Tissue-specific roles of de novo DNA methyltransferases

Abstract

DNA methylation, catalyzed by DNA methyltransferases (DNMT), plays pivotal role in regulating embryonic development, gene expression, adaption to environmental stress, and maintaining genome integrity. DNMT family consists of DNMT1, DNMT3A, DNMT3B, and the enzymatically inactive DNMT3L. DNMT3A and DNMT3B establish novel methylation patterns maintained by DNMT1 during replication. Genetic variants of DNMT3A and DNMT3B cause rare diseases such as Tatton-Brown-Rahman and ICF syndromes. Additionally, somatic mutations cause common conditions such as osteoarthritis, osteoporosis, clonal hematopoiesis of indeterminate potential (CHIP), hematologic malignancies, and cancer. While DNMTs have been extensively studied in vitro, in early development and in disease, their detailed physiologic roles remain less understood as in vivo investigations are hindered by the embryonic or perinatal lethality of the knockout mice. To circumvent this problem, tissue-specific Dnmt3a and Dnmt3b knockouts were engineered. This review explores their diverse molecular roles across various organs and cell types and characterizes the phenotype of the knockout mice. We provide a comprehensive collection of over forty tissue-specific knockout models generated by cre recombinase. We highlight the distinct functions of DNMT3A and DNMT3B in germ cells, early development, uterus, hematopoietic differentiation, musculoskeletal development, visceral organs, and nervous system. Our findings indicate that DNMT3A primarily regulates hematopoietic differentiation, while DNMT3B is crucial for cartilage homeostasis and ossification. We emphasize the context-dependent roles of DNMT3A and DNMT3B and demonstrate that they also complement DNMT1 maintenance methyltransferase activity. Overall, the expression patterns of DNMTs across tissues provide insights into potential therapeutic applications for treating neurologic diseases, cancer, and osteoporosis.

Keywords: De novo methyltransferase; Cre recombinase; DNA methylation; Development; Differentiation; Dnmt3a; Dnmt3b; Knockout; LoxP; Stem cells; Tissue-specific.

Fig. 1

DNMT3A and DNMT3B isoforms in mouse. Conserved regions are colored, and their respective functions are indicated below. The conserved domain IV in the catalytic region of Dnmt3a is flanked by the loxP sites is indicated in red. HSJS stands for Heyn-Sproul-Jackson syndrome, associated with mutations in the DNMT3A PWWP domain. The W326R mutation is homologous to the human W330R disease-causing mutation and recapitulates the disease phenotype (see under “Medical Relevance” chapter). TBRS represents Tattoon-Brown Rahman Syndrome, AML indicates acute myeloid leukemia. Both can develop due to the dominant R382H mutation in human. The corresponding mouse mutation R878H is indicated. DNMT3B isoforms are shown on the panel below. The conserved domains I - VI in the catalytic region of Dnmt3b flanked by the loxP sites are indicated in red. A609T and D823G mutations cause similar phenotype than the human ICF syndrome (immunodeciency-centromeric instability-facial dismorphism). Homologous A603T and D817G mutations were reported in ICF patients

Fig. 2

DNMT3A plays pivotal role in germ cell maturation and during preimplantation. Dnmt3a knockout primordial germ cells (PGC) are unable to differentiate to mature germ cells and can not establish parental-specific methylation patterns required for genomic imprinting. The PGCs maintain their ability to replicate and remain PGCs. DNMT3B plays minor role in complementing of the maintainance methylation activity of DNMT1 in mouse embryonic fibroblasts (MEF). ICRs are imprinting control regions. Lollipops represent CpG sites. Black CpGs are methylated, white CpGs are unmethylated

Fig. 3

DNMT3A and DNMT3B are involved in fetal development. DNMT3B is required for the decidualization of the endometrium and the absence of the gene from progesterone-sensitive cells disturbs this process and leads to embryonic loss. Both DNMT3A and DNMT3B are required for labyrinth formation. The maintenance of Dnmt3b expression in trophoblast cells is crucial for embryonic development and survival

Fig. 4

Hematopoietic stem cells (HSC) maintain their stemness or undergo differentiation after replication. Differentiation is primarily regulated by DNMT3A. DNMT3B has a complementary role in this process. Hematopoietic differentiation also requires TET2 demethylase activity. The methylome is characterized by kb long canyons with very low methylation levels enriched by methylation sensitive transcription factor (TF) binding sites involved in erythroid differentiation. The edges of the canyons are enriched in 5hmC (grey lollipops), regulating myeloid differentiation. TFs binding these regions are less sensitive to DNA methylation. Dnmt3a knockout mice have skewed erythroid, Tet2 knockout mice have skewed myeloid differentiation. CHIP: clonal hematopoiesis of indeterminate potential; MDS: myelodysplastic syndrome

Fig. 5

De novo methyltransferases play primordial role in ossification. Mesenchymal progenitor cells (MPC) differentiate to chondroblasts (CB) then chondrocytes (CC) and osteoblasts (OB) then osteocytes (OC). DNMT3B is a key regulator of this process. In the absence of Dnmt3b delayed CC maturation, osteoarthritis, impaired fracture healing is observed. Osteoclasts (OCl), responsible for bone resorption and maintaining homeostasis of ossification are differentiating from MPC (macrophages) themselves originating from myeloblasts (MB). In the absence of Dnmt3a increased bone mass was reported. Skeletal muscle development also requires DNMT3A. In knockouts decreased body weight was reported. MPSC: myogenic progenitor stem cells; MB: myoblasts; MF: myofibrillul; MT: myotube