficient to rescue runx1 expression in silent heart morpholino-injected embryos lacking blood circulation, SNAP exposure was unable to rescue runx1 expression in tet2/3DM larvae (Figures S3A–S3E). These results suggest that the HSC defects observed in tet2/3DM larvae are not secondary to defects in vascular development or aberrant blood flow

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Introduction

The epigenetically modified base 5-methylcytosine (5mC) is associated with transcriptional repression and is essential for normal development (Goll and Bestor, 2005). Growing evidence suggests that conversion of 5mC to 5hmC and its derivatives can provide a first step toward DNA demethylation through active base excision or passive dilution of oxidized bases (Kohli and Zhang, 2013). Consistent with a role in regulating gene expression through DNA demethylation, 5hmC levels are most abundant in euchromatic regions, including transcription start sites, enhancers, and exons (Pastor et al, 2011; Stroud et al, 2011; Williams et al, 2011). Homozygous mutation of Tet, Tet, or Tet is compatible with mouse embryonic development, Tet mutant mice die perinatally (Kohli and Zhang, 2013). In contrast to single mutants, embryonic stem cells (ESCs) mutated for all three Tet genes contribute poorly to chimeras, suggesting that Tet family members have overlapping functions in promoting embryonic development (Dawlaty et al, 2014). Tet1/2 double-homozygous-mutant mice can survive into adulthood, but other Tet mutant combinations have yet to be described (Dawlaty et al, 2013)

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