Primitive Erythropoiesis in the Mouse is Independent of DOT1L Methyltransferase Activity

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

doi:10.3389/fcell.2021.813503

Cited by 7 publications

(21 citation statements)

References 33 publications

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“…The Dot1L -KO mice were generated and maintained as described previously ( Feng et al, 2010 ). To produce the Dot1L -MM mouse, we generated mutant mESC ( Malcom et al, 2022 ). Briefly, mESCs (E14TG2a) were targeted with CRISPR/Cas9 to introduce a point mutation (Asp241Ala) within exon 9 of Dot1L.…”

Section: Methodsmentioning

confidence: 99%

“…The methyltransferase mutant, Dot1l -MM mice, were also embryonic lethal. The embryos died around mid-gestation ( Malcom et al, 2022 ). The mice also displayed defects in embryonic hematopoiesis, including a decreased ability to form definitive myeloid, and oligopotent (mixed) blood progenitors in ex vivo cultures ( Malcom et al, 2022 ).…”

Section: Introductionmentioning

confidence: 99%

“…The embryos died around mid-gestation ( Malcom et al, 2022 ). The mice also displayed defects in embryonic hematopoiesis, including a decreased ability to form definitive myeloid, and oligopotent (mixed) blood progenitors in ex vivo cultures ( Malcom et al, 2022 ). However, unlike the Dot1L knockout ( Dot1l- KO) ( Feng et al, 2010 ), Dot1L- MM embryos were not anemic at E10.5 and hematopoietic progenitor cells (HPC) from Dot1l- MM yolk sacs were able to produce erythroid colonies in numbers similar to the wildtype ( Malcom et al, 2022 ).…”

Section: Introductionmentioning

confidence: 99%

“…DOT1L is the only known methyltransferase in eukaryotic cells to methylate lysine 79 of histone H3 (H3K79) ( Feng et al, 2002 ). We generated a mouse line carrying a point mutation (Asn241Ala) in the mouse Dot1l gene ( Dot1l -MM) that renders its catalytic domain inactive ( Min et al, 2003 ; Malcom et al, 2022 ). Dot1l -MM mice expressed an intact DOT1L protein that lacked only the H3K79 methyltransferase activity ( Malcom et al, 2022 ).…”

Section: Introductionmentioning

confidence: 99%

“…We generated a mouse line carrying a point mutation (Asn241Ala) in the mouse Dot1l gene ( Dot1l -MM) that renders its catalytic domain inactive ( Min et al, 2003 ; Malcom et al, 2022 ). Dot1l -MM mice expressed an intact DOT1L protein that lacked only the H3K79 methyltransferase activity ( Malcom et al, 2022 ). The methyltransferase mutant, Dot1l -MM mice, were also embryonic lethal.…”

Section: Introductionmentioning

confidence: 99%

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DOT1L Mediated Gene Repression in Extensively Self-Renewing Erythroblasts

et al. 2022

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DOT1L is essential for embryonic hematopoiesis but the precise mechanisms of its action remain unclear. The only recognized function of DOT1L is histone H3 lysine 79 (H3K79) methylation, which has been implicated in both transcriptional activation and repression. We observed that deletion of the mouse Dot1L gene (Dot1L-KO) or selective mutation of its methyltransferase domain (Dot1L-MM) can differentially affect early embryonic erythropoiesis. However, both mutations result in embryonic lethality by mid-gestation and growth of hematopoietic progenitor cells (HPCs) is similarly affected in extensively self-renewing erythroblast (ESRE) cultures established from yolk sac cells. To understand DOT1L-mediated gene regulation and to clarify the role of H3K79 methylation, we analyzed whole transcriptomes of wildtype and Dot1L-mutant ESRE cells. We observed that more than 80% of the differentially expressed genes (DEGs) were upregulated in the mutant ESRE cells either lacking the DOT1L protein or the DOT1L methyltransferase activity. However, approximately 45% of the DEGs were unique to either mutant group, indicating that DOT1L possesses both methyltransferase-dependent and -independent gene regulatory functions. Analyses of Gene Ontology and signaling pathways for the DEGs were consistent, with DEGs that were found to be common or unique to either mutant group. Genes related to proliferation of HPCs were primarily impacted in Dot1L-KO cells, while genes related to HPC development were affected in the Dot1L-MM cells. A subset of genes related to differentiation of HPCs were affected in both mutant groups of ESREs. Our findings suggest that DOT1L primarily acts to repress gene expression in HPCs, and this function can be independent of its methyltransferase activity.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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DOT1L Mediated Gene Repression in Extensively Self-Renewing Erythroblasts

et al. 2022

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A familial rearrangement resulting in pure duplication of distal 19p13.3

Bain,

Koulouris,

Scott

et al. 2023

Clinical Dysmorphology

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Developmental regulation of primitive erythropoiesis

Rossmann,

Palis

2024

Current Opinion in Hematology

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Purpose of review In this review, we present an overview of recent studies of primitive erythropoiesis, focusing on advances in deciphering its embryonic origin, defining species-specific differences in its developmental regulation, and better understanding the molecular and metabolic pathways involved in terminal differentiation. Recent findings Single-cell transcriptomics combined with state-of-the-art lineage tracing approaches in unperturbed murine embryos have yielded new insights concerning the origin of the first (primitive) erythroid cells that arise from mesoderm-derived progenitors. Moreover, studies examining primitive erythropoiesis in rare early human embryo samples reveal an overall conservation of primitive erythroid ontogeny in mammals, albeit with some interesting differences such as localization of erythropoietin (EPO) production in the early embryo. Mechanistically, the repertoire of transcription factors that critically regulate primitive erythropoiesis has been expanded to include regulators of transcription elongation, as well as epigenetic modifiers such as the histone methyltransferase DOT1L. For the latter, noncanonical roles aside from enzymatic activity are being uncovered. Lastly, detailed surveys of the metabolic and proteomic landscape of primitive erythroid precursors reveal the activation of key metabolic pathways such as pentose phosphate pathway that are paralleled by a striking loss of mRNA translation machinery. Summary The ability to interrogate single cells in vivo continues to yield new insights into the birth of the first essential organ system of the developing embryo. A comparison of the regulation of primitive and definitive erythropoiesis, as well as the interplay of the different layers of regulation – transcriptional, epigenetic, and metabolic – will be critical in achieving the goal of faithfully generating erythroid cells in vitro for therapeutic purposes.

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Primitive Erythropoiesis in the Mouse is Independent of DOT1L Methyltransferase Activity

Cited by 7 publications

References 33 publications

DOT1L Mediated Gene Repression in Extensively Self-Renewing Erythroblasts

DOT1L Mediated Gene Repression in Extensively Self-Renewing Erythroblasts

A familial rearrangement resulting in pure duplication of distal 19p13.3

Developmental regulation of primitive erythropoiesis

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