Epigenetic Protection of Vertebrate Lymphoid Progenitor Cells by Dnmt1

Iwanami, Norimasa; Takeshita, Keizo; Lawir, Divine-Fondzenyuy; Suetake, Isao; Tsuji, Shoji; Sikora, Katarzyna; Trancoso, Inês; O’Meara, Connor P.; Siamishi, Iliana; Takahama, Yousuke; Furutani-Seiki, Makoto; Kondoh, Hisato; Yonezawa, Yasushige; Schorpp, Michael; Boehm, Thomas

doi:10.1016/j.isci.2020.101260

Cited by 9 publications

(11 citation statements)

References 71 publications

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“…While a mechanistic evaluation of this phenomenon might be worthwhile, it certainly is beyond the scope of this study. Given that the function of DNMT1 is thought to be maintaining the methylation pattern [ 19 ] a reduced mitochondrial abundance of this enzyme could lead to a reduced mtDNA methylation.…”

Section: Discussionmentioning

confidence: 99%

LPS-Induced Endotoxemia Evokes Epigenetic Alterations in Mitochondrial DNA That Impacts Inflammatory Response

Koos

Moderegger

Rump

et al. 2020

Cells

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Mitochondrial DNA (mtDNA) plays a vital role as a damage-associated molecular pattern in sepsis being able to shape the immune response. Since pathogen recognition receptors of innate immune cells are activated by demethylated DNA only, we set out to investigate the amount of DNA methyltransferase 1 (DNMT1) in mitochondria and the extent of mtDNA methylation in a human endotoxin model. Peripheral blood mononuclear cells of 20 healthy individuals were isolated from whole blood and stimulated with lipopolysaccharide (LPS) for 48 h. Subsequently, DNMT1 protein abundance was assessed in whole cells and a mitochondrial fraction. At the same time, methylation levels of mtDNA were quantified, and cytokine expression in the supernatant was measured. Despite increased cellular expression of DNMT1 after LPS stimulation, the degree of mtDNA methylation slightly decreased. Strikingly the mitochondrial protein abundance of DNMT1 was reduced by 50% in line with the lower degree of mtDNA methylation. Although only modest alterations were seen in the degree of mtDNA methylation, these strongly correlated with IL-6 and IL-10 expression. Our data may hint at a protein import problem for DNMT1 into the mitochondria under LPS stimulation and suggest a role of demethylated mtDNA in the regulation of the inflammatory immune response.

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Section: Discussionmentioning

confidence: 99%

LPS-Induced Endotoxemia Evokes Epigenetic Alterations in Mitochondrial DNA That Impacts Inflammatory Response

Koos

Moderegger

Rump

et al. 2020

Cells

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“…The ligation mixtures were dialyzed and transformed into E.coli DH5α by electroporation. Culture of transformants, plasmid extraction and in vitro transcription of guide RNAs were carried out as described 52 . Purified CRISPR guide RNAs were tested for specificity by in vitro digestion of target DNA (80 ng PCR amplicon containing target sequence, 600 ng Cas9 protein from Streptococcus pyogenes [PNA Bio], 300 ng guide RNA, 1X CutSmart buffer [New England Biolabs]) at 37 °C for 1 h. Guide RNA was removed by adding 4 μg of RNAse A to the reaction for 15 min at 37 °C prior to visualization of cleavage products by agarose gel electrophoresis.…”

Section: Methodsmentioning

confidence: 99%

Stage-specific and cell type-specific requirements of ikzf1 during haematopoietic differentiation in zebrafish

Hess

Sagar

Meara

et al. 2022

Sci Rep

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The zinc finger transcription factor Ikaros1 (Ikzf1) is required for lymphoid development in mammals. Four zinc fingers constitute its DNA binding domain and two zinc fingers are present in the C-terminal protein interaction module. We describe the phenotypes of zebrafish homozygous for two distinct mutant ikzf1 alleles. The IT325 variant lacks the C-terminal two zinc fingers, whereas the fr105 variant retains only the first zinc finger of the DNA binding domain. An intact ikzf1 gene is required for larval T cell development, whereas low levels of adult lymphoid development recover in the mutants. By contrast, the mutants exhibit a signature of increased myelopoiesis at larval and adult stages. Both mutations stimulate erythroid differentiation in larvae, indicating that the C-terminal zinc fingers negatively regulate the extent of red blood cell production. An unexpected differential effect of the two mutants on adult erythropoiesis suggests a direct requirement of an intact DNA binding domain for entry of progenitors into the red blood cell lineage. Collectively, our results reinforce the biological differences between larval and adult haematopoiesis, indicate a stage-specific function of ikzf1 in regulating the hierarchical bifurcations of differentiation, and assign distinct functions to the DNA binding domain and the C-terminal zinc fingers.

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“…The construct was linearized and injected into fertilized eggs of FVB mice to generate the FVB/N-tg(Foxn1-Gcm2)1 Tbo /Mpie transgenic line. A Gcm2 null allele (FVB/N- Gcm2 em1Tbo /Mpie) was generated by CRISPR/Cas9-mediated internal deletion of nucleotides 124,954 to 127,882 in Genbank accession number AC158538.2 in the Gcm2 gene using two sgRNAs (nucleotides 124,953 to 124,971; nucleotides 127,878 to 127,896 in Genbank accession number AC158538.2) as described 42 ; note that nucleotides 125,018 to 125,035 in Genbank accession number AC158538.2 are retained in the deletion. A Gcm2:Foxn1 knock-in allele (FVB/N- Gcm2 em1(Foxn1)Tbo /Mpie) was generated by homologous recombination in fertilized oocytes primed by simultaneous CRISPR/Cas9-induced internal deletion as described for the Gcm2 null allele.…”

Section: Methodsmentioning

confidence: 99%

Limits to in vivo fate changes of epithelia in thymus and parathyroid by ectopic expression of transcription factors Gcm2 and Foxn1

Nagakubo

Hirakawa

Iwanami

et al. 2022

Sci Rep

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The development of the parathyroid and the thymus from the third pharyngeal pouch depends on the activities of the Gcm2 and Foxn1 transcription factors, respectively, whose expression domains sharply demarcate two regions in the developing third pharyngeal pouch. Here, we have generated novel mouse models to examine whether ectopic co-expression of Gcm2 in the thymic epithelium and of Foxn1 in the parathyroid perturbs the establishment of organ fates in vivo. Expression of Gcm2 in the thymic rudiment does not activate a parathyroid-specific expression programme, even in the absence of Foxn1 activity. Co-expression of Foxn1 in the parathyroid fails to impose thymopoietic capacity. We conclude that the actions of Foxn1 and Gcm2 transcription factors are cell context-dependent and that they each require permissive transcription factor landscapes in order to successfully interfere with organ-specific cell fate.

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Epigenetic Protection of Vertebrate Lymphoid Progenitor Cells by Dnmt1

Cited by 9 publications

References 71 publications

LPS-Induced Endotoxemia Evokes Epigenetic Alterations in Mitochondrial DNA That Impacts Inflammatory Response

LPS-Induced Endotoxemia Evokes Epigenetic Alterations in Mitochondrial DNA That Impacts Inflammatory Response

Stage-specific and cell type-specific requirements of ikzf1 during haematopoietic differentiation in zebrafish

Limits to in vivo fate changes of epithelia in thymus and parathyroid by ectopic expression of transcription factors Gcm2 and Foxn1

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