Altered lipid catabolism in the vitamin A deficient liver.

The transcriptome pattern of metabolic genes in vitamin A deficient (VAD) liver has been compared to the vitamin A-sufficient (VAS) state using the Mouse 32k oligonucleotide (70mer) array. In VAD liver there was a decrease in expression of genes encoding enzymes of mitochondrial fatty acid (FA) oxidation; these genes included fatty acyl CoA ligase, carnitine o-palmitoyl transferase 1, medium chain acyl-CoA dehydrogenase, 3-ketoacyl CoA thiolase, and citrate synthase. Particularly affected was peroxisome metabolism, as genes encoding enzymes of peroxisomal FA oxidation and transport proteins were differentially expressed. These genes included those encoding acyl-CoA oxidase 1, the peroxisomal bifunctional enzyme, peroxisomal thiolase, and carnitine o-octanoyl transferase, the enzyme involved in shuttling FAs from the peroxisome to the mitochondrion. Most genes that were differentially expressed with chronic vitamin A depletion were responsive to treatment with all-trans retinoic acid (RA). Consistent with the decreased expression of genes involved in FA oxidation, we found an increase in hepatic macrocytic lipid accumulation and triglyceride levels. The relevant nuclear receptor gene that was differentially expressed in the VAD liver was that encoding the peroxisome proliferator-activated receptor (PPAR) alpha, the mRNA levels for which were decreased in VAD liver and increased with all-trans RA treatment. Down regulation of the PPAR alpha gene is the likely cause of the altered expression pattern of the above metabolic genes in VAD liver.

Nuclear receptor binding to the retinoic acid response elements of the phosphoenolpyruvate carboxykinase gene in vivo: effects of vitamin A deficiency.

Vitamin A deficiency decreases hepatic phosphoenolpyruvate carboxykinase (PEPCK) gene expression in mice and expression is restored with retinoic acid treatment in vivo. This report examines further the mechanism of retinoid regulation of the PEPCK gene in vivo. We have identified nuclear receptors that bind to retinoic acid response elements (RAREs) in the PEPCK promoter by electrophoretic mobility shift assay and have verified these in vivo using chromatin immunoprecipitation (ChIP) in mouse liver. Based on the results of our ChIP assay, hepatic nuclear factor (HNF)-4alpha, retinoid X receptor (RXR) alpha, retinoic acid receptor (RAR) alpha, peroxisome proliferator-activated receptor (PPAR) alpha and chicken ovalbumin upstream promoter transcription factor (COUP-TF) II bind to the downstream retinoic acid response unit RARE1/RARE2, and PPARalpha and RXRalpha bind to the upstream RARE3 of the PEPCK gene. HNF-4alpha, RXRalpha, RARalpha, PPARalpha and COUP-TFII bind PEPCK RAREs in a specific pattern that, with the exception of PPARalpha, does not change significantly with vitamin A deficiency. PPARalpha binding to the upstream retinoic acid response element is decreased in the vitamin A-deficient liver, when compared to the vitamin A-sufficient state. These results provide the first in vivo measures of nuclear receptor binding to the upstream and downstream RAREs of the PEPCK gene under conditions where the nucleosomal structure of the chromatin is maintained and the nuclear receptors are physically cross-linked in situ to the PEPCK DNA in intact liver.

Vitamin A status in mice affects the histone code of the phosphoenolpyruvate carboxykinase gene in liver.

Vitamin A deficiency decreases hepatic phosphoenolpyruvate carboxykinase (PEPCK) gene expression in mice, and expression is restored with retinoic acid (RA) treatment in vivo. In the studies reported here, we examined changes in histone modification and coregulator association with the regulatory domains of the PEPCK gene in response to alterations in vitamin A status. We identified nuclear receptors that bind to retinoic acid response elements (RAREs) in the PEPCK promoter by electrophoretic mobility shift assay and verified these in vivo using chromatin immunoprecipitation in mouse liver. Hypothetically, nuclear receptors at PEPCK RAREs recruit specific coactivator molecules that contribute to the acetylation of core histones and/or serve as bridging molecules between nuclear receptors and basal transcription factors at the transcription start site. We identified 3 coactivator molecules, cAMP-response element binding protein (CBP), steroid receptor coactivator (SRC)-1, and peroxisome-proliferator activated receptor (PPAR)-gamma-coactivator (PGC)-1alpha, that bound in association with the PEPCK RAREs in vivo. Furthermore, there was differential binding of these coactivators in vitamin A-deficient mice. Related to this, specific lysine residues were acetylated on histones H3 and H4 at the 3 RAREs of the PEPCK promoter, consistent with the action of the above coactivators, and acetylation of certain lysines was significantly decreased with vitamin A deficiency. These results demonstrate the associated changes that occur in nuclear receptor binding, coactivator recruitment, and histone acetylation in response to vitamin A status, identified at specific RAREs in the PEPCK gene in vivo.

Vitamin A depletion is associated with low phosphoenolpyruvate carboxykinase mRNA levels during late fetal development and at birth in mice.

Expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene is repressed during fetal liver development and activated at birth. It has been shown that the PEPCK gene is a retinoid-responsive gene, but whether it is regulated by vitamin A in the fetus has not been established. In this study, we found that PEPCK mRNA can be detected in the murine fetal liver as early as gestational d 17. In addition, expression and cAMP induction of the PEPCK gene during late gestation and at birth require vitamin A sufficiency in the fetus and neonate. The PEPCK promoter contains several regulatory elements that bind a diverse array of transcription factors and nuclear coregulators, although it is largely unknown which of these factors are expressed early in liver development. Expression of some of these nuclear factors in livers of fetal mice was investigated by immunohistochemistry (IHC). Fetuses were from dams that were fed from the beginning of gestation diets that were adequate or devoid of vitamin A. Hepatocyte nuclear factor 4alpha (HNF4alpha) was expressed at the earliest stage of liver development on d 11, whereas retinoid X receptor alpha (RXRalpha) and nuclear coactivator CREB-binding protein (CBP) were expressed from d 16 onward. Although expressions of RXRalpha and CBP in livers of vitamin A-sufficient and vitamin A-depleted fetal mice did not differ, the level of HNF4alpha was consistently lower in the latter. Our findings strongly suggest that vitamin A is required during liver development for staged expression of the PEPCK gene and that HNF4alpha may be involved in mediating vitamin A regulation of the PEPCK gene at these critical periods.

Retinoid regulation of the phosphoenolpyruvate carboxykinase gene in liver.

The cytosolic PEPCK gene is a model gene for assessing retinoid regulation of liver-specific genes encoding enzymes of carbohydrate metabolism. In vivo, we have demonstrated that the PEPCK gene is inhibited by vitamin A deficiency. Specifically, under conditions of food deprivation, induction of the PEPCK gene is inhibited in the vitamin A deficient mouse. Inhibition of the PEPCK gene by vitamin A deficiency is reversed by all-trans or 9-cis retinoic acid (RA) treatment. In a transgenic mouse model, a -460 and -355 bp PEPCK promoter fragment confers susceptibility to inhibition by vitamin A deficiency and responsiveness to all-trans RA treatment. However, there is a differential effect of 9-cis RA on the PEPCK promoter; the -460 fragment confers responsiveness to 9-cis RA, but the -355 fragment does not. Taken together, these results indicate that the PEPCK retinoic acid response element (RARE)1 is required for 9-cis RA induction-but not all-trans RA induction-of the PEPCK gene. In order to determine if vitamin A deficiency alters specific localized expression of the PEPCK gene in the periportal cells of the liver, the effect of vitamin A status on PEPCK localization in the liver was also measured. The PEPCK transgenes were expressed specifically in the periportal region of the liver acinus and although vitamin A deficiency caused a decrease in PEPCK transgene mRNA levels in periportal cells, it did not alter the periportal cell-specific pattern of expression. Retinoid treatment induced PEPCK transgene mRNA levels in the same population of cells, however, the -355 bp PEPCK promoter fragment did not respond to 9-cis RA treatment. In order to determine the nuclear transcription factor(s) responsible for retinoid regulation of the PEPCK gene in the liver, we investigated retinoic acid receptor (RAR)alpha and beta and the retinoid X receptor (RXR)alpha-the major retinoid receptors in liver-in terms of expression and the ability of the receptors to bind the PEPCK RAREs. Vitamin A deficiency significantly decreased hepatic RAR beta, but not RAR alpha or RXR alpha mRNA levels. In situ hybridization showed that RAR alpha, RAR beta and RXR alpha mRNAs were localized in the periportal region, however, immunohistochemistry showed that RAR alpha and RXR alpha were distributed evenly across the liver acinus, whereas only RAR beta levels were higher in periportal cells. The binding of nuclear receptors to PEPCK RARE1, RARE2 and RARE3 indicates a complex pattern of retinoid receptor and orphan nuclear receptor binding.

Using the two-hybrid screen in the classroom laboratory.

The National Science Foundation and others have made compelling arguments that research be incorporated into the learning of undergraduates. In response to these arguments, a two-hybrid research project was incorporated into a molecular biology course that contained both a lecture section and a laboratory section. The course was designed around specific goals for educational outcomes, including introducing research to a wide range of students, teaching students experimental design and data analysis, and enhancing understanding of course material. Additional goals included teaching students to search genomic databases, to access scientific articles, and to write a paper in scientific format. Graded events tested these goals, and a student evaluation indicated student perception of the project. According to our analysis of the data, the yeast two-hybrid screen was a success: several novel clones were identified; students met expectations on graded lab reports, the poster session, and the final paper; and evaluations indicated that students had achieved the outlined goals. Students indicated on the evaluations that the research project increased their interest in research and greatly improved understanding of the course material. Finally, several students in the course intend to submit the findings of the research project to an undergraduate research journal.