Dataset Information

Humanizing mouse folate metabolism: conversion of the dual-promoter mouse folylpolyglutamate synthetase gene to the human single-promoter structure.

ABSTRACT: The mouse is extensively used to model human folate metabolism and therapeutic outcomes with antifolates. However, the folylpoly-?-glutamate synthetase (fpgs) gene, whose product determines folate/antifolate intracellular retention and antifolate antitumor activity, displays a pronounced species difference. The human gene uses only a single promoter, whereas the mouse uses two: P2, akin to the human promoter, at low levels in most tissues; and P1, an upstream promoter used extensively in liver and kidney. We deleted the mouse P1 promoter through homologous recombination to study the dual-promoter mouse system and to create a mouse with a humanized fpgs gene structure. Despite the loss of the predominant fpgs mRNA species in liver and kidney (representing 95 and 75% of fpgs transcripts in these tissues, respectively), P1-knockout mice developed and reproduced normally. The survival of these mice was explained by increased P2 transcription due to relief of transcriptional interference, by a 3-fold more efficient translation of P2-derived than P1-derived transcripts, and by 2-fold higher stability of P2-derived FPGS. In combination, all 3 effects reinstated FPGS function, even in liver. By eliminating mouse P1, we created a mouse model that mimicked the human housekeeping pattern of fpgs gene expression.

SUBMITTER: Yang C

PROVIDER: S-EPMC3986845 | biostudies-other | 2014 May

REPOSITORIES: biostudies-other

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Humanizing mouse folate metabolism: conversion of the dual-promoter mouse folylpolyglutamate synthetase gene to the human single-promoter structure.

Yang Chen C Xie Lin-Ying LY Windle Jolene J JJ Taylor Shirley M SM Moran Richard G RG

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20140214 5

The mouse is extensively used to model human folate metabolism and therapeutic outcomes with antifolates. However, the folylpoly-γ-glutamate synthetase (fpgs) gene, whose product determines folate/antifolate intracellular retention and antifolate antitumor activity, displays a pronounced species difference. The human gene uses only a single promoter, whereas the mouse uses two: P2, akin to the human promoter, at low levels in most tissues; and P1, an upstream promoter used extensively in liver a ...[more]

PMID: 24532667

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Project description:Abnormal uptake or metabolism of folate increases risk of human pregnancy complications, though the mechanism is unclear. Here, we explore how defective folate metabolism influences early development by analysing mice with the hypomorphic Mtrr gt mutation. MTRR is necessary for methyl group utilisation from folate metabolism, and the Mtrr gt allele disrupts this process. We show that the spectrum of phenotypes previously observed in Mtrr gt/gt conceptuses at embryonic day (E) 10.5 is apparent from E8.5 including developmental delay, congenital malformations, and placental phenotypes. Notably, we report misalignment of some Mtrr gt conceptuses within their implantation sites from E6.5. The degree of misorientation occurs across a continuum, with the most severe form visible upon gross dissection. Additionally, some Mtrr gt/gt conceptuses display twinning. Therefore, we implicate folate metabolism in blastocyst orientation and spacing at implantation. Skewed growth likely influences embryo development since developmental delay and heart malformations (but not defects in neural tube closure or trophoblast differentiation) associate with severe misalignment of Mtrr gt/gt conceptuses. Typically, the uterus is thought to guide conceptus orientation. To investigate a uterine effect of the Mtrr gt allele, we manipulate the maternal Mtrr genotype. Misaligned conceptuses were observed in litters of Mtrr +/+ , Mtrr +/gt , and Mtrr gt/gt mothers. While progesterone and/or BMP2 signalling might be disrupted, normal decidual morphology, patterning, and blood perfusion are evident at E6.5 regardless of conceptus orientation. These observations argue against a post-implantation uterine defect as a cause of conceptus misalignment. Since litters of Mtrr +/+ mothers display conceptus misalignment, a grandparental effect is explored. Multigenerational phenotype inheritance is characteristic of the Mtrr gt model, though the mechanism remains unclear. Genetic pedigree analysis reveals that severe conceptus skewing associates with the Mtrr genotype of either maternal grandparent. Moreover, the presence of conceptus skewing after embryo transfer into a control uterus indicates that misalignment is independent of the peri- and/or post-implantation uterus and instead is likely attributed to an embryonic mechanism that is epigenetically inherited. Overall, our data indicates that abnormal folate metabolism influences conceptus orientation over multiple generations with implications for subsequent development. This study casts light on the complex role of folate metabolism during development beyond a direct maternal effect.

Dataset Information

Humanizing mouse folate metabolism: conversion of the dual-promoter mouse folylpolyglutamate synthetase gene to the human single-promoter structure.

Publications

Humanizing mouse folate metabolism: conversion of the dual-promoter mouse folylpolyglutamate synthetase gene to the human single-promoter structure.

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