Gene expression profiling in phosphatidylethanolamine N-methyltransferase knockout mice

Molecular Brain Research 134 (2005) 239 – 255 www.elsevier.com/locate/molbrainres

Research report

Gene expression profiling in phosphatidylethanolamine N-methyltransferase knockout mice Xiaonan Zhu, Steven H. Zeisel* Department of Nutrition, CB #7461, School of Public Health, School of Medicine, University of North Carolina, Chapel Hill, NC 27599, USA Accepted 24 October 2004 Available online 13 February 2005

Abstract Choline is derived from the diet as well as from de novo methylation of phosphatidylethanolamine catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT). Pemt knockout mice have no endogenous synthesis of choline molecules. We previously reported that these mice have excess S-adenosylmethionine and hypermethylated DNA in brain, as well as increased mitosis in neural progenitor cells of the hippocampus in embryonic day 17 (E17) brain. In the present study, E17 fetal brains and adult brains were harvested and total RNA was extracted. In fetal brain, using gene expression profiling and Significance Analysis of Microarrays, we identified 107 significant genes with increased expression and 379 significant genes with decreased expression. In adult brain, we identified 381 significant genes with increased expression and 1037 significant genes with decreased expression. We observed significant changes in expression of genes regulating cell cycle (such as TP53, Fgf4, and Ing1), differentiation and neurogenesis (such as S100A4 and D14Ws), and phospholipid metabolism (such as Pip5k1a, Pitpn, and Pla2g6) as well as in a number of methyltransferase genes (including Gnmt). Some genes with expression known to be regulated by promoter methylation were suppressed in Pemt knockout brain (such as S100a4 and TP53). These findings are consistent with the biochemical changes that we previous reported in fetal brains from Pemt knockout mice. This is the first report of gene profiling in Pemt / mouse brain. D 2004 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Nutritional and prenatal factors Keywords: PEMT; Brain; Mouse; Choline; Gene expression profiling

1. Introduction Choline is derived not only from the diet, but as well from de novo synthesis involving methylation of phosphatidylethanolamine catalyzed by the enzyme phosphatidylethanolamine N-methyltransferase (PEMT; EC 2.1.1.17) using S-adenosylmethionine (AdoMet) as the methyl donor [13]. Choline, or its metabolites, are needed for the structural integrity and signaling functions of cell membranes; it is the major source of methyl groups in the diet (one of choline’s metabolites, betaine, participates in the * Corresponding author. Fax: +1 919 966 7216. E-mail address: [email protected] (S.H. Zeisel). 0169-328X/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.molbrainres.2004.10.040

methylation of homocysteine to form methionine), and it directly affects cholinergic neurotransmission, transmembrane signaling, and lipid transport/metabolism [27,78]. Pemt / mice have two disrupted alleles of the pemt2 gene at exon 2 and these mice do not express any PEMT activity [69]. Therefore, they completely depend on dietary choline intake to meet daily choline requirements. When fed a diet deficient in choline and insufficient in methionine, Pemt / mice developed decreased phosphatidylcholine concentrations in hepatic membranes, severe liver damage, and died; a choline-supplemented diet prevented this [70] and reversed hepatic damage if begun early enough [68]. Recently, we showed that Pemt / mice were choline deficient despite being fed sufficient or

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supplemental amounts of dietary choline [79]. At the same time, we found that concentrations of AdoMet in brain were increased in brains of Pemt / mice (presumably because AdoMet was not being used to form phosphatidylcholine) and DNA was hypermethylated [80]. Additionally, we found that brain development was altered. Embryonic day 17 Pemt / mice had increased mitosis in hippocampal ventricular epithelium and decreased calretinin (a neuronal marker) concentrations in dentate gyrus [80]. These selected examples suggest that there might be widespread changes in gene expression in the Pemt / mouse brain due to altered gene methylation, altered brain development, or due to specific responses to the deletion of the Pemt gene. Gene expression profiling has never been performed on these knockout mice. Here we report the identification of seven distinct functional categories with altered gene expression profiles associated with Pemt deletion. Our data confirm some previous findings and also reveal novel information regarding the role of PEMT in developing and developed brain.

2. Materials and methods 2.1. Animals Pemt / mice were a generous gift from Dr. Dennis Vance, University of Alberta, Edmonton, Canada. Wild

type mice of the same strain from which the knockout mice were generated were used (mixed genetic background of 129/J and C57BL/6). All animal procedures were approved by the University of North Carolina Institutional Animal Care and Use Committee. Mice were housed in cages in a climate controlled room (24 8C) exposed to light during 06:00–18:00 daily. Timed-pregnant Pemt / and wild type mice, and 12-week-old Pemt / and wild type mice were given free access to water and to AIN-76A diet (1.1 g choline chloride/kg of diet). Mice ingested an average of 3 mg choline/day. 2.2. Tissue collection On gestational day 17, pregnant mice were anesthetized with a single injection of 0.03 mL ketamine (100 mg/mL) and 0.02 mL xylazine (20 mg/mL) (Henry Schein Inc., Melville, NY, USA) subcutaneously and the mice were kept on a heating pad to maintain body temperature. The uterine horns were exposed by a midline abdominal incision and the fetuses were removed individually. The fetuses were decapitated and the brains were collected and quick-frozen in liquid nitrogen and stored at 80 8C prior to RNA and protein extraction. We pooled male and female fetuses for this study. We chose to study brains on embryonic day 17 because there is existing published data about developmental changes in the hippocampus of the mouse [6,16,17]. By choosing this day we are able to compare our results to those in experiments where wild

Fig. 1. Confirmation of representative gene expression in Pemt / mouse brain compared to wild type. Detection of Chk, Ptdss1, Gnmt, Cdkn1a, Cdk4, Msh2, S100a4, and Pdk4 mRNA levels was performed by a real-time RT-PCR assay. Relative quantitation of mRNA expression was calculated by the comparative the threshold cycle (Ct) method described in Materials and methods. The relative quantitation value of target, normalized to an endogenous control (h-actin) gene and relative to a calibrator, is expressed as 2 DDCt, where DCt = Ct of target Ct of h-actin, and DDCt = DCt of Pemt / DCt of wild type for the target gene. Expression of a gene is increased with a value of qRT-PCR power N1, and decreased with a value b1. Representative protein expression was analyzed by Western blotting using anti-PiP5K1A, GNMT, P21, P53, MSH2, and PDK4 serum with horseradish peroxidase secondary antibodies and developed with chemiluminescent reagents. Representative blots are shown. Lane 1: Pemt +/+ brain; lane 2: Pemt / brain. Each lane contained 75-Ag protein. The direction of change in gene expression assessed by oligoarray analysis is indicated by an arrow in the last column.

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type mice were fed choline- or folate-supplemented or -deficient diets during pregnancy. There is also a great deal of data available about development of the hippocampus in rats on day 18 of gestation (which is equivalent to day 17 in the mouse) [2,4,5,24]. Additionally, we found that brain development was altered in embryonic day 17 Pemt / mice; they had increased mitosis in hippocampal ventricular epithelium and decreased calretinin (a neuronal marker) concentrations in dentate gyrus [80]. Twelve-week-old mice were anesthetized with a single injection of 0.03 mL ketamine (100 mg/mL) and 0.02 mL xylazine (20 mg/mL) (Henry Schein Inc., Melville, NY, USA) subcutaneously and the brains were collected and quick-frozen in liquid nitrogen and stored at 80 8C prior to RNA and protein extraction. 2.3. RNA extraction Total RNA was extracted from E17 fetal and adult brain using TRIzol reagent (Invitrogen, Darmstadt, Germany) accoring to the manufacture’s instructions. RNA content and purity were determined by measuring the optical density at 260 nm and 280 nm. The quality of the RNA samples was assessed by using the Agilent 2100 Bioanalyzer (RNA chip; Palo Alto, CA, USA), which uses capillary electrophoresis with fluorescence detection to characterize size distribution. Table 1 Changes in gene expression in fetal brains of Pemt Fetal

/

241

2.4. Microarray hybridization Purification of probes, hybridization, and washing were performed according to a protocol provided by University of North Carolina at Chapel Hill (UNC) Genomics Core (http:// genomicscore.unc.edu). Briefly, 40 Ag of total RNA was reverse transcribed into cDNA with Cy3 or Cy5 monoreactive fluorophors using CyScribe First-Strand cDNA Labeling Kit (Amersham Biosciences, Inc., Piscataway, NJ) and Superscript II RT (Invitrogen Life Technologies, Carlsbad, CA) primed with a T7-(dT24) oligonucleotide. Wild type and Pemt / labeled cDNA were combined and cleaned up using Qiaquick PCR Purification Kit (Qiagen; Valencia, CA, USA) according to the manufacturer’s instructions. Both cDNA samples were hybridized to a mouse 16K+ DNA oligonucleotide microarray printed by the UNC Genomics Core and Microarray Facility followed by washing and staining. 2.5. Image analysis Fluorescent images of the microarrays were collected using a GenePix 4000 fluorescent scanner (Axon Instruments, Inc., Union City, CA). Gridding and primary data acquisition were performed using GenePix Pro Microarray Acquisition software. The data were then imported into the UNC Microarray database (UMD, http://genome.unc.edu) and automatically normalized.

versus wild type mice

Term

GO ID

Total

Under

Over

Change

P value (under)

P value (over)

P value (changed)

Antigen binding Organismal physiological process Ion transport Plasma membrane Integral to plasma membrane Immune response Protein kinase C activity Response to biotic stimulus Defense response Channel/pore class transporter activity Response to stimulus Cation transport Phorbol ester receptor activity Response to external stimulus Monovalent inorganic cation transport Calcium ion binding G-protein-coupled receptor protein signaling pathway Neurophysiological process Neurogenesis Methyltransferase activity Phospholipid metabolism Apoptosis Cell cycle Cell differentiation

3823 50874 6811 5886 5887 6955 4697 9607 6952 15267 50896 6812 1565 9605 15672 5509 7186

26 680 343 857 428 341 4 438 403 235 769 236 5 646 142 321 386

6 59 39 75 43 31 3 38 35 28 55 27 3 50 19 25 30

3 13 2 7 4 8 0 8 8 0 17 1 0 12 0 6 6

9 72 41 82 47 39 3 46 43 28 72 28 3 62 19 31 36

0.0016 b0.0001 b0.0001 b0.0001 b0.0001 0.001 0.0005 0.0007 0.0011 b0.0001 0.0046 0.0001 0.0012 0.0014 0.0001 0.0203 0.0119

0.01 0.4067 0.9848 0.9949 0.9463 0.2438 1 0.4993 0.4055 1 0.1815 0.9854 1 0.4541 1 0.491 0.6725

b0.0001 0.0001 0.0002 0.0006 0.0006 0.0008 0.0012 0.0018 0.0019 0.0025 0.0026 0.0026 0.0028 0.0029 0.0033 0.0292 0.0316

50877 7399 8168 6644 6915 7049 30154

215 199 46 27 208 393 194

19 15 2 2 9 15 5

2 4 2 0 5 9 6

21 19 4 2 14 24 11

0.0124 0.0789 0.6844 0.4001 0.7338 0.9035 0.9718

0.894 0.4593 0.1911 1 0.297 0.2464 0.1227

0.0587 0.0831 0.383 0.5573 0.558 0.7435 0.7782

The extent of changes within gene ontology classes was assessed using GoMiner. A selected list is presented, where P values were computed for over expressed, under expressed, and the total number of changed genes within each class. Classes are sorted in ascending order, based on the P value (changed). Genes in boldface were altered in both fetal and adult brains in same direction.

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2.6. Data analysis The Loess normalized data were used for downstream data analysis. The Loess normalized red/green ratio was then queried to identify those genes that gave a signal intensity 30 units above background intensity. Once bfiltered,Q gene sets were exported and gene selection, after the initial filtering for expressed genes, was performed using supervised analyses, significance analysis of microarrays (SAM) [64], to identify differentially expressed genes. SAM not only produced a list of bsignificantlyQ differentially expressed genes, but also provided an estimation of the false significance rate using permutation testing. 2.7. SAM parameters used For E17 fetal brain microarrays, 11 separate oligoarrays (comparing WT and Pemt / ) including 4 dye-flip experiments were performed and we identified differentially expressed genes: 107 significant genes with increased expression and 379 significant genes with decreased expression. Input parameters used for SAM analysis were as follows: imputation engine was b10-Nearest Neighbor ImputerQ; data type was bOne class ResponseQ; number of permutations was b1000Q; no blocked permutations; RNG seed was b1234567Q; delta was b0.534Q; (Upper Cutoff, Lower Cutoff) was (1.69256, 1.43765). Computed quantities were as follows: computed exchangeability factor S0 was Table 2 Changes in gene expression in adult brains of Pemt Adult

/

b0.07053991Q; S0 percentile was b0.49Q; false significant number (median, 90 percentile) was (50.93508, 228.71266); false discovery rate (median, 90 percentile) was (4.66438, 20.94438); Pi0Hat was b0.70743Q. For adult brain microarrays, 8 separate oligoarrays (comparing WT and Pemt / ) including 4 dye-flip experiments were performed and we identified differentially expressed genes: 381 significant genes with increased expression and 1037 significant genes with decreased expression. Input parameters for SAM analysis were as follows: imputation engine was b10-Nearest Neighbor ImputerQ; data type was bOne class ResponseQ; number of permutations was b1000Q; no blocked permutations; RNG seed was b1234567Q; delta was b0.61114Q; (Upper Cutoff, Lower Cutoff) was (1.56700, 1.40890). Computed quantities were as follows: computed exchangeability factor S0 was b0.061515024Q; S0 percentile was b0.25Q; false significant number (median, 90 percentile) was (146.33373, 862.64036); false discovery rate (median, 90 percentile) was (4.85674, 28.63061); Pi0Hat was b0.60719Q. 2.8. Biological process categorization by gene ontology Data from SAM output files were analyzed using GoMiner (version 1.22; http://www.miblab.gatech.edu/gominer/) which generated a list of genes classified by gene ontology (GO) classes based on the default database (com.mysql.jdbc. Driver at jdbc:mysql://discover.nci.nih.gov/GEEVS). Fish-

versus wild type mice

Term

GO ID

Total

Under

Over

Change

P value (under)

P value (over)

P value (changed)

Antigen binding Response to pest/pathogen/parasite Response to stress Immune response Acute-phase response Organismal physiological process Defense response Extracellular Signal transducer activity Response to stimulus Extracellular space Humoral immune response Receptor activity Response to biotic stimulus Axon guidance receptor activity Neuropeptide receptor activity Calcium ion binding Phospholipid metabolism Neurogenesis G-protein-coupled receptor protein signaling pathway Cell cycle Apoptosis Methyltransferase activity Cell differentiation

3823 9613 6950 6955 6953 50874 6952 5576 4871 50896 5615 6959 4872 9607 8046 8188 5509 6644 7399 7186

26 196 386 341 22 680 403 1692 1289 769 1577 62 826 438 5 37 321 27 199 386

6 29 53 49 3 101 59 243 212 108 224 8 141 63 3 8 47 5 37 65

8 28 45 39 8 56 40 113 65 64 107 13 41 40 1 3 22 3 7 14

14 57 98 88 11 157 99 356 277 172 331 21 182 103 4 11 69 8 44 79

0.0808 0.1271 0.1481 0.0919 0.4975 0.0091 0.052 0.0003 b0.0001 0.0331 0.001 0.4634 b0.0001 0.0615 0.014 0.0661 0.0763 0.2131 0.0036 0.0021

0.0002 0.0001 0.0001 0.0005 b0.0001 0.0461 0.0058 0.465 0.9958 0.0282 0.3921 0.0002 0.9847 0.0214 0.2896 0.4461 0.4613 0.2628 0.9808 0.9967

0.0001 0.0002 0.0004 0.0004 0.0008 0.001 0.0011 0.0014 0.0016 0.0026 0.0027 0.0028 0.0038 0.0041 0.005 0.0668 0.0941 0.1111 0.1116 0.1734

7049 6915 8168 30154

393 208 46 194

45 24 5 26

23 11 2 6

68 35 7 32

0.6415 0.602 0.6557 0.2908

0.7616 0.8197 0.8176 0.9906

0.7598 0.7655 0.7745 0.7962

The extent of changes within gene ontology classes was assessed using GoMiner. A selected list is presented, where P values were computed for over expressed, under expressed, and the total number of changed genes within each class. Classes are sorted in ascending order, based on the P value (changed). Genes in boldface were altered in both fetal and adult brains in same direction.

X. Zhu, S.H. Zeisel / Molecular Brain Research 134 (2005) 239–255 Table 3 Changes in expression of genes of phospholipid metabolism in Pemt versus wild type fetal mouse brains

Fetal

Symbol

Gene name

Pip5k1a

phosphatidylinositol4-phosphate 5-kinase, type 1 alpha phosphatidylinositol 3-kinase, C2 domain containing, alpha polypeptide ATPase, aminophospholipid transporter (APLT), class I, type 8A, member 1 phosphatidylinositol membrane associated inositol polyphosphate4-phosphatase, type II, 105 kDa phosphatidylethanolamine N-methyltransferase inositol polyphosphate-5phosphatase B phospholipase A2, group VI

Pik3c2a

Atp8a1

Pitpnm INPP4B

pemt Inpp5b Pla2g6

Score (d)

/

q value (%)

3.6297

0.3451

1.8068

1.4968

1.5869

3.1210

threshold, was determined. Relative quantification of target gene mRNA expression was calculated by the comparative Ct method described elsewhere [37]. The relative quantification value of target, normalized to an endogenous control h-actin gene and relative to a calibrator, was expressed as Table 4 Changes in expression of genes of phospholipid metabolism in Pemt versus wild type adult mouse brains Symbol Adult Ptdss1 Pip5k1a

1.5786

3.1210

1.5015

4.2389

1.0883

33.0691

1.7810

3.8009

1.7995

3.8009

/

and wild type E17 fetal Gene expression arrays were used to study Pemt brains. SAM was employed to identify differentially expressed genes. Genes in boldface were altered in both fetal and adult brains in same direction. Score (d) is assigned to each gene on the basis of change in gene expression relative to the standard deviation of repeated measurements. Genes with positive scores were over expressed in Pemt / ; those with negative scores were under expressed. q value is the lowest false discovery rate at which gene expression change is significant. Input parameters for SAM analysis and computed quantities were described in the text.

er’s exact tests were performed to assess the extent of change within the total number of genes in each GO class. Three significance tests were performed for each class, based on the number of genes that were over expressed, under expressed, or on the total number of changed genes. 2.9. Real-time RT-PCR For selected genes, expression results were confirmed with a real-time RT-PCR assay using an ABI prism 7700 sequence detection system (Foster, CA, USA). Primers and probes for murine target genes (Chk, Ptdss1, Gnmt, Cdkn1a, Cdk4, Msh2, S100a4, and Pdk4) and h-actin were designed; the target gene’s probes were labeled with a reporter dye (FAM, 6-carboxyfluorescein) at the 5V end and a quencher dye (TAMRA, 6-carboxytetramethyrhodamine) at the 3V end. The h-actin probe was labeled with a reporter dye (TET, tetramethylrhodamine) at the 5V end. A 30-AL reaction mixture contained 500 ng of total RNA and 0.5 Amol/L of each primer. The reaction conditions were RT at 48 8C for 30 min and initial denaturation at 95 8C for 10 min followed by 40 cycles with 15 s at 95 8C for denaturing and 1 min at 60 8C for annealing and extension. The threshold cycle (Ct), i.e., the cycle number at which the amount of amplified gene of interest reached a fixed

243

Gene name

phosphatidylserine synthase 1 phosphatidylinositol4-phosphate 5-kinase, type 1 alpha Pla2g1br phospholipase A2, group IB, pancreas, receptor Frap1 FK506 binding protein 12-rapamycin associated protein 1 Pla2g1b phospholipase A2, group IB, pancreas Pitpnm phosphatidylinositol membrane associated Trhr2 thyrotropin releasing hormone receptor 2 Pik3ca phosphatidylinositol 3-kinase, catalytic, alpha polypeptide Pps putative phosphatase Oc90 otoconin 90 Pip5k1c phosphatidylinositol4-phosphate 5-kinase, type 1 gamma 4933424M23Rik phosphatidylglycero phosphate synthase 1 Ptger3 prostaglandin E receptor 3 (subtype EP3) Edg3 endothelial differentiation, sphingolipid G-proteincoupled receptor, 3 Inpp5e inositol polyphosphate5-phosphatase E pemt phosphatidylethanolamine N-methyltransferase Inpp5d inositol polyphosphate5-phosphatase D D8Ertd319e DNA segment, Chr 8, ERATO Doi 319, expressed Pla2g6 phospholipase A2, group VI Plcg1 phospholipase C, gamma 1 Gpam glycerol-3-phosphate acyltransferase, mitochondrial Pigm phosphatidylinositol glycan, class M

Score (d)

/

q value (%)

3.3620 3.1621

0.1124 0.1124

2.0606

0.8268

1.8321

1.4956

1.8264

1.4956

1.7802

1.6052

1.7153

1.9178

1.6652

2.2388

1.6273 1.5796 1.5126

2.9767 3.4760 3.8468

1.5056

3.8468

1.4981

4.3740

1.4492

4.8567

1.4363

4.8567

0.1307 58.1837 1.6218

4.3740

1.6800

3.8468

1.9712 2.1643 2.4880

1.6052 0.9740 0.3256

3.2890

0.1124

Gene expression arrays were used to study Pemt / and wild type adult brains. SAM was employed to identify differentially expressed genes. Genes in boldface were altered in both fetal and adult brains in same direction. Score (d) is assigned to each gene on the basis of change in gene expression relative to the standard deviation of repeated measurements. Genes with positive scores were over expressed in Pemt / ; those with negative scores were under expressed. q value is the lowest false discovery rate at which gene expression change is significant. Input parameters for SAM analysis and computed quantities were described in the text.

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Table 5 Changes in expression of genes encoding for methyltransferases in Pemt versus wild type fetal mouse brains

Fetal

Gene symbol

Gene name

Cntn1 Ezh2

contactin 1 enhancer of zeste homolog 2 (Drosophila) glycine N-methyltransferase

Gnmt

Score (d)

/

q value (%)

2.1628 1.7696

0.3451 3.8009

1.7783

3.8009

Gene expression arrays were used to study Pemt / and wild type E17 fetal brains. SAM was employed to identify differentially expressed genes. Genes in boldface were altered in both fetal and adult brains in same direction. Score (d) is assigned to each gene on the basis of change in gene expression relative to the standard deviation of repeated measurements. Genes with positive scores were over expressed in Pemt / ; those with negative scores were under expressed. q value is the lowest false discovery rate at which gene expression change is significant. Input parameters for SAM analysis and computed quantities were described in the text.

2 DDCt (power/folds), where DCt=Ct of target gene Ct of endogenous control gene (h-actin), and DDCt=DCt of samples for target gene (Pemt / ) DCt of the calibrator (wild type) for the target gene. Primers used for real-time RT-PCR were as follows: Chk (forward, 5V-CACTTGGGCCAAAACTCTATG-3V; reverse, 5V-ACTCTTCAGTGTCCAATCGC-3V), Ptdss1 (forward, 5V-TACCACTGGGCAAGCTTCAA-3V; reverse, 5VGTGAACTGCAGCACAGCTC-3V), Gnmt (forward, 5VTable 6 Changes in expression of genes encoding for methyltransferases in Pemt versus wild type adult mouse brains

Adult

Gene symbol

Gene name

Suv39h1

suppressor of variegation 3–9 homolog 1 (Drosophila) O-6-methylguanineDNA methyltransferase thioether S-methyltransferase nuclear receptor binding SET-domain protein 1 DNA (cytosine-5-)methyltransferase 3-like catechol-Omethyltransferase glycine Nmethyltransferase

Mgmt

Temt Nsd1

Dnmt3l

Comt Gnmt

Score (d) 3.9890

ACTTGCCAAACTGCAAAGGT-3V; reverse, 5V-GCACCATGCTTGCAATGTTC-3V), Cdkn1a (forward, 5VAACATCTCAGGGCCGAAAAC-3V; reverse, 5V-AAGACCAATCTGCGCTTGGA-3V), Cdk4 (forward, 5V-TACGCAACACCCGTGGACA-3V; reverse, 5V-TTCCACAGAAGAGAGGCTTC-3V), Msh2 (forward, 5V-TATTAACCAGCTCCCCAGC-3V; reverse, 5V-CCAACAACAGGCCTGGTGT-3V), S100a4 (forward, 5V-AAGACAGAGCTCAAGGAGCT-3V; reverse, 5V-CTGGAATGCAGCTTCATCTG-3V), Pdk4 (forward, 5V-TCCAACACCTGTGATGGcACA-3V; reverse, 5V-AGACGAGAAATTGGCAAGCC-3V), h-actin (forward, 5V-CTGCCTGACGGCCAAGTC-3V; reverse, 5V-CAAGAAGGAAGGCT.GAAAAGA-3V). Table 7 Changes in expression of genes involved in cell cycle regulation in Pemt versus wild type fetal mouse brains Symbol Gene name Fetal TP53 Cdkn1a Trp63 UBE2C PCNA Fgf5 Nmyc1

/

q value (%) 0.1124

Cdk5r Fgf4 PLK1 Cxcl1

1.9700

0.9740

1.6615

2.5724

1.6277

2.5724

Cdk4 Ing1

1.5127

3.8468

Brca1 Cdkap1

1.4821

4.3740

2.2045

0.8268

Tlk2 Cdkn2a

Gene expression arrays were used to study Pemt / and wild type adult brains. SAM was employed to identify differentially expressed genes. Genes in boldface were altered in both fetal and adult brains in same direction. Score (d) is assigned to each gene on the basis of change in gene expression relative to the standard deviation of repeated measurements. Genes with positive scores were over expressed in Pemt / ; those with negative scores were under expressed. q value is the lowest false discovery rate at which gene expression change is significant. Input parameters for SAM analysis and computed quantities were described in the text.

Cks1 Msh2 Cdca3 Cks2

tumor protein p53 (Li–Fraumeni syndrome) cyclin-dependent kinase inhibitor 1A (P21) transformation-related protein 63 ubiquitin-conjugating enzyme E2C proliferating cell nuclear antigen fibroblast growth factor 5 neuroblastoma myc-related oncogene 1 cyclin-dependent kinase 5, regulatory subunit (p35) fibroblast growth factor 4 polo-like kinase (Drosophila) chemokine (C-X-C motif) ligand 1 tousled-like kinase 2 (Arabidopsis) cyclin-dependent kinase inhibitor 2A cyclin-dependent kinase 4 inhibitor of growth family, member 1 breast cancer 1 CDK2 (cyclin-dependent kinase 2)-associated protein 1 CDC28 protein kinase 1 mutS homolog 2 (E. coli) cell division cycle associated 3 CDC28 protein kinase regulatory subunit 2

/

Score (d) q value (%) 2.7157

0.3451

2.4967

0.3451

1.9043

1.0917

1.8088

1.4968

1.7979

1.4968

1.6572 1.6058

2.3006 3.1210

1.5802

3.1210

1.5579 1.5314

3.6355 3.6355

1.5162

3.8009

1.4628

4.6644

1.4563

4.6644

1.7168 1.7289

4.6644 4.2389

1.7858 1.8443

3.8009 3.1210

2.1985 2.3100 2.3583 2.9082

1.0917 0.5970 0.5970 0.3451

Gene expression arrays were used to study Pemt / and wild type E17 fetal brains. SAM was employed to identify differentially expressed genes. Genes in boldface were altered in both fetal and adult brains in same direction. Score (d) is assigned to each gene on the basis of change in gene expression relative to the standard deviation of repeated measurements. Genes with positive scores were over expressed in Pemt / ; those with negative scores were under expressed. q value is the lowest false discovery rate at which gene expression change is significant. Input parameters for SAM analysis and computed quantities were described in the text.

X. Zhu, S.H. Zeisel / Molecular Brain Research 134 (2005) 239–255 Table 8 Changes in expression of genes involved in cell cycle regulation in Pemt Adult

/

versus wild type adult mouse brains

Symbol

Gene name

UBE2C MCM6

ubiquitin-conjugating enzyme E2C MCM6 minichromosome maintenance deficient 6 (MIS5 homolog, S. pombe) (S. cerevisiae) tousled-like kinase 2 (Arabidopsis) cell division cycle 45 homolog (S. cerevisiae)-like myeloblastosis oncogene v-erb-a erythroblastic leukemia viral oncogene homolog 4 (avian) mutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli) E2F transcription factor 3 CD40 ligand-activated specific transcript 3 Harvey rat sarcoma oncogene, subgroup R ELK4, member of ETS oncogene family fibroblast growth factor 4 c-fos-induced growth factor RAN, member RAS oncogene family cyclin B2 transcription factor Dp 1 Mdm2, transformed 3T3 cell double minute 2, p53 binding protein (mouse) par-6 partitioning defective 6 homolog gamma (C. elegans) neurofibromatosis 2 tumor protein p53 (Li–Fraumeni syndrome) vascular endothelial growth factor C retinoblastoma binding protein 4 thrombopoietin G1 to phase transition 1 chemokine (C-X-C motif) ligand 1 serum-inducible kinase Hus1 homolog (S. pombe) vav 1 oncogene growth arrest specific 1 minichromosome maintenance deficient 3 (S. cerevisiae) RAD1 homolog (S. pombe) nuclear factor of activated T-cells, cytoplasmic 1 signal-induced proliferation associated gene 1 geminin cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4) ZW10 homolog (Drosophila), centromere/kinetochore protein mitogen-activated protein kinase 1 mitogen-activated protein kinase kinase 7 cell division cycle 2 homolog (S. pombe)-like 2 minichromosome maintenance deficient 7 (S. cerevisiae) small protein effector 1 of Cdc42 mitogen-activated protein kinase 13 inhibitor of growth family, member 1 transformation-related protein 73 CDC like kinase 4 cell division cycle 37 homolog (S. cerevisiae) transforming growth factor beta regulated gene 4 stratifin E2F transcription factor 5 raf-related oncogene vascular endothelial growth factor A cytochrome P450, family 3, subfamily a, polypeptide 16 CDC-like kinase 3 RAD9 homolog (S. pombe) thymoma viral protooncogene 1 par-3 (partitioning defective 3) homolog (C. elegans) v-maf musculoaponeurotic fibrosarcoma oncogene family, protein G (avian) mutS homolog 2 (E. coli) cytochrome P450, family 3, subfamily a, polypeptide 41

Tlk2 Cdc45l Myb Erbb4 MLH1 E2f3 Clast3 Rras Elk4 Fgf4 Figf Ran Ccnb2 Tfdp1 MDM2 Pard6g Nf2 TP53 Vegfc Rbbp4 Thpo Gspt1 Cxcl1 Plk2 Hus1 Vav1 Gas1 Mcm3 Rad1 Nfatc1 Sipa1 Gmnn Cdkn2b Zw10 Mapk1 Map2k7 Cdc2l2 Mcm7 1300002M12Rik Mapk13 Ing1 Trp73 Clk4 Cdc37 Tbrg4 Sfn E2f5 Araf Vegfa Cdk8 Clk3 Rad9 Akt1 Pard3 Mafg Msh2 Cdk8

245

Score (d)

q value (%)

4.7219 3.6927

0.1124 0.1124

3.2248 3.2244 3.1787 3.0393 2.8536 2.7588 2.5416 2.4079 2.3346 2.2434 2.1911 2.1604 2.1156 1.9430 1.7900

0.1124 0.1124 0.1124 0.1124 0.1124 0.1124 0.1124 0.2711 0.3256 0.4660 0.4660 0.4907 0.6753 1.0976 1.6052

1.7729 1.7592 1.7256 1.7173 1.6704 1.6173 1.6145 1.5807 1.5774 1.5484 1.5474 1.5456 1.5368 1.5340 1.5170 1.4931 1.4714 1.4677 1.4412 1.4114 1.5763 1.6463 1.6876 1.7185 1.7926 1.8369 1.8536 1.8984 1.9077 1.9651 1.9875 1.9932 2.1693 2.2361 2.4240 2.4562 2.5929 2.6739 3.2464 3.5252

1.6052 1.9178 1.9178 1.9178 2.2388 2.9767 2.9767 3.4760 3.4760 3.4760 3.4760 3.4760 3.8468 3.8468 3.8468 4.3740 4.3740 4.3740 4.8567 4.8567 4.8567 4.3740 3.8468 3.4760 2.9767 2.5724 2.2388 2.2388 1.9178 1.6052 1.4956 1.4956 0.9740 0.6753 0.3684 0.3684 0.2011 0.1124 0.1124 0.1124

3.9693 4.6121

0.1124 0.1124

Gene expression arrays were used to study Pemt / and wild type E17 adult brains. SAM was employed to identify differentially expressed genes. Genes in boldface were altered in both fetal and adult brains in same direction. Score (d) is assigned to each gene on the basis of change in gene expression relative to the standard deviation of repeated measurements. Genes with positive scores were over expressed in Pemt / ; those with negative scores were under expressed. q value is the lowest false discovery rate at which gene expression change is significant. Input parameters for SAM analysis and computed quantities were described in the text.

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2.10. Western blot Tissue was homogenized in Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA) and protein was extracted according to the manufacturer’s instruction. Protein content was measured [38] using bovine serum albumin (Sigma, St. Louis, MO) as the standard. The protein was diluted in SDS sample buffer, boiled for 5 min, loaded onto a gradient polyacrylamide/SDS gel, and transferred onto a nitrocellulose membrane. Then, the nitrocellulose membrane was blocked with 5% non-fat milk powder in phosphate-buffered saline (PBS) and incubated with rabbit polyclonal anti-PIP5K1A (Abgent, San Diego, CA, USA) at a dilution of 1:100, with rabbit polyclonal antiGNMT antibody at a dilution of 1:500, with mouse monoclonal anti-p21WAF1 (Oncogene, San Diego, CA, USA) at a dilution of 1:30, with mouse monoclonal antip53 (Oncogene, San Diego, CA, USA) at a dilution of 1:40, with rabbit polyclonal anti-MSH2 (Santa Cruz biotechnology, Santa Cruz, CA, USA) at a dilution of 1:30, and with rabbit polyclonal anti-PDK4 (Abgent, San Diego, CA, USA) at a dilution of 1:100 at 4 8C overnight. After extensive washing with PBS–Tween-20, the nitrocellulose membrane was incubated with HRP-conjugated donkey anti-rabbit IgG or sheep anti-mouse IgG (Amersham, Little Chalfont, Buckinghamshire, England) at room temperature at a dilution of 1:5000 for 45 min and visualized with a chemiluminescent reagent (Pierce; Rockford, IL, USA) and film (Kodak; Rochester, NY, USA). Molecular weight was calibrated using Benchmark molecular weight markers (Invitrogen Life Technologies, Carlsbad, CA, USA).

of Pemt / mice [80]. Genes regulating cell cycling were examined because of our previous observation of increased mitosis in brains of Pemt / mice [80]. In addition, PEMT expression negatively regulates cell division in rat liver and in cultured hepatoma cells [18,65]. Genes of neurogenesis and differentiation were expected to change because of our observation that markers of neuronal differentiation are perturbed in Pemt / and in choline-deficient mice [1,4,6,16,80]. Genes regulating apoptosis were expected to be differentially expressed because previously we had shown that choline deficiency induces apoptosis in mouse brain [16]. Genes of phospholipid metabolism were expected to change because synthesis of phosphatidylcholine is perturbed in Pemt / mice [69,79]. 3.1. Choline metabolism Gene expression for choline kinase, phosphocholine cytidylyltransferase 1, choline acetyltransferase, and betaine–homocysteine methyltransferase were not changed Table 9 Changes in expression of genes involved in apoptosis in Pemt wild type fetal mouse brains

Fetal

Symbol

Gene name

TP53

tumor protein p53 (Li–Fraumeni syndrome) cyclin-dependent kinase inhibitor 1A (P21) tumor necrosis factor receptor superfamily, member 5 Tnf receptor-associated factor 5 glutathione peroxidase 1 CASP8 and FADDlike apoptosis regulator programmed cell death 8 EGL nine homolog 3 (C. elegans) protein kinase C, alpha programmed cell death protein 7 caspase 6 baculoviral IAP repeat-containing 5 Cd27 binding protein (Hindu God of destruction) programmed cell death protein 11

Cdkn1a Tnfrsf5

Traf5

3. Results In fetal brain, of the 16,192 oligonucleotides analyzed on the array, 5645 unique genes were expressed and passed the filter selection, of which 107 were upregulated and 379 were down-regulated in Pemt / mice. Among the differentially expressed genes, positive and negative significant genes accounted for 22.0% and 78%, respectively. In adult brain, of the 16,192 oligonucleotides analyzed on the array, 5619 unique genes were expressed and passed the filter selection, of which 381 genes were up-regulated and 1037 genes were down-regulated in Pemt / mice. Among the differentially expressed genes, positive and negative significant genes account for 26.9% and 73.1%, respectively. Prior to the start of our studies, we prospectively identified groups of genes which we expected might vary in expression due to deletion of Pemt. We reasoned that genes of choline metabolism might be perturbed because of the lack of endogenous synthesis of choline [79]. Methyltransferases were expected to change because of the increased concentrations of AdoMet present in tissues

Gpx1 Cflar Pdcd8 Egln3 Prkca Pdcd7 Casp6 Birc5 Siva

Pdcd11

Score (d)

/

versus

q value (%)

2.7157

0.3451

2.4967

0.3451

2.1555

0.3451

1.7124

1.9542

1.5910 1.5806

3.1210 3.1210

1.5742 1.5126

3.1210 3.8009

1.4829 1.7116

4.2389 4.6644

1.7714 1.8133

3.8009 3.6355

2.0027

1.9542

2.0498

1.6879

Gene expression arrays were used to study Pemt / and wild type E17 fetal brains. SAM was employed to identify differentially expressed genes. Genes in boldface were altered in both fetal and adult brains in same direction. Score (d) is assigned to each gene on the basis of change in gene expression relative to the standard deviation of repeated measurements. Genes with positive scores were over expressed in Pemt / ; those with negative scores were under expressed. q value is the lowest false discovery rate at which gene expression change is significant. Input parameters for SAM analysis and computed quantities were described in the text.

X. Zhu, S.H. Zeisel / Molecular Brain Research 134 (2005) 239–255

in fetal or adult brains. Choline kinase gene expression was confirmed by RT-PCR (Fig. 1). 3.2. Phospholipid metabolism PEMT is the key enzyme for endogenous biosynthesis of phosphatidylcholine. It is known to be expressed in liver, and though there was expression in fetal and adult brain (Tables 3 and 4), this was not significantly changed in Pemt / fetal and adult brain. The false discovery rate for both values was high because the level of expression was low. The disrupted site of Pemt gene in Pemt / mice was not included in the oligonucleotides on the microarray but the probe for Pemt detected the RNA product. As expected, expression of some of the genes of phospholipid metabolism were altered. Using the GO phospholipid classification, in fetal brain out of 27 genes, 2 were under expressed and 0 over expressed; in adult brain 3 genes were over expressed and 5 under expressed (Tables 1 and 2). Using SAM analysis (Tables 3 and 4) phosphatidylinositol-4-phosphate 5-kinase, type 1 alpha (Pip5k1a), phosphatidylinositol membrane-associated, phos-

Table 10 Changes in expression of genes involved in apoptosis in Pemt Adult

/

pholipase A2, group VI (Pla2g6) were differentially expressed in both fetal and adult brains. In adult brain, phosphatidylserine synthase 1 (Ptdss1) expression was decreased. Decreased expression of Pip5k1a was confirmed by Western blot (Fig. 1). Expression of Ptdss1 was confirmed by real-time RT-PCR (Fig. 1). 3.3. Methyltransferases A number of methyltransferases were differentially expressed in brains of Pemt / mice (Tables 5 and 6). Using the GO methyltransferases classification, in fetal brain out of 46 genes, 2 were under expressed and 2 were over expressed; in adult brain 5 were under expressed, and 2 were over expressed (Tables 1 and 2). Using SAM analysis, glycine N-methyltransferase (Gnmt) was induced in both the fetal and adult brain. Protein expression and mRNA for Gnmt were analyzed by Western blot (Fig. 1) and real-time RT-PCR (Fig. 1), respectively, and both were increased in Pemt / brain, in agreement with microarray data.

versus wild type adult mouse brains

Symbol

Gene name

Muc2 Traf5 Tnfrsf5 Tnfsf6 Casp1 Pdcd8 Prss25 Fastk Gpx1 Casp8ap2 Birc1a Map3k5 Slc37a3

mucin 2 Tnf receptor-associated factor 5 tumor necrosis factor receptor superfamily, member 5 tumor necrosis factor (ligand) superfamily, member 6 caspase 1 programmed cell death 8 protease, serine, 25 Fas-activated serine/threonine kinase glutathione peroxidase 1 caspase 8 associated protein 2 baculoviral IAP repeat-containing 1a mitogen-activated protein kinase kinase kinase 5 Mus musculus clone TSAP7 p53-induced apoptosis differentially expressed mRNA sequence tumor protein p53 (Li–Fraumeni syndrome) programmed cell death 6 interacting protein baculoviral IAP repeat-containing 4 DNA fragmentation factor, beta subunit protein kinase, DNA activated, catalytic polypeptide tumor necrosis factor (ligand) superfamily, member 10 caspase 14 CASP8 and FADD-like apoptosis regulator Fas (TNFRSF6)-associated via death domain tumor necrosis factor receptor superfamily, member 12a BCL2/adenovirus E1B 19kDa-interacting protein 3-like death-associated protein 3 Bcl-associated death promoter programmed cell death protein 11 apoptosis regulator BCL-G WW domain-containing oxidoreductase

TP53 Pdcd6ip Birc4 Dffb Prkdc Tnfsf10 Casp14 Cflar FADD Tnfrsf12a Bnip3l Dap3 Bad Pdcd11 9030625M01Rik Wwox

247

Score (d)

q value (%)

3.1589 3.0206 2.7543 2.5393 2.3731 2.3534 2.3135 2.2318 2.0289 1.9459 1.9417 1.8862 1.7974

0.1124 0.1124 0.1124 0.1124 0.2711 0.3256 0.3684 0.4660 0.8268 1.0976 1.0976 1.3023 1.4956

1.7256 1.6026 1.5842 1.5738 1.5610 1.5407 1.4887 1.4879 1.6961 1.7184 1.7353 1.8040 1.8410 2.4094 2.5106 3.1732

1.9178 2.9767 2.9767 3.4760 3.4760 3.8468 4.3740 4.3740 3.8468 3.4760 3.4760 2.9767 2.5724 0.3684 0.3256 0.1124

Gene expression arrays were used to study Pemt / and wild type E17 adult brains. SAM was employed to identify differentially expressed genes. Genes in boldface were altered in both fetal and adult brains in same direction. Score (d) is assigned to each gene on the basis of change in gene expression relative to the standard deviation of repeated measurements. Genes with positive scores were over expressed in Pemt / ; those with negative scores were under expressed. q value is the lowest false discovery rate at which gene expression change is significant. Input parameters for SAM analysis and computed quantities were described in the text.

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3.4. Cell cycle regulation Tables 7 and 8 show the differentially expressed genes that are known to regulate cell cycling. Using the GO cell cycle classification, in fetal brain out of 393 genes, 15 were under expressed and 9 were over expressed; in adult brain 45 were under expressed and 23 were over expressed (Tables 1 and 2). Using SAM analysis, among the downregulated genes in both adult and fetal brain were tousledlike kinase 2 (Arabidopsis), tumor protein p53 (Li– Fraumeni syndrome), chemokine (C-X-C motif) ligand 1, fibroblast growth factor 4, and ubiquitin-conjugating enzyme E2C. Among the up-regulated genes in both adult and fetal brain were mutS homolog 2 (Escherichia coli) and inhibitor of growth family, member 1. In addition, cyclindependent kinase inhibitor 1A (P21) was down-regulated and cyclin-dependent kinase 4 was up-regulated in fetal Pemt / brain. Gene expression of mutS homolog 2 (E. coli), tumor protein p53 (Li–Fraumeni syndrome), and cyclin-dependent kinase inhibitor 1A were confirmed by both Western blot (Fig. 1) and real-time RT-PCR (Fig. 1), cyclin-dependent kinase 4 expression was confirmed by real-time RT-PCR (Fig. 1), and the results were in agreement with microarray data. 3.5. Apoptosis Using the GO apoptosis classification, in fetal brain out of 208 genes, 9 were under expressed and 5 were over expressed; in adult brain 24 were under expressed and 11 were over expressed (Tables 1 and 2). Using SAM analysis (Tables 9 and 10) we identified differentially expressed genes that are known to regulate apoptosis. Programmed cell death protein 11, programmed cell death 8, CASP8 and FADD-like apoptosis regulator, glutathione peroxidase 1, Tnf receptor-associated factor 5, tumor necrosis factor receptor superfamily, member 5, tumor protein p53 (Li– Fraumeni syndrome) were differentially expressed in Pemt / fetal and adult brains compared to wild type controls. Gene expression of tumor protein p53 (Li– Fraumeni syndrome) was confirmed by Western blot (Fig. 1). In addition, cyclin-dependent kinase inhibitor 1A (P21) was down-regulated in fetal Pemt / brain, and this was confirmed by both Western blot (Fig. 1) and real-time RT-PCR (Fig. 1). 3.6. Differentiation and neurogenesis Using the GO differentiation classification, in fetal brain out of 194 genes, 5 were under expressed and 6 were over expressed; in adult brain 26 were under expressed and 6 were over expressed. Using the GO neurogenesis classification, in fetal brain out of 199 genes, 15 were under expressed and 4 were over expressed; in adult brain 37 were under expressed and 7 were over expressed (Tables 1 and 2).

Table 11 Changes in expression of genes involved in differentiation and neurogenesis in Pemt / versus wild type fetal mouse brains Gene symbol

Fetal Cdh18

Gene name

cadherin 18 L1cam L1 cell adhesion molecule IL5 interleukin 5 (colonystimulating factor, eosinophil) Sema7a sema domain, immunoglobulin domain (Ig), and GPI membrane anchor, (semaphorin) 7A Efna5 ephrin A5 Sema3c sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3C Notch4 Notch gene homolog 4 (Drosophila) Cdh17 cadherin 17 Ccr1 chemokine (C-C motif) receptor 1 Calb2 calbindin 2 Mef2c myocyte enhancer factor 2C Cdh11 cadherin 11 STAT4 signal transducer and activator of transcription 4 A830016G23Rik cadherin 10 Hes5 hairy and enhancer of split 5 (Drosophila) Rorc RAR-related orphan receptor gamma/receptor S100a4 S100 calcium binding protein A4 Ascl2 achaete-scute complex homolog-like 2 (Drosophila) RARRES3 retinoic acid receptor responder (tazarotene induced) 3 Tlx1 T cell leukemia, homeobox 1 D14Wsu89e erythroid differentiation regulator Dll4 delta-like 4 (Drosophila) Pirb paired-Ig-like receptor A11 AI098020 ATP binding protein associated with cell differentiation IL13 interleukin 13

Score (d)

q value (%)

2.8237 0.3451 2.1638 0.3451 2.1547 0.3451

2.0373 0.7607

1.8383 1.3276 1.6940 2.3006

1.9135 1.0917 1.8668 1.3276 1.8333 1.3276 1.7099 1.9542 1.5918 3.1210 1.5886 3.1210 1.5496 3.6355 1.4998 4.2389 1.4808 4.2389 1.4690 4.6644 1.4559 4.6644 1.4515 4.6644

1.4399 4.6644

1.7434 4.2389 1.8085 3.8009 1.8141 3.6355 1.8457 3.1210 1.8745 2.6085

3.1398 0.3451

Gene expression arrays were used to study Pemt / and wild type E17 fetal brains. SAM was employed to identify differentially expressed genes. Genes in boldface were altered in both fetal and adult brains in same direction. Score (d) is assigned to each gene on the basis of change in gene expression relative to the standard deviation of repeated measurements. Genes with positive scores were over expressed in Pemt / ; those with negative scores were under expressed. q value is the lowest false discovery rate at which gene expression change is significant. Input parameters for SAM analysis and computed quantities were described in the text.

X. Zhu, S.H. Zeisel / Molecular Brain Research 134 (2005) 239–255

Using SAM analysis (Tables 11 and 12) we identified differentially expressed genes that are known to regulate neurogenesis and neural differentiation. S100 calcium binding protein A4, erythroid differentiation regulator, delta-like 4 (Drosophila), paired-Ig-like receptor A11, interleukin 13, hairy and enhancer of split 5 (Drosophila), ephrin A5, sema domain, immunoglobulin domain (Ig), and GPI membrane anchor, (semaphorin) 7A, L1 cell adhesion molecule were differentially expressed in Pemt / fetal and

adult brains in similar pattern compared to wild type controls. Calb2 (calretinin) expression was decreased in fetal brain. Gene expression of S100 calcium binding protein A4 was confirmed by real-time RT-PCR (Fig. 1), and the result was consistent with microarray data. Gene expression of calretinin was measured in fetal brain by real-time RT-PCR in our previous report [80], and it was consistent with microarray data.

Table 12 Changes in expression of genes involved in differentiation and neurogenesis in Pemt Adult

/

versus wild type adult mouse brains

Gene symbol

Gene name

Etd Cbfb S100a4 S100a5 Myf5 Xdh Rqcd1 AW319487 Nhlh1 Traf6 Myd88 Dll4 Pappa Lgals1 Lifr Pirb D14Wsu89e IL13 Sema7a

embryonic testis differentiation core binding factor beta S100 calcium binding protein A4 S100 calcium binding protein A5 myogenic factor 5 xanthine dehydrogenase rcd1 (required for cell differentiation) homolog 1 (S. pombe) myocyte induction differentiation originator nescient helix loop helix 1 Tnf receptor-associated factor 6 myeloid differentiation primary response gene 88 delta-like 4 (Drosophila) pregnancy-associated plasma protein A lectin, galactose binding, soluble 1 leukemia inhibitory factor receptor paired-Ig-like receptor A11 erythroid differentiation regulator interleukin 13 sema domain, immunoglobulin domain (Ig), and GPI membrane anchor, (semaphorin) 7A hairy and enhancer of split 5 (Drosophila) L1 cell adhesion molecule Notch gene homolog 4 (Drosophila) ephrin A5 sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5A NK2 transcription factor related, locus 2 (Drosophila) achaete-scute complex homolog-like 2 (Drosophila) peroxisomal membrane protein 3 nucleoporin 50 single-minded 2 numb-like sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5B epilepsy, progressive myoclonic epilepsy, type 2 gene alpha neurogenin 2 transcription factor AP-2 beta (activating enhancer binding protein 2 beta) galanin receptor 2 sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3E neuropilin 2 Down syndrome cell adhesion molecule zinc finger homeobox 1b

Hes5 L1cam Notch4 Efna5 Sema5a

Nkx2-2 Ascl2 Pxmp3 Nup50 Sim2 Numbl Sema5b

Epm2a Neurog2 TFAP2B Galr2 Sema3e Nrp2 Dscam ZFHX1B

249

Score (d)

q value (%)

3.2287 2.7825 2.6939 2.4434 1.9571 1.8660 1.7973 1.7653 1.6230 1.4098 1.8228 1.8372 2.0084 2.2333 2.4183 3.6209 3.6806 5.9996 4.4004

0.1124 0.1124 0.1124 0.2011 0.9740 1.3023 1.4956 1.6052 2.9767 4.8567 2.5724 2.5724 1.4956 0.6753 0.3684 0.1124 0.1124 0.1124 0.1124

3.8904 3.3055 2.9742 2.7324 2.5896

0.1124 0.1124 0.1124 0.1124 0.1124

2.2642 2.0671 2.0452 2.0144 1.9557 1.9419 1.8590

0.3684 0.8268 0.8268 0.9740 0.9740 1.0976 1.3023

1.8579 1.8443 1.5268

1.3023 1.3023 3.8468

1.4949 1.4233

4.3740 4.8567

1.4160 2.3514 2.4760

4.8567 0.4660 0.3256

Gene expression arrays were used to study Pemt / and wild type E17 adult brains. SAM was employed to identify differentially expressed genes. Genes in boldface were altered in both fetal and adult brains in same direction. Score (d) is assigned to each gene on the basis of change in gene expression relative to the standard deviation of repeated measurements. Genes with positive scores were over expressed in Pemt / ; those with negative scores were under expressed. q value is the lowest false discovery rate at which gene expression change is significant. Input parameters for SAM analysis and computed quantities were described in the text.

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Table 13 Changes in expression of genes known to be regulated by methylation in Pemt

Fetal

/

versus wild type fetal mouse brains

Gene symbol

Gene name

Score (d)

Cdkn1a TP53 Cdkn2a S100a4 Brca1 Tfpi2 Msh2 Pax6

cyclin-dependent kinase inhibitor 1A (P21) tumor protein p53 (Li–Fraumeni syndrome) cyclin-dependent kinase inhibitor 2A S100 calcium binding protein A4 breast cancer 1 tissue factor pathway inhibitor 2 mutS homolog 2 (E. coli) paired box gene 6

2.4967 1.4757 1.4563 1.4559 1.7858 1.8735 2.3100 2.4788

q value (%)

Reported change with methylation and references

0.3451 4.2389 4.6644 4.6644 3.8009 2.6085 0.5970 0.3451

Down [52] Down [50] Down [56,67] Down [45,54] Down [41] Down [25,34] Down [71] Variable [7,57,63]

Gene expression arrays were used to study Pemt / and wild type E17 fetal brains. SAM was employed to identify differentially expressed genes. Genes in boldface were altered in both fetal and adult brains in same direction. Score (d) is assigned to each gene on the basis of change in gene expression relative to the standard deviation of repeated measurements. Genes with positive scores were over expressed in Pemt / ; those with negative scores were under expressed. q value is the lowest false discovery rate at which gene expression change is significant. The direction of change in gene expression with increased methylation, as reported in the indicated references, is included in the last column. Input parameters for SAM analysis and computed quantities were described in the text.

3.7. Genes regulated by methylation

3.8. Other miscellaneous genes

Increased AdoMet concentration and DNA hypermethylation observed in Pemt / fetal brain [80] should result in the silencing or induction of genes known to be regulated by methylation of their promoter region [48]. Tables 13 and 14 show altered expression of genes previously described to be regulated by methylation. As expected, many of these genes are suppressed by methylation, among them S100 calcium binding protein A4 and tumor protein p53. Gene expression of mutS homolog 2 (E. coli) and cyclindependent kinase inhibitor 1A was confirmed by both Western blot (Fig. 1) and real-time RT-PCR (Fig. 1). Expression of tumor protein p53 was confirmed by Western blot. Expression of the S100 calcium binding protein A4 gene was confirmed by real-time RT-PCR (Fig. 1).

Pyruvate dehydrogenase kinase, isoenzyme 4 was downregulated in both fetal and adult brain (in adult brain d= 11.7138, q=0.1124; in fetal brain d= 4.5124, q=0.3451) and these large expression changes were confirmed by both Western blot (Fig. 1) and real-time RTPCR (Fig. 1), the result was consistent with the microarray data. Statistical analysis using Fisher’s exact test revealed significant changes within other GO classes (Tables 1 and 2). These included gene ontology categories of antigen binding, organismal physiological process, immune response, defense response, response to stimulus, and calcium ion binding that were most significantly changed in Pemt / fetal and adult brain.

Table 14 Changes in expression of genes known to be regulated by methylation in Pemt

Adult

/

versus wild type adult mouse brains

Gene symbol

Gene name

Muc2 MLH1 S100a4 Cftr Fgf4 Mgmt Rarb Cdh13 TERT Nf2 TP53 Tgfbr1 Esr1 Cdkn2b Sfn Msh2

mucin 2 mutL homolog 1, colon cancer, nonpolyposis type 2 (E. coli) S100 calcium binding protein A4 cystic fibrosis transmembrane conductance regulator homolog fibroblast growth factor 4 O-6-methylguanine-DNA methyltransferase retinoic acid receptor, beta heat shock factor binding protein 1 telomerase reverse transcriptase neurofibromatosis 2 tumor protein p53 (Li–Fraumeni syndrome) transforming growth factor, beta receptor I AJ272161 cyclin-dependent kinase inhibitor 2B (p15, inhibits CDK4) stratifin mutS homolog 2 (E. coli)

Score (d) 3.1589 2.8536 2.6939 2.5510 2.2434 1.9700 1.8523 1.8206 1.8199 1.7592 1.7256 1.7108 1.6838 1.4677 1.9875 3.9693

q value (%)

Reported change with methylation and references

0.1124 0.1124 0.1124 0.1124 0.4660 0.9740 1.3023 1.4956 1.4956 1.9178 1.9178 1.9178 2.2388 4.3740 1.4956 0.1124

Down Down Down Down Down Down Down Down Down Down Down Down

[23] [36,61] [45,54] [32] [14] [9] [20] [53] [30,59] [31] [50] [49]

Down [73] Down [10,62] Down [71]

Gene expression arrays were used to study Pemt / and wild type E17 adult brains. SAM was employed to identify differentially expressed genes. Genes in boldface were altered in both fetal and adult brains in same direction. Score (d) is assigned to each gene on the basis of change in gene expression relative to the standard deviation of repeated measurements. Genes with positive scores were over expressed in Pemt / ; those with negative scores were under expressed. q value is the lowest false discovery rate at which gene expression change is significant. The direction of change in gene expression with increased methylation, as reported in the indicated references, is included in the last column. Input parameters for SAM analysis and computed quantities were described in the text.

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4. Discussion We previously reported that, in the hippocampus of this Pemt knockout mouse, we observed increased mitosis in neural progenitor cells, decreased expression of calretinin, increased concentrations of AdoMet, and increased methylation of DNA and histones [80]. We now investigated changes in gene expression in Pemt / fetal and adult brains. The expression of a large number of genes was changed in both adult and fetal Pemt / mice compared with wild type mice (indicated in boldface in tables). We compared expression between knockouts and wild type mice that were selected from different litters, and we recognize that the individual dam’s behaviors and metabolism might differ depending on genotype. The concordance between the fetal and adult knockout brains provided a high degree of confidence in our overall data set. In addition, there were numerous genes that had changed expression in the fetal or adult brain alone. In all, expression of more than 1000 genes was changed in the fetus, and more than 3000 genes were changed in the brains of adults. PEMT catalyzes the synthesis of an important phospholipid, phosphatidylcholine. Phosphatidylcholine is a source of choline moiety, and can spare the dietary requirement for choline [78]. Thus, deletion of the Pemt gene could be expected to alter the expression of genes associated with phospholipids or choline metabolism. We did not observe differential expression of any of the major genes associated with choline metabolism. In this study we examined brain tissue, yet the most active organ for metabolism and synthesis of choline is the liver [78]; perhaps if we had studied liver we might have observed changes in these genes. We did observe significant changes in the expression of several genes associated with phospholipid metabolism. Phosphatidylinositol biphosphate is an important signaling compound in membranes [26]. In both fetal and adult Pemt / brain, we observed changes in the expression of two genes involved in phosphatidylinositol metabolism (Pip51ka and Pitpnm; Tables 3 and 4). Phospholipase A2 (PLA2) belongs to a family of enzymes that catalyze the cleavage of fatty acids from the sn-2 position of phospholipids. PLA2s acting on membrane phospholipids have also been implicated in intracellular membrane trafficking, differentiation, proliferation, and apoptotic processes [35,60]. In adult brain, we observed down-regulation of phosphatidylserine synthase 1 (Pss1) which produces phosphatidylserine by exchanging serine for the head groups of other phospholipids. Pss1 uses phosphatidylcholine for this serine exchange reaction [8]. Thus, the observed suppression of expression of this gene might be due to the decreased substrate phosphatidylcholine available for the exchange reaction. PEMT is a methyltransferase, and its deletion results in increased availability of S-adenosylmethionine (AdoMet) as a substrate for other methyltransferases [80]. Glycine Nmethyltransferase (GNMT) acts to waste AdoMet and thus

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regulates its availability for other methylations [39,44,55, 76,77]. GNMT is mainly distributed in liver, kidney, and pancreas [76]. In wild type brain, constitutive expression of GNMT was small, but in Pemt / brain expression was significantly increased (Fig. 1, Tables 5 and 6). It is possible that increased AdoMet levels also modulate GNMT gene expression in brain. As discussed earlier, we previously observed that proliferation of neural precursor cells was increased in Pemt / fetal brain [80]. We never examined cell proliferation in adult brain. In fetal brain from Pemt / mice, we observed inhibition of expression of cyclin-dependent kinase inhibitor 1A (p21; Table 7). Cdkn1a functions as an inhibitor of D cyclin–cdk4 activity and it induces G1/S arrest [51]. In fetal brain, we also saw decreased expression of other cyclin-dependent kinase inhibitors (cyclin-dependent kinase inhibitor 2A) and increased expression of cyclindependent kinase 2 associated protein (Table 7). All of the above would act to enhance progress through the cell cycle. In both fetal brain and adult brain from Pemt / mice, we observed decreased expression of tumor protein p53 (TP53; Tables 7 and 8) which can induce both G1 and G2/M arrest, in a p21-dependent manner in cell culture [33]. Up-regulated Msh2 was observed in both fetal and adult Pemt / brain. Msh2 mediates mismatch repair in eukaryotic cells, the absence of which increases the frequency of mutation [29]. Also, Msh2 is a key component of an earlyacting DNA damage-activated G2 checkpoint [29], Msh2 deficiency can result in the premature commitment to mitosis [40]. These changes in gene expression may explain why we observed increased progenitor cell proliferation in the brains of Pemt / fetuses. We previously reported that choline deficiency resulted in increased apoptosis in cultured neurons, hepatocytes, and rat liver [3,75]. In Pemt / brain, we observed changes in expression of a number of genes involved in apoptosis. P53 was down-regulated in both fetal and adult Pemt / brain and p21 was down-regulated in fetal Pemt / brain. As discussed earlier, p53 is an inducer of both cell cycle arrest and apoptosis; the transition to apoptosis is mediated by p21. Like p53 and p21, a number of genes changed in Pemt / brain have dual roles in both cell cycle progression and apoptosis. For instance, ubiquitin-conjugating enzyme E2C was down-regulated in both fetal and adult Pemt / brain. Recent studies found that ubiquitination regulated many molecules of the cell death machinery, such as p53, caspases, and Bcl-2 family members [74]. We previously reported that expression of a protein associated with neural cell differentiation (calretinin) was decreased in fetal brain of Pemt / mice [80]. We now show that expression of the gene for calretinin (Calb2) is significantly decreased in fetal brain of Pemt / mice (Table 11). Decreased expression of calretinin might contribute to changes in synaptic plasticity in mouse brain [58]. In both adult and fetal brain from Pemt / mice, we

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observed decreased expression of S100A4. The oligomeric form of S100A4 induces differentiation of cultured hippocampal neurons [46]. In addition, expression of the gene for L1 cell adhesion molecule was down-regulated in both fetal and adult Pemt / brain; this protein plays an important role in cell proliferation and migration, and in neuritic elongation and guidance [22,66]. Mutations of L1 cell adhesion molecule in humans produce a spectrum of neurologic abnormalities and mental retardation [72]. These data suggest that differentiation of neural cells in Pemt / fetal brain is perturbed and is in agreement with our earlier study using immunohistochemical approaches [80]. As noted earlier, the increased AdoMet levels and DNA hypermethylation observed in Pemt / fetal brain [80] should result in the silencing or induction of genes known to be regulated by methylation of their promoter region [28,48]. We attempted to identify, from the literature, genes that are known to have expression regulated by methylation (Tables 13 and 14). Our data are consistent with the hypothesis that increased brain AdoMet concentrations in brains of Pemt / mice increased DNA methylation and altered the expression of many genes. In fetal and adult brain (Tables 13 and 14), decreased expression of TP53 and S100a4 was consistent with that predicted by the literature. However, Msh2 was induced in both fetal and adult brain. We know that genes have multiple CpG sites, and that there can be differential methylation of hotspots in the promoter regions [19]. It is possible that critical CpG sites were not over methylated in this gene. In addition, genes that are normally highly methylated genes may not be more methylated in Pemt / mice. Finally, genes are also regulated by binding transcription factors to sites other than methylatable CpG sites. In that case, changes in methylation could be overridden. There were remarkable changes (in terms of magnitude) among several genes. Sema domain, immunoglobulin domain (Ig), and GPI membrane anchor, (semaphorin) 7A (Sema7a) gene expression in both fetal and adult Pemt / brain, and Sema5a gene expression in adult Pemt / brain (Tables 11 and 12) were significantly decreased. Semaphorins/collapsins, a family of genes with a semaphorin domain conserved from insects through to mammals, are involved in axon guidance during neuronal development [42,43]. Unlike many other semaphorins, which act as repulsive guidance cues, Sema7A enhances central and peripheral axon growth and is required for proper axon tract formation during embryonic development [47]. Interleukin 13 (IL-13) expression increased significantly in both fetal and adult Pemt / brain (Tables 11 and 12). IL-13 acts as a proinflammatory cytokine in the brain [12]. WW domain-containing oxidoreductase (Wwox) gene expression was significantly increased in adult Pemt / brain (Table 10). WOX1 is a proapoptotic protein that is differentially distributed in developing murine nervous system and may participate in multiple signaling pathways involved in the neuronal development [15]. Suppressor of variegation 3–9 homolog

1 (Suv39h1) gene expression in adult Pemt / brain was significantly decreased (Table 6). Suv39h2 and Suv39h1 govern methylation of histone H3 Lys9 (H3-Lys9) in heterochromatic regions. Suv39h2 plays an important role in epigenetic regulation of telomere length in mammals [21]. Contactin 1 (Cntn1) gene expression in fetal Pemt / brain was significantly decreased (Table 5). Cntn1 and Cntn2 are closely related axonal glycoproteins that are differentially regulated during development and the precise spatiotemporal regulation of Cntn2 and Cntn1 expression is critical for normal cerebellar morphogenesis [11]. We note that there were very large changes in expression of pyruvate dehydrogenase kinase, a gene of glucose metabolism. This interesting change has no clear mechanism at this time. Also, in Table 1, we present most significantly changed and selective gene categories identified by GoMiner analysis. Genes involved in antigen binding, immune response, and defense response were significantly altered in fetal and adult Pemt / brain (Table 1). Genes involved in G-protein-coupled signaling were also significantly under expressed in Pemt / fetal and adult brain (Tables 1 and 2). It should be emphasized that since Pemt ablation causes multiple changes in brain such as hypermethylation, increased AdoMet, increased progenitor cell mitosis, and decreased calretinin, the alteration in gene expression may be attributed to direct Pemt regulation, to changes of upstream genes (e.g., transcriptional factors), or to other physiological, cellular, and molecular abnormalities associated with Pemt ablation. Therefore, not all of the altered genes should be considered as direct Pemt target genes. Further investigations are needed to fully understand the molecular bases underlying brain development abnormalities seen as a consequence of Pemt ablation, and the gene expression data presented in this study may have provided a foundation for such investigations.

Acknowledgments Supported by the National Institutes of Health (DK55865, AG09525). Support for this work was also provided by grants from the NIH to the UNC Clinical Nutrition Research Unit (DK56350) and the Center for Environmental Health (ES10126). We thank Dr. Charles M. Perou for advice and technical support in SAM analysis. We thank Dr. Dennis E. Vance for kindly providing us with Pemt / mice. We thank Dr. Conrad Wagner for kindly providing us with GNMT antibody.

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