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Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase
Published on: October 15, 2018
[Functional analysis of TET2 using a knockdown mouse model]
Kotaro Shide1, Takuro Kameda, Kazuya Shimoda
1Department of Gastroenterology and Hematology, Faculty of Medicine, University of Miyazaki.
Abstract:
In myeloid malignancies, mutations have occurred in epigenetic regulator genes, including Ten-Eleven-Translocation 2 (TET2). TET2 is an enzyme that catalyzes the conversion of 5-methylcytosine into 5-hydroxymethylcytosine (5-hmC) which is a key intermediate for oxidative DNA demethylation. We analyzed the in vivo phenotype of TET2 failure using Ayu17-449 (TET2(trap/trap)) mice created by the gene-trap method in which TET2 mRNA levels were decreased to about 20% of the level in wild-type (WT) mice. In TET2(trap/trap) mice the levels of 5-hmC in genomic DNA from bone marrow (BM) cells were decreased in comparison to WT mice. TET2(trap/trap) mice were born at an expected Mendelian frequency but died at a high rate by postnatal day 3, indicating TET2 to be essential for survival. In analysis of the hematopoietic system, transplantation of TET2(trap/trap), but not WT fetal liver cells, led to mild myeloid hyperplasia and splenomegaly in WT recipient mice, but no onsets of lethal hematological malignancies were observed during a follow-up period of 12 months. TET2 knockdown led to an increased serial replating capacity of BM cells in vitro and increased hematopoietic stem cell (HSC) self-renewal in vivo in competitive repopulation and serial transplantation assays. These data indicate that TET2 has a critical role in survival and HSC homeostasis.
Insights
Ten-Eleven-Translocation 2 (TET2) is crucial for survival and hematopoietic stem cell (HSC) homeostasis. TET2 deficiency in mice leads to reduced 5-hydroxymethylcytosine levels and impaired HSC function, highlighting its essential role.
Area of Science:
- Epigenetics
- Hematopoiesis
- Molecular Biology
Background:
- Mutations in epigenetic regulators like Ten-Eleven-Translocation 2 (TET2) are implicated in myeloid malignancies.
- TET2 enzyme activity is critical for DNA demethylation via 5-hydroxymethylcytosine (5-hmC) production.
Purpose of the Study:
- To investigate the in vivo consequences of TET2 deficiency on survival and hematopoietic stem cell (HSC) function.
- To characterize the phenotype of mice with reduced TET2 expression.
Main Methods:
- Generation and analysis of TET2(trap/trap) gene-trap mice with reduced TET2 mRNA levels.
- Measurement of genomic 5-hmC levels in bone marrow (BM) cells.
- Hematopoietic system analysis via fetal liver cell transplantation and competitive repopulation assays.
- In vitro serial replating assays of BM cells.
Main Results:
- TET2(trap/trap) mice exhibited decreased 5-hmC levels in BM cells and high postnatal mortality, indicating essential survival function.
- Transplantation of TET2(trap/trap) fetal liver cells caused mild myeloid hyperplasia and splenomegaly in recipients.
- TET2 knockdown enhanced BM cell replating capacity and HSC self-renewal in vivo.
Conclusions:
- TET2 is essential for organismal survival and proper hematopoietic stem cell (HSC) homeostasis.
- TET2 deficiency impairs HSC function and promotes myeloid hyperplasia, suggesting a role in preventing hematological malignancies.
