| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Journal of Lipid Research, Vol. 44, 2311-2319, December 2003
Copyright © 2003 by American Society for Biochemistry and Molecular Biology
,
* Department of Biochemistry, University of Iowa, Iowa City, IA 52242
Department of Radiation Oncology, University of Iowa, Iowa City, IA 52242
The Holden Comprehensive Cancer Center, University of Iowa, Iowa City, IA 52242
Published, JLR Papers in Press, September 1, 2003. DOI 10.1194/jlr.M300273-JLR200
1 To whom correspondence should be addressed. e-mail: frederick-domann{at}uiowa.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
These findings indicate that fatty acid delta-6-desaturase deficiency and decreased FADS2 transcription are caused by a nucleotide insertion in the transcriptional regulatory region of the human FADS2 gene.
Abbreviations: C/EBP, CCAAT enhancer binding protein; DF, delta-6-desaturase deficient human skin fibroblasts; DHA, docosahexaenoic acid; FADS2, fatty acid delta-6-desaturase gene; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NF, normal human skin fibroblasts; SCD, stearoyl-CoA-desaturase (delta-9-desaturase); SC5D, sterol C5-desaturase-like (delta-5-desaturase)
Supplementary key words fatty acid metabolism • polyunsaturated fatty acid • gene expression • insertion polymorphism • CCAAT enhancer binding protein
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
We previously identified a female patient with an inborn error of lipid metabolism in which cellular delta-6-desaturase enzyme activity was deficient compared with cells from normal individuals (5). This patient developed clinical symptoms characteristic of essential fatty acid deficiency shortly after birth, and GLC analysis of her plasma fatty acid composition indicated low levels of both arachidonic acid and DHA. We demonstrated that cultured skin fibroblasts obtained from this patient converted 85% to 95% less [1-14C]linoleic acid to arachidonic acid than corresponding normal fibroblast cultures (5). Our results also indicated that the conversion of radiolabeled PUFA precursors to DHA was markedly reduced in the patient's fibroblasts. This patient's clinical symptoms, including corneal ulceration, feeding intolerance, growth failure, marked photophobia, and skin abnormalities all significantly improved when her diet was supplemented with black currant seed oil and fish oil, subsequently replaced by a mixture of 20:4n-6 and DHA (5).
The human delta-6-desaturase gene was recently cloned by searching the human expressed sequence tag database for a human homolog cDNA with the nucleotide sequence of mouse liver delta-6-desaturase (6). Marquardt et al. successfully identified three members of the human fatty acid desaturase gene family (FADS1, FADS2, and FADS3) by direct selection of cDNA fragments within a 1.4 mb region in chromosome 11q12-q13.1 (7). FADS1 and FADS2 both encode proteins of 444 amino acids and were found to share 61% nucleotide sequence identity. In addition, the FADS2 cDNA extends approximately 140 nt upstream in the 5' untranslated region (UTR) (7). Although the precise function of FADS3 remains unknown, this gene encodes a 445 amino acid protein that is highly homologous to FADS1 and FADS2 and which contains all the motifs common to desaturase enzymes. FADS1 and FADS2 encode predicted peptides with two membrane-spanning domains and a cytochrome b5-like domain characteristic of nonmammalian delta-6-desaturases. This evolutionarily conserved sequence structure is suggestive of the crucial role played by delta-6-desaturase in PUFA biosynthesis. This is supported by the fact that it is ubiquitously expressed, especially in brain and liver, as well as in heart, skeletal muscle, testes, kidney, lung, prostate, ovary, and adipose tissue (6, 7).
It was previously reported that delta-6 fatty acid desaturation is defective in the Sjogren-Larsson syndrome (OMIM #270200), a human autosomal recessive disease characterized by a deficiency in the oxidation of fatty alcohols to fatty acids (8). However, the fatty aldehyde dehydrogenase activity in the delta-6-desaturase deficient patient's fibroblasts was 20 times higher than those reported for the Sjogren-Larsson syndrome (9, 10), indicating that this was not the cause of the delta-6-desaturase deficiency in this patient. Thus, this appears to be the first observation of a defect in human PUFA metabolism associated with decreased expression of the delta-6-desaturase gene. In the present study, we have determined the mechanism of this decrease at the molecular level.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
RNA isolation and RT-PCR
Total RNA was isolated from 85–90% confluent cells using the Qiagen RNeasy kit (Qiagen, Chatsworth, CA). Quantitation was by absorbance measurement at 260 nm with a DU-64 spectrophotometer (Beckman Instruments Inc., Fullerton, CA).
Reverse transcription employed the Superscript kit (Gibco-BRL/Life Technologies, Grand Island, NY). One microgram of total RNA from each sample was used for synthesis of first strand, single-stranded cDNA in the presence of 200 U Superscript II reverse transcriptase under the reaction conditions stipulated by the manufacturer. The PCR reaction was performed with 1.5–2.0 µl of the first strand cDNA reaction product in a total of 50 µl reaction mixture. Included in the reaction mixture were: 20 mM Tris-Cl buffer (pH 8.4); 50 mM KCl; 0.2 mM of each of the four deoxynucleotide triphosphates; 4 mM MgCl2; 2.5 U Amplitaq DNA polymerase (Perkin-Elmer Corporation, Foster City, CA); and each specific pair of primers at a final concentration of 0.2 µM. A 3 min denaturation step was performed at 94°C (hot start PCR), and then 30 amplification cycles consisting of denaturation for 1 min at 94°C, annealing for 1 min at 52°C, and elongation for 1.5 min at 72°C (FADS2 coding sequence fragments), or annealing at 60°C and elongation for 2.5 min at 72°C (FADS2 3'- and 5'-UTR sequence fragments), were performed using the Perkin Elmer thermocycler (GeneAmp PCR System 2400).
The FADS2 coding sequence was amplified in two fragments using the primers: sense 5'-ATGGGGAAGGGAGGGAAC-3' (positions 13 to 31 of gene sequence, GenBank Accession number NM 004265), antisense 5'-AGGTAGGCGTCCAGCCAC-3' (positions 1,338 to 1,321) for an amplification product of 1,326 bp as the first fragment, and sense 5'-CCCGCTGGTGAAGTCTCTAT-3' (positions 1,213 to 1,232), antisense 5'-GCCACCTCTCTGCTCCTT-3' (positions 1,625 to 1,608) amplification product of 413 bp for the second fragment. Primers for the 3'-UTR region were sense 5'-AAGGAGCAGAGAGGTGGC-3' (positions 1,608 to 1,625) and antisense 5'-AATGTAGGGGTGGGGAAGT-3' (positions 2,986 to 2,968), giving a product of 1,379 bp; while those for the 5'-UTR sequence were sense 5'-GCCAGTTCCTCATCGCCCCC-3' (positions -1,047 to -1,028, human chromosome 11q12.2 gene sequence, GenBank Accession number AC004228) and antisense 5'-TCCCTTCCCCATGCTGCCTG-3' (positions +12 to -8) for a product of 1,059 bp. Primers for FADS2 (delta-6-desaturase) (6), SC5D (delta-5-desaturase) (15), SCD (delta-9-desaturase) (16), and the house-keeping gene glyceraldehyde 3-phosphate dehydrogenase (GADPH) (17) were obtained according to the published information. GAPDH transcript levels were assessed at the same time as those of SCD and FADS2 to serve as positive controls in the RT-PCR experiments and to enable semiquantitation of product bands. Amplification products were analyzed in a 1.2% agarose gel and stained with ethidium bromide, and semiquantitative analysis of band intensities was determined by image analysis using the AlphaImagerTM 950 Documentation and Analysis System (Alpha Innotech Corporation, San Leandro, CA).
Northern blot analysis
Ten micrograms of total RNA from each sample was separated by electrophoresis in 1.2% agarose/2.2 M formaldehyde gels, transferred to nylon membranes (GeneScreen, New England Nuclear, Boston, MA) in a 10x saline-sodium citrate solution, and cross-linked by exposure to ultraviolet light (Stratalinker 1800, Stratagene, LA Jolla, CA). cDNA probes for FADS2 and GAPDH were 204 and 876 base pair sequences, respectively, synthesized by RT-PCR using published primers as detailed previously. Sequences were cloned using a TA Cloning kit (Qiagen Inc., Valencia, CA). The sequences of the cloned cDNAs were confirmed by sequence analysis in the University of Iowa DNA Core Laboratory. The plasmids were restricted with EcoRI, and the inserted cDNA fragments were purified from 1.5% agarose gels using a Qiagen gel extraction kit. The gel-purified cDNAs were 32P-labeled using a Prime-it II random priming kit (Stratagene) according to the manufacturer's instructions.
Northern blots were prehybridized and hybridized (overnight) at 42°C in 10 ml of hybridization solution containing 10% dextran sulfate, 49.0% formamide, 5x saline-sodium phosphate-EDTA (SSPE), 0.5% SDS, 2x Denhardt's reagent, and 0.1 mg/ml sheared salmon sperm DNA. Blots were initially washed with 2x SSPE containing 0.1% SDS and subsequently with 0.1% SSPE at 50°C before exposing to X-ray film at -80°C. The FADS2 probe detected a predominant band at 2.9 kb. Careful inspection of the autoradiograms indicated the presence of additional minor transcripts at 3.3 and 1.7 kb, but these bands were very faint and accounted for only a very small percentage of the radioactivity. Following autoradiography with the FADS2 probe, each blot was stripped according to the manufacturer's instructions and probed again with the 32P-labeled GAPDH sequence to enable relative quantification of the FADS2 transcripts.
mRNA stability analysis
The normal and delta-6-desaturase-deficient fibroblasts were grown to 80% confluence in T-75 flasks and then were incubated with 10 µg/ml actinomycin D to arrest new RNA synthesis. Cells were harvested for RNA extraction at 0 h, 4 h, 8 h, 12 h, 16 h, and 20 h after treatment with actinomycin D. Northern analysis was performed with isolated RNA as described previously to determine the level of FADS2 and GAPDH transcripts at the indicated time-points. FADS2 mRNA levels were normalized to GAPDH levels, and transcript half lives were determined from plots of band intensities versus time.
Southern blot analysis
DNA was extracted from confluent normal and delta-6-desaturase-deficient fibroblasts in T-75 flasks using the Qiagen DNeasy kit. Fifteen micrograms of DNA from each cell strain was restricted with HindIII and EcoRI endonucleases separately and in combination for 16 h, and the digested fragments were size fractionated by electrophoresis in a 0.8% agarose gel in 1x Tris-borate-EDTA (TBE) (Tris, 89 mM; boric acid, 89 mM; EDTA, 2 mM) buffer, pH 8.0. The gel was soaked in 0.25 M HCl for 30 min with gentle shaking before soaking twice for 20 min each in 500 ml denaturation solution containing 1.5 M NaCl and 0.5 M NaOH. This was followed by 500 ml neutralization solution containing 1.5 M NaCl and 0.5 M Tris-HCl (pH 7.0). DNA was transferred from the gel to a nitrocellulose filter by blotting in 10x saline sodium citrate (SSC) and cross-linked by exposure to UV light in a Stratalinker as described above for Northern blots. The filters were prehybridized and hybridized (overnight) at 42°C in 10 ml of hybridization solution (10% dextran sulfate, 49.0% formamide, 5x SSPE, 0.5% SDS, 2x Denhardt's reagent, and 0.1 mg/ml sheared salmon sperm DNA) containing the 32P-labeled FADS2 probe to identify bands containing FADS2 gene sequences. Blots were initially washed with 2x SSPE containing 0.1% SDS and subsequently with 0.1% SSPE at 50°C before exposing to X-ray film at -80°C.
In vitro transcription assay
The method of Tetradis (18) as modified (19) was employed for preparing nuclei from cells and the subsequent labeling of RNA transcripts with 32P-UTP. Briefly, confluent normal and delta-6-desaturase deficient fibroblasts in T-75 flasks were rinsed once with PBS, collected by scraping into PBS, and harvested by centrifugation at 1,500 g for 2 min. The cell pellet from each flask was resuspended in 1 ml ice-cold hypotonic buffer [10 mM KCl, 10 mM Tris-HCl (pH 7.5), 1.5 mM MgCl2, 0.3 M sucrose, 0.25% NP-40, 1 mM DTT, and 0.5 mM phenylmethoxysulfonyl fluoride (PMSF)] and gently disrupted in a Dounce homogenizer with pestle B (Kontes Scientific Glassware, Vineland, NJ). The nuclear pellet was obtained by centrifugation of the lysate for 15 min at 1,000 g and then immediately resuspended in 200 µl of transcription reaction buffer [50 mM Tris-HCl (pH 7.5), 0.1 M ammonium sulfate, 1.8 mM DTT, 1.8 mM MnCl2, 2 µl RNasin, 300 µM each of ATP, CTP, GTP, and 100 µCi of [32P]UTP]. Transcription was carried out for 30 min at room temperature and terminated by the addition of 5 µl RNase-free DNase and 2 µl of tRNA (20 mg/ml). After 15 min, 20 µl of 10% SDS and 2 µl proteinase K (0.1 mg/ml) were added, and the incubation was continued for 45 min at 37°C. RNA was then extracted by the Qiagen RNeasy method. Five micrograms of cDNAs prepared from the genes of interest (SC5D, FADS2, and GAPDH) were obtained by PCR as previously described and blotted onto prepared nitrocellulose membranes.
Membranes were prehybridized for 1 h at 52°C in 3 ml of a solution containing 49.3% formamide, 4.93x SSC, 0.1% SDS, 1 mM EDTA, 10 mM Tris-HCl (pH 7.5), 4x Denhardt's solution, 0.34 mg/ml yeast tRNA, and 0.34 mg/ml sheared salmon sperm DNA. Radioactive RNA (107 cpm) was added to each vial containing membranes from the different cells, and the membranes were hybridized at 52°C for 72 h in a solution containing 58% formamide, 5.8x SSC, 0.12% SDS, 1.2 mM EDTA, 12 mM Tris-HCl (pH 7.5), 4.7x Denhardt's solution, 0.4 mg/ml yeast tRNA, and 0.4 mg/ml sheared salmon sperm DNA. Membranes were then washed twice at 65°C for 1 h in 25 ml of 2x SSC, air-dried, and exposed to X-ray film at -80°C.
Nuclear protein extraction and electrophoretic mobility shift assays
Confluent NIH/3T3 mouse embryo fibroblasts in T-75 flasks were harvested by scraping in 0.5 ml cold buffer A [10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, and 0.5 mM DTT]. Cells were incubated on ice for 30 min, lysed using a Dounce homogenizer, and centrifuged for 30 s at 500 g. The supernatant was removed and the cell pellet resuspended in 15 µl cold buffer C [20 mM HEPES (pH 7.9), 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and 0.5 mM DTT]. The suspended nuclei were incubated on ice for 30 min and centrifuged at 10,000 g for 5 min at 4°C. The supernatant was harvested and diluted 1:3 in cold buffer D [20 mM HEPES (pH 7.9), 20% glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.5 mM PMSF, and 0.5 mM DTT]. The protein concentration of the nuclear extracts was determined by the Bradford method.
For the electrophoretic mobility shift assays, double-stranded synthetic oligodeoxynucleotides encompassing the mutated and normal CCAAT enhancer binding protein (C/EBP) consensus sites from positions -949 to -934 of the human delta-6-desaturase upstream regulatory region were used as 32P-labeled probes in the gel shift assays. The oligos for the normal and mutant sequences were therefore, respectively, as follows:
5'-AGCTTCTTTTCAAGATTG-3'
3'-AGAAAAGTTCTAACCTAG-5'
5'-AGCTTCTTTTCTAAGATTG-3'
3'-AGAAAAGATTCTAACCTAG-5'
For the gel shift assays, probes were 32P-labeled on the 5' overhangs with Klenow DNA polymerase in the presence of [
32P] dCTP. Nuclear protein (10 µg) was incubated at room temperature for 20 min with 1 µg poly dIdC (Pharmacia) and 500,000 cpm of the probes per binding reaction in 1x gel shift buffer [10 mM Tris (pH 7.5), 50 mM NaCl, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, and 4% glycerol]. The bound DNA-protein complexes were separated from free probe by gel electrophoresis in 5% native polyacrylamide gels in 1x TBE. Electrophoresis was conducted at a constant current of 25 mA for 1.5 h. The gel was wrapped in plastic wrap and exposed to X-ray film at -80°C overnight.
Plasmid construction, transfections, and reporter assays
To create the 1 kb human FADS2 promoter-pGL3 basic luciferase reporter vectors, the FADS2 promoters from position -1,047 to +12 relative to the translation initiation site were PCR amplified from genomic DNA from normal and delta-6-desaturase deficient fibroblasts. The sequence from mutant fibroblasts harbored an insertion of a thymidine nucleotide between positions -941 and -942 as identified by sequence analysis. These were initially cloned into the pCR 2.1 plasmid vector using the TA Cloning kit (Invitrogen, Carlsbad, CA) and then directionally subcloned into the pGL3 basic reporter plasmid using KpnI and XhoI restriction enzymes.
For transfection with the FADS2 wild-type and mutant promoter-reporter constructs, NIH 3T3 cells were seeded at a density of 1 x 105 cells per well in 6-well plates (Corning Inc., Corning, NY) in DMEM containing 10% FBS for 24 h. The cells were transfected at 50% confluency in serum-free transfection medium using Superfect reagent (Life Technologies Inc., Rockville, MD) according to the manufacturer's instructions. The plasmid CMV-ß galactosidase was cotransfected to measure transfection efficiency (Clontech Laboratories, Inc., Palo Alto, CA). Each well was transfected with 7.5 µl Superfect reagent, 1 µg of the promoter-luciferase vector construct, and 0.25 µg of the CMV-ß-galactosidase plasmid in a total of 500 µl transfection medium and maintained for 6 h at 37°C in a 5% CO2 incubator. The transfection medium was replaced with normal medium for a further 18 h before cells were harvested with 1x luciferase assay lysis buffer (Promega, Madison, WI), centrifuged at 10,000 g for 2 min at 4°C, and the supernatant was collected for the assays. Luciferase activities were measured using 20 µl lysate supernatant and adding 100 µl Luciferase Assay Reagent (Promega) according to the manufacturer's instructions in a MLX Microtiter plate luminometer (Dynex, Chantilly, VA).
For the ß-galactosidase assay, 30 µl of supernatant was incubated with 3 µl 100x magnesium buffer (0.1 M MgCl2 and 4.5 M ß-mercaptoethanol), 66 µl 1x o-nitrophenyl ß-D-galactopyranoside (50 U/ml; Sigma), and 191 µl 0.1 M sodium phosphate (pH 7.5). Samples were incubated at 37°C for 30 min, and 500 µl of 1 M Na2CO3 (pH 12) was added to stop the reaction. The ß-galactosidase activity was determined by absorbance measurements at 420 nm.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
80% to 90% decrease in FADS2 transcript levels in the delta-6-desaturase deficient fibroblasts (Fig. 1A
, lane 3) compared with human fibroblasts from three different normal individuals (Fig. 1A, lanes 1, 2, and 4). These results are consistent with our previously described metabolic biochemical findings and FADS2 expression analysis from this delta-6-desaturase deficient patient (5). To confirm and extend these findings, we performed Northern blot analyses on these same RNAs. Results of this experiment, shown in Fig. 1B, are consistent with the RT-PCR results. Densitometry of these Northern blots, shown in the lower panel, confirmed the magnitude of the decrease in the delta-6-desaturase deficient cells as suggested by the RT-PCR analysis.
|
|
Cloning and sequencing of the 3'-UTR consisting of 1,676 bp revealed four sequence variations in the FADS2 gene from the DF (Table 1), raising the possibility that decreased mRNA stability of the FADS2 transcript might account for the decreased levels of FADS2 mRNA in the DF compared with the NF. Although the nucleotide environment of these mutations did not appear to be crucial for message stability, we nevertheless tested the possible contribution of the identified sequence variations in the 3'-UTR of the gene by a mRNA stability assay following actinomycin D treatment (Fig. 3) . The half-life of the FADS2 mRNA was not significantly different in the DF and NF cells. Although the absolute amount of mRNA was less in the DF compared with the NF as previously shown (Fig. 1), the rate of decay of the transcripts in both normal and delta-6-desturase deficient cells was insignificantly different (Fig. 3). Transcripts from both cell types decayed at the same rate, leading to 34% (NF) and 32% (DF) of the starting levels by 20 h, the final assay time point.
|
|
|
|
|
6-fold increased promoter activity for the normal FADS2 relative to the mutated FADS2 promoter sequence (P < 0.01). These results confirm the functional relevance of the thymidine insertion polymorphism in the 5' regulatory region of the FADS2 gene to the decreased FADS2 gene expression and delta-6-desaturase enzyme activity observed in the patient-derived fibroblasts harboring the newly described polymorphic variant. The 6-fold difference in promoter activity is also consistent with results from mRNA expression analyses where 5- to 6-fold decrease in steady-state transcript levels were observed in the mutant fibroblast cells compared with the wild-type cells. Moreover, these results are consistent with our data from the nuclear run-on analyses that demonstrated an 5-fold decrease in the rate of transcription initiation in the mutant as compared with the wild-type cells. |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Detection of a decreased transcription rate in delta-6-desaturase deficient fibroblasts implied the potential existence of mutational differences in the 5'-UTR encoding promoter and other regulatory gene sequence elements of the FADS2 gene. The FADS2 gene is located on human chromosome 11q12–q13.1 (6, 7, 15). The Human Genome Project provided sequence information for human chromosome 11q12 that enabled us to design primers for PCR amplification from genomic DNA of 1 kb upstream from the translation initiation codon of the FADS2 gene. Sequence analysis of this upstream regulatory region revealed the insertion of an additional thymidine nucleotide between positions -941 and -942, coinciding with a putative C/EBP response element. Gel mobility shift assays employing end-labeled, double-stranded, normal, and T-insertion DNA sequences showed modest differences in binding of a putative C/EBP protein from nuclear extracts of 3T3 cells, suggesting a causal role for the insertion mutation in the decreased FADS2 gene expression.
This insertion polymorphism has also been proven to be functionally relevant to the decreased expression of the FADS2 gene by luciferase reporter construct assays. The same degree of difference was obtained in luciferase activity between the normal and mutant sequences as were obtained for the in vitro transcriptional rate assay and steady-state transcript levels; the single nucleotide insertion caused a 6-fold decrease in activity for the mutant sequence. In fact, the correlation between the decreases in steady-state mRNA levels, in vitro transcription rates, and promoter activities obtained from the FADS2 mutant compared with the normal controls was remarkable.
We have further analyzed the 5' regulatory region of the FADS2 gene and identified several putative transcriptional regulatory sequence elements in the region examined (Fig. 7) . The promoter is GC-rich and encodes a TATA-less region immediately upstream from the transcriptional initiation site. This region has multiple putative cis-regulatory elements, a common feature of versatile multi-functional promoters (22). Lipid-specific metabolic regulatory elements that are present include the C/EBP, sterol regulatory element binding protein (SREBP), and the PUFA-response elements. The C/EBPs have been characterized as basic region/leucine zipper transcription factors that regulate growth and differentiation in many cell types (23). They have been further identified as crucial factors in the transcriptional enhancement of the human (24) and mouse (24–26) SCD genes that encode SCDs. The C/EBP-response elements identified in the FADS2 promoter were particularly abundant; six such sites were identified as compared with three in a similar region of the human SCD promoter. This is probably indicative of the prominent role played by this factor in the transcriptional regulation of FADS2 gene expression. Two putative C/EBP binding sites contained overlapping sequence elements with 5' to 3' ends in opposite directions, and one such site occurred immediately upstream from the C/EBP site affected by the insertion of a thymidine at position –942. The proximity and juxtaposition of these C/EBP binding sites strongly suggests the possibility that they comprise a bona fide response element, the disruption of which could lead to transcriptional repression of the FADS2 locus as is seen in the patient with delta-6-desaturase deficiency. This could account for the severity of interference with transcriptional enhancement function caused by the insertion of an extra nucleotide in the FADS2 promoter of the delta-6-desaturase deficient fibroblasts. Further confirmation for the functional significance of the C/EBP binding sites was obtained from a comparison of the human FADS2 promoter region to its mouse and rat (putative) orthologs (Fig. 7A). This comparison revealed a remarkable similarity in location and frequency of the C/EBP transcriptional enhancer factor binding sites, including familiar PUFA metabolism-associated regulatory sites such as peroxisome proliferator-activated receptor and SREBP. Of particular interest is the conservation of the –942 C/EBP site in all three mammalian species compared (Fig. 7B). This is the only such site to retain not only the functional regulatory sequence but also the core nucleotide base specificity of this transcriptional enhancer factor binding site. This finding underscores the apparent crucial role of this regulatory site and the deleterious effect of the T-insertion in its sequence.
|
|
ACKNOWLEDGMENTS |
|---|
Manuscript received June 20, 2003 and in revised form August 14, 2003.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
6-desaturase deficiency in human skin fibroblasts. J. Lipid Res. 42: 501–508.-6-desaturase. J. Biol. Chem. 274: 471–477.
-Linolenic acid (GLA) is cytotoxic to 36B10 malignant rat astrocytoma cells but not to normal rat astrocytes. Br. J. Cancer. 77: 1612–1620.[Medline]
-5 desaturase. J. Biol. Chem. 274: 37335–37339., ß, and , and cellular retinol binding protein-1 expression in breast fibrocystic disease and cancer. Eur. J. Endocrinol. 137: 410–414.[Abstract]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  M. A. Schirmer and S. D. Phinney {gamma}-Linolenate Reduces Weight Regain in Formerly Obese Humans J. Nutr., June 1, 2007; 137(6): 1430 - 1435. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Baylin, E. Ruiz-Narvaez, P. Kraft, and H. Campos {alpha}-Linolenic acid, {Delta}6-desaturase gene polymorphism, and the risk of nonfatal myocardial infarction Am. J. Clinical Nutrition, February 1, 2007; 85(2): 554 - 560. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Journal of Biological Chemistry |
