Choline is an essential nutrient for humans, though some of the requirement can be met by endogenous synthesis catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT). Premenopausal women are relatively resistant to choline deficiency compared with postmenopausal women and men. Studies in animals suggest that estrogen treatment can increase PEMT activity. In this study we investigated whether the PEMT gene is regulated by estrogen. PEMT transcription was increased in a dose-dependent manner when primary mouse and human hepatocytes were treated with 17-beta-estradiol for 24 h. This increased message was associated with an increase in protein expression and enzyme activity. In addition, we report a region that contains a perfect estrogen response element (ERE) approximately 7.5 kb from the transcription start site corresponding to transcript variants NM_007169 and NM-008819 of the human and murine PEMT genes, respectively, three imperfect EREs in evolutionarily conserved regions and multiple imperfect EREs in nonconserved regions in the putative promoter regions. We predict that both the mouse and human PEMT genes have three unique transcription start sites, which are indicative of either multiple promoters and/or alternative splicing. This study is the first to explore the underlying mechanism of why dietary requirements for choline vary with estrogen status in humans.

Drosophila dendritic arborization (da) neurons contain subclasses of neurons with distinct dendritic morphologies. We investigated calcium/calmodulin-dependent protein kinase II (CaMKII) regulation of dendritic structure and dynamics in vivo using optically transparent Drosophila larvae. CaMKII increases the dynamic nature and formation of dendritic filopodia throughout larval development but only affects neurons that normally contain dendritic filopodia. In parallel, we examined the effects of Rac1 activity on dendritic structure to explore signaling specificity. In contrast to CaMKII activity, Rac1 does not alter filopodia stability but instead causes de novo filopodia formation on all da neurons. Although both mediators increase cytoskeletal turnover, measured by fluorescence recovery after photobleaching experiments, only CaMKII increases the dynamic nature of dendritic filopodia. CaMKII signaling thus appears to use mechanisms and machinery distinct from Rac1 signaling. This study illustrates a molecular means of uncoupling cytoskeletal regulation from morphological regulation. Our results suggest that Drosophila dendritic filopodia may share some cytoskeletal regulatory mechanisms with mammalian dendritic filopodia. Furthermore, general dendrite cytoskeletal compartmentalization is conserved in multipolar neurons.

When dietary choline is restricted, most men and postmenopausal women develop multiorgan dysfunction marked by hepatic steatosis (choline deficiency syndrome (CDS)). However, a significant subset of premenopausal women is protected from CDS. Because hepatic PEMT (phosphatidylethanolamine N-methyltransferase) catalyzes de novo biosynthesis of choline and this gene is under estrogenic control, we hypothesized that there are SNPs in PEMT that disrupt the hormonal regulation of PEMT and thereby put women at risk for CDS. In this study, we performed transcript-specific gene expression analysis, which revealed that estrogen regulates PEMT in an isoform-specific fashion. Locus-wide SNP analysis identified a risk-associated haplotype that was selectively associated with loss of hormonal activation. Chromatin immunoprecipitation, analyzed by locus-wide microarray studies, comprehensively identified regions of estrogen receptor binding in PEMT. The polymorphism (rs12325817) most highly linked with the development of CDS (p < 0.00006) was located within 1 kb of the critical estrogen response element. The risk allele failed to bind either the estrogen receptor or the pioneer factor FOXA1. These data demonstrate that allele-specific ablation of estrogen receptor-DNA interaction in the PEMT locus prevents hormone-inducible PEMT expression, conferring risk of CDS in women.

Choline is an essential nutrient for humans, though some part of this requirement can be met by endogenous synthesis catalyzed by phosphatidylethanolamine N‐methyltransferase (PEMT). Human diets which lack sufficient choline intake can result in organ and muscle dysfunction. Pre‐menopausal women are more resistant to choline deficiency than post‐menopausal women and men. There is evidence in animal models which suggests that estrogen can increase PEMT activity. The goal of this study is to investigate whether and how the PEMT gene is regulated by estrogen. Real Time RT‐PCR revealed gene transcription was increased in a dose‐dependent manner in cultured primary mouse hepatocytes treated with various concentrations of 17B‐estradiol for 24 hours. Protein levels and enzyme activity were also increased. The human PEMT promoter contains eight putative estrogen response elements (EREs) and an Electrophoretic Mobility Shift Assay (EMSA) revealed that a 41 bp segment, which contains two putative EREs (2 and 3), can bind estrogen receptor complexes. Transfection studies, in which the human PEMT promoter is linked to a promoterless luciferase reporter gene, will be used to determine whether estrogen regulates promoter activity. This work supported by NIH grants to SZ(DK55865, AG09525, DK56350).

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