Nicholas Wagner

DGAT2 Is a New Diacylglycerol Acyltransferase Gene Family PURIFICATION, CLONING, AND EXPRESSION IN INSECT CELLS OF TWO POLYPEPTIDES FROM MORTIERELLA RAMANNIANA WITH DIACYLGLYCEROL ACYLTRANSFERASE ACTIVITY

Acyl CoA:diacylgycerol acyltransferase (EC2.3.1.20; DGAT) catalyzes the final step in the production of triacylglycerol. Two polypeptides, which co-purified with DGAT activity, were isolated from the lipid bodies of the oleaginous fungusMortierella ramanniana with a procedure consisting of dye affinity, hydroxyapatite affinity, and heparin chromatography. The two enzymes had molecular masses of 36 and 36.5 kDa, as estimated by gel electrophoresis, and showed a broad activity maximum between pH 6 and 8. Based on partial peptide sequence information, polymerase chain reaction techniques were used to obtain full-length cDNA sequences encoding the purified proteins. Expression of the cDNAs in insect cells conferred high levels of DGAT activity on the membranes isolated from these cells. The two proteins share 54% homology with each other but are unrelated to the previously identified DGAT gene family (designated DGAT1), which is related to the acyl CoA:cholesterol acyltransferase gene family, or to any other gene family with ascribed function. This report identifies a new gene family, including members in fungi, plants and animals, which encode enzymes with DGAT function. To distinguish the two unrelated families we designate this new class DGAT2 and refer to the M. ramanniana genes asMrDGAT2A and MrDGAT2B.

Gestational Choline Deficiency Causes Global and Igf2 Gene DNA Hypermethylation by Up-regulation of Dnmt1 Expression

During gestation there is a high demand for the essential nutrient choline. Adult rats supplemented with choline during embryonic days (E) 11-17 have improved memory performance and do not exhibit age-related memory decline, whereas prenatally choline-deficient animals have memory deficits. Choline, via betaine, provides methyl groups for the production of S-adenosylmethionine, a substrate of DNA methyltransferases (DNMTs). We describe an apparently adaptive epigenomic response to varied gestational choline supply in rat fetal liver and brain. S-Adenosylmethionine levels increased in both organs of E17 fetuses whose mothers consumed a choline-supplemented diet. Surprisingly, global DNA methylation increased in choline-deficient animals, and this was accompanied by overexpression of Dnmt1 mRNA. Previous studies showed that the prenatal choline supply affects the expression of multiple genes, including insulin-like growth factor 2 (Igf2), whose expression is regulated in a DNA methylation-dependent manner. The differentially methylated region 2 of Igf2 was hypermethylated in the liver of E17 choline-deficient fetuses, and this as well as Igf2 mRNA levels correlated with the expression of Dnmt1 and with hypomethylation of a regulatory CpG within the Dnmt1 locus. Moreover, mRNA expression of brain and liver Dnmt3a and methyl CpG-binding domain 2 (Mbd2) protein as well as cerebral Dnmt3l was inversely correlated to the intake of choline. Thus, choline deficiency modulates fetal DNA methylation machinery in a complex fashion that includes hypomethylation of the regulatory CpGs within the Dnmt1 gene, leading to its overexpression and the resultant increased global and gene-specific (e.g. Igf2) DNA methylation. These epigenomic responses to gestational choline supply may initiate the long term developmental changes observed in rats exposed to varied choline intake in utero.