Primitive Erythropoiesis in the Mouse is Independent of DOT1L Methyltransferase Activity.

Carrie A Malcom,Joanna Piasecka-Srader,Timothy A Fields,Nehemiah S Alvarez,M.A Karim Rumi,Patrick E Fields,Shaon Borosha,Anamika Ratri,Jay L Vivian,V Praveen Chakravarthi

doi:10.3389/fcell.2021.813503

Carrie A Malcom, Joanna Piasecka-Srader + Show 8 more

Open Access

https://doi.org/10.3389/fcell.2021.813503

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Abstract

DOT1-like (DOT1L) histone methyltransferase is essential for mammalian erythropoiesis. Loss of DOT1L in knockout (Dot1l-KO) mouse embryos resulted in lethal anemia at midgestational age. The only recognized molecular function of DOT1L is its methylation of histone H3 lysine 79 (H3K79). We generated a Dot1l methyltransferase mutant (Dot1l-MM) mouse model to determine the role of DOT1L methyltransferase activity in early embryonic hematopoiesis. Dot1l-MM embryos failed to survive beyond embryonic day 13.5 (E13.5), similarly to Dot1l-KO mice. However, when examined at E10.5, Dot1l-MM embryos did not exhibit overt anemia like the Dot1l-KO. Vascularity and the presence of red blood cells in the Dot1l-MM yolk sacs as well as in the AGM region of Dot1l-MM embryos appeared to be similar to that of wildtype. In ex vivo cultures of yolk sac cells, Dot1l-MM primitive erythroblasts formed colonies comparable to those of the wildtype. Although ex vivo cultures of Dot1l-MM definitive erythroblasts formed relatively smaller colonies, inhibition of DOT1L methyltransferase activity in vivo by administration of EPZ-5676 minimally affected the erythropoiesis. Our results indicate that early embryonic erythropoiesis in mammals requires a DOT1L function that is independent of its intrinsic methyltransferase activity.

Highlights

DOT1-like (DOT1L) histone H3 lysine methyltransferase is essential for a number of biological processes during embryonic development, including early hematopoiesis (Feng et al, 2010; Cattaneo et al, 2016; Pursani et al, 2018; Sutter et al, 2021)
Methyl mutant E14 mouse embryonic stem (mES) cells were generated by targeted replacement of the coding sequence of Asn 241 in Dot1l gene to Ala (Figure 1A) using CRISPR/Cas9 system and the mutant sequences were verified by Sanger sequencing (Figure 1B)
We previously reported that DOT1L histone methyltransferase is essential for early embryonic hematopoiesis in mice (Feng et al, 2010)

Summary

Introduction

DOT1-like (DOT1L) histone H3 lysine methyltransferase is essential for a number of biological processes during embryonic development, including early hematopoiesis (Feng et al, 2010; Cattaneo et al, 2016; Pursani et al, 2018; Sutter et al, 2021). Dot1l-KO erythroid-myeloid progenitors failed to develop normally, exhibiting slowed cell-cycle progression in vitro associated with downregulation of GATA2, a transcription factor essential for erythropoiesis, and upregulation of PU., another factor that inhibits erythropoiesis (Feng et al, 2010). A number of cellular processes including transcriptional elongation (Steger et al, 2008), cell-cycle progression

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