MicroRNA-129-5p is regulated by choline availability and controls EGF receptor synthesis and neurogenesis in the cerebral cortex.

Choline availability modulates neurogenesis and cerebral cortex development through the regulation of neural progenitor cell (NPC) proliferative and differentiation capacity. In this study, we demonstrated that cortical NPC self-renewal is controlled by choline via the expression of a microRNA (miR-129-5p), whose role in the developing brain has not been examined, and which, in turn, inhibits synthesis of the epidermal growth factor receptor (EGFR) protein. Specifically, we found that low choline (LC) availability led to the upregulation of miR-129-5p expression in cortical NPCs in vitro and in vivo, causing the downregulation of EGFR and thereby disrupting NPC self-renewal and cortical neurogenesis. Furthermore, in response to LC availability, methylation potential (the S-adenosylmethionine: S-adenosylhomocysteine ratio) in the developing brain was reduced. Restoring methylation potential in LC cortical NPCs led to the re-establishment of normal miR-129-5p expression. We concluded that inhibiting miR-129-5p function and restoring EGFR protein levels in vivo is sufficient to reverse LC-induced defects in cortical NPC self-renewal. For the first time, to our knowledge, we have identified the molecular links that explain how a change in the availability of the diet metabolite choline impacts the essential cellular processes underlying brain development.-Trujillo-Gonzalez, I., Wang, Y., Friday, W. B., Vickers, K. C., Toth, C. L., Molina-Torres, L., Surzenko, N., Zeisel, S. H. MicroRNA-129-5p is regulated by choline availability and controls EGF receptor synthesis and neurogenesis in the cerebral cortex.

Low availability of choline in utero disrupts development and function of the retina.

Adequate supply of choline, an essential nutrient, is necessary to support proper brain development. Whether prenatal choline availability plays a role in development of the visual system is currently unknown. In this study, we addressed the role of in utero choline supply for the development and later function of the retina in a mouse model. We lowered choline availability in the maternal diet during pregnancy and assessed proliferative and differentiation properties of retinal progenitor cells (RPCs) in the developing prenatal retina, as well as visual function in adult offspring. We report that low choline availability during retinogenesis leads to persistent retinal cytoarchitectural defects, ranging from focal lesions with displacement of retinal neurons into subretinal space to severe hypocellularity and ultrastructural defects in photoreceptor organization. We further show that low choline availability impairs timely differentiation of retinal neuronal cells, such that the densities of early-born retinal ganglion cells, amacrine and horizontal cells, as well as cone photoreceptor precursors, are reduced in low choline embryonic d 17.5 retinas. Maintenance of higher proportions of RPCs that fail to exit the cell cycle underlies aberrant neuronal differentiation in low choline embryos. Increased RPC cell cycle length, and associated reduction in neurofibromin 2/Merlin protein, an upstream regulator of the Hippo signaling pathway, at least in part, explain aberrant neurogenesis in low choline retinas. Furthermore, we find that animals exposed to low choline diet in utero exhibit a significant degree of intraindividual variation in vision, characterized by marked functional discrepancy between the 2 eyes in individual animals. Together, our findings demonstrate, for the first time, that choline availability plays an essential role in the regulation of temporal progression of retinogenesis and provide evidence for the importance of adequate supply of choline for proper development of the visual system.-Trujillo-Gonzalez, I., Friday, W. B., Munson, C. A., Bachleda, A., Weiss, E. R., Alam, N. M., Sha, W., Zeisel, S. H., Surzenko, N. Low availability of choline in utero disrupts development and function of the retina.

SIP1/NHERF2 enhances estrogen receptor alpha transactivation in breast cancer cells.

The estrogen receptor alpha (ERα) is a ligand-activated transcription factor that possesses two activating domains designated AF-1 and AF-2 that mediate its transcriptional activity. The role of AF-2 is to recruit coregulator protein complexes capable of modifying chromatin condensation status. In contrast, the mechanism responsible for the ligand-independent AF-1 activity and for its synergistic functional interaction with AF-2 is unclear. In this study, we have identified the protein Na+/H+ Exchanger RegulatoryFactor 2 (NHERF2) as an ERα-associated coactivator that interacts predominantly with the AF-1 domain of the nuclear receptor. Overexpression of NHERF2 in breast cancer MCF7 cells produced an increase in ERα transactivation. Interestingly, the presence of SRC-1 in NHERF2 stably overexpressing MCF7 cells produced a synergistic increase in ERα activity. We show further that NHERF2 interacts with ERα and SRC-1 in the promoter region of ERα target genes. The binding of NHERF2 to ERα in MCF7 cells increased cell proliferation and the ability of MCF7 cells to form tumors in a mouse model. We analyzed the expression of NHERF2 in breast cancer tumors finding a 2- to 17-fold increase in its mRNA levels in 50% of the tumor samples compared to normal breast tissue. These results indicate that NHERF2 is a coactivator of ERα that may participate in the development of estrogen-dependent breast cancer tumors.

Holocarboxylase synthetase acts as a biotin-independent transcriptional repressor interacting with HDAC1, HDAC2 and HDAC7.

In human cells, HCS catalyzes the biotinylation of biotin-dependent carboxylases and mediates the transcriptional control of genes involved in biotin metabolism through the activation of a cGMP-dependent signal transduction pathway. HCS also targets to the cell nucleus in association with lamin-B suggesting additional gene regulatory functions. Studies from our laboratory in Drosophila melanogaster showed that nuclear HCS is associated with heterochromatin bands enriched with the transcriptionally repressive mark histone 3 trimethylated at lysine 9. Further, HCS was shown to be recruited to the core promoter of the transcriptionally inactive hsp70 gene suggesting that it may participate in the repression of gene expression, although the mechanism involved remained elusive. In this work, we expressed HCS as a fusion protein with the DNA-binding domain of GAL4 to evaluate its effect on the transcription of a luciferase reporter gene. We show that HCS possesses transcriptional repressor activity in HepG2 cells. The transcriptional function of HCS was shown by in vitro pull down and in vivo co-immunoprecipitation assays to depend on its interaction with the histone deacetylases HDAC1, HDAC2 and HDAC7. We show further that HCS interaction with HDACs and its function in transcriptional repression is not affected by mutations impairing its biotin-ligase activity. We propose that nuclear HCS mediates events of transcriptional repression through a biotin-independent mechanism that involves its interaction with chromatin-modifying protein complexes that include histone deacetylases.

Choline metabolites and incident cardiovascular disease in a prospective cohort of adults: Coronary Artery Risk Development in Young Adults (CARDIA) Study.

BACKGROUND: The potential role for choline metabolite trimethylamine N-oxide (TMAO) in cardiovascular disease (CVD) has garnered much attention, but there have been limited data from diverse population-based cohorts. Furthermore, few studies have included circulating choline and betaine, which can serve as precursors to TMAO and may independently influence CVD. OBJECTIVE: We quantified prospective associations between 3 choline metabolites and 19-y incident CVD in a population-based cohort and tested effect modification of metabolite-CVD associations by kidney function. METHODS: Data were from the Coronary Artery Risk Development in Young Adults (CARDIA) Study, a prospective cohort with recruitment from 4 US urban centers (year 0: 1985-1986, n = 5115, ages 18-30). The analytic sample included 3444 White and Black males and females, aged 33 to 45, who attended the year 15 follow-up exam and did not have prevalent CVD. TMAO, choline, and betaine were quantitated from stored plasma (-70°C) using liquid-chromatography mass-spectrometry. Nineteen-year incident CVD events (n = 221), including coronary heart disease and stroke, were identified through adjudicated hospitalization records and linkage with the National Death Register. RESULTS: Plasma choline was positively associated with CVD in Cox proportional hazards regression analysis adjusted for demographics, health behaviors, CVD risk factors, and metabolites (hazard ratio: 1.24; 95% CI: 1.09, 1.40 per standard deviation-unit choline). TMAO and betaine were not associated with CVD in an identically adjusted analysis. There was statistical evidence for effect modification by kidney function with CVD positively associated with TMAO and negatively associated with betaine at lower values of estimated glomerular filtration rate (interaction P values: 0.0046 and 0.020, respectively). CONCLUSIONS: Our findings are consistent with a positive association between plasma choline and incident CVD. Among participants with lower kidney function, TMAO was positively, and betaine negatively, associated with CVD. These results further our understanding of the potential role for choline metabolism on CVD risk.

Choline Regulates SOX4 through miR-129-5p and Modifies H3K27me3 in the Developing Cortex.

Choline availability regulates neural progenitor cell proliferation and differentiation in the developing cerebral cortex. Here, we investigated the molecular mechanism underlying this process and demonstrated that choline regulates the transcription factor SOX4 in neural progenitor cells. Specifically, we found that low choline intake during neurogenesis reduces SOX4 protein levels, causing the downregulation of EZH2, a histone methyltransferase. Importantly, we demonstrate that low choline is not involved in SOX4 protein degradation rate and established that protein reduction is caused by aberrant expression of a microRNA (miR-129-5p). To confirm the role of miR-129-5p, we conducted gain-of-function and loss-of-function assays in neural progenitor cells and demonstrated that directly altering miR-129-5p levels could affect SOX4 protein levels. We also observed that the reduction in SOX4 and EZH2 led to decreased global levels of H3K27me3 in the developing cortex, contributing to reduced proliferation and precocious differentiation. For the first time, to our knowledge, we demonstrate that a nutrient, choline, regulates a master transcription factor and its downstream targets, providing a novel insight into the role of choline in brain development.

Homocysteine-induced endoplasmic reticulum stress activates FGF21 and is associated with browning and atrophy of white adipose tissue in Bhmt knockout mice.

Betaine-homocysteine methyltransferase (BHMT) catalyzes the transfer of methyl groups from betaine to homocysteine (Hcy), producing methionine and dimethylglycine. In this work, we characterize Bhmt wild type (Bhmt-WT) and knockout (Bhmt-KO) mice that were fully backcrossed to a C57Bl6/J background. Consistent with our previous findings, Bhmt-KO mice had decreased body weight, fat mass, and adipose tissue weight compared to WT. Histological analyses and gene expression profiling indicate that adipose browning was activated in KO mice and contributed to the adipose atrophy observed. BHMT is not expressed in adipose tissue but is abundant in liver; thus, a signal must originate from the liver that modulates adipose tissue. We found that, in Bhmt-KO mice, homocysteine-induced endoplasmic reticulum (ER) stress is associated with activation of the hepatic transcription factor cyclic AMP response element binding protein (CREBH), and an increase in hepatic and plasma concentrations of fibroblast growth factor 21 (FGF21), which is known to induce adipose browning. Our data indicate that the deletion of a single gene in one-carbon metabolism modifies adipose biology and energy metabolism. Future studies could focus on identifying if functional polymorphisms in BHMT result in a similar adipose atrophy phenotype.