From the Department of Food Science and Human Nutrition and the Division of Nutritional Sciences, University of Illinois, Urbana, Illinois 61801
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ABSTRACT |
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We previously showed that rat liver betaine-homocysteine methyltransferase (BHMT) mRNA content and activity increased 4-fold when rats were fed a methionine-deficient diet containing adequate choline, compared with rats fed the same diet with control levels of methionine (Park, E. I., Renduchintala, M. S., and Garrow, T. A. (1997) J. Nutr. Biochem. 8, 541-545). A further 2-fold increase was observed in rats fed the methionine-deficient diet with supplemental betaine. The nutrition studies reported here were designed to determine whether other methyl donors would induce rat liver BHMT gene expression when added to a methionine-deficient diet and to define the relationship between the degree of methionine restriction and level of methyl donor intake on BHMT expression. Therefore, rats were fed amino acid-defined diets varying in methionine and methyl donor composition. The effect of diet on BHMT expression was evaluated using Northern, Western, and enzyme activity analyses. Similar to when betaine was added to a methionine-deficient diet, choline or sulfonium analogs of betaine induced BHMT expression. The diet-induced induction of hepatic BHMT activity was mediated by increases in the steady-state level of its mRNA and immunodetectable protein. Using methyl donor-free diets, we found that methionine restriction was required but alone not sufficient for the high induction of BHMT expression. Concomitant with methionine restriction, dietary methyl groups were required for high levels of BHMT induction, and a dose-dependent relationship was observed between methyl donor intake and BHMT induction. Furthermore, the severity of methionine restriction influenced the magnitude of BHMT induction.
To study the molecular mechanisms that regulate the expression of BHMT,
we have cloned the human BHMT gene. This gene spans about 20 kilobases
of DNA and contains 8 exons and 7 introns. Using RNA isolated from
human liver and hepatoma cells, a major transcriptional start site has
been mapped using the 5' rapid amplification of cDNA ends
technique, and this start site is 26 nucleotides downstream from a
putative TATA box.
Betaine-homocysteines S-methyltransferase
(BHMT)1 (EC 2.1.1.5)
catalyzes a methyl transfer from betaine to homocysteine (Hcy), forming dimethylglycine and Met, respectively. Betaine is an
intermediate of choline oxidation, and the enzymes of this pathway are
primarily found in the liver and kidney of mammals (1), although we
have recently shown that BHMT is also expressed in the lenses of rhesus monkeys and humans (2). This enzyme has recently been shown to be
zinc-dependent (3), and based on protein purification reports, it has been estimated that BHMT represents 0.5-2% of the
total soluble protein in mammalian liver (4). Although these studies
presumably used livers from animals that were consuming diets
containing adequate Met, it is possible that BHMT can represent an even
greater proportion of liver protein, because as reported here, when
choline-containing diets are deficient in Met, there occur dramatic
increases in hepatic BHMT protein content. However, regardless of
dietary conditions, it is clear that BHMT is a major zinc metalloenzyme
in liver.
Interest in the nutrient and genetic factors that influence Hcy
metabolism has increased because elevated concentrations of this amino
acid in blood have been correlated to the incidence of arteriosclerotic
vascular disease and thrombosis. BHMT is one of two known mammalian
enzymes that methylate Hcy, the other being the folate/vitamin
B12-dependent Met synthase (EC 2.1.1.13). Of
these two methyltransferases, it is known that genetic defects that
reduce the flux through the Met synthase catalyzed reaction, or a
deficient intake of either of its coenzyme vitamin precursors, result
in elevated concentrations of blood Hcy (5, 6). Whether BHMT is
required for normal Hcy metabolism is not known, because choline, the
metabolic precursor of betaine, is not an essential nutrient unless the
level of Met in the diet is severely deficient. Under normal
nutritional status, the choline moiety can be synthesized by the
S-adenosylmethionine-dependent conversion of
phosphatidylethanolamine to phosphatidylcholine. A naturally
occurring combined deficiency of Met and choline has not been described
in humans and is not likely to occur because choline, primarily as
phosphatidylcholine, is abundant in human diets. Furthermore, to date,
there have been no inherited defects in the BHMT-catalyzed reaction
described, and therefore, it has not been possible to ascertain whether
such defects perturb Hcy metabolism in humans.
Although the significance of BHMT in Hcy homeostasis is not clear, it
is known that the relative contribution of BHMT to Hcy remethylation
can be influenced by diet. For example, it has been shown that the rate
of Hcy remethylation is increased when choline or betaine is added to
the diet of humans (7-9), and treating non-vitamin-responsive forms of
homocystinuria with supplemental choline or betaine elicits a plasma
Hcy-lowering response (5, 6, 10). As previously indicated by Mudd (11),
these studies suggest that betaine concentrations in human liver are
below that required to saturate BHMT, and present knowledge of hepatic
betaine concentrations and the Michaelis constants of betaine for the human enzyme support this idea (3). In vitro studies
designed to simulate Hcy metabolism in rat liver suggest that BHMT and Met synthase contribute equally to the conversion of Hcy to Met in that
organ (12). Furthermore, we have shown that when a diet is otherwise
nutritionally adequate, Met restriction dramatically elevates rat liver
BHMT gene expression (13). This dietary induction of BHMT gene
expression presumably enhances the methylation of the available Hcy
in vivo, thus conserving the nutritionally essential carbon
backbone of Hcy under conditions of Met deficiency. All of these
studies suggest that BHMT has a quantitatively significant role in the
hepatic conversion of Hcy to Met, although its significance in
whole-body Hcy remethylation remains speculative.
The purpose of the nutrition studies reported here is to further
clarify the interaction between dietary Met restriction and methyl
donor intake on rat liver BHMT gene expression. We report here that
dramatic changes in BHMT expression can be elicited when rats consume
diets deficient in Met yet rich in methyl donor, i.e.
choline, betaine, or sulfonium analogs of betaine. As an initial step
to elucidate the molecular mechanisms that mediate any of the
nutritional, hormonal, and tissue-specific expression of BHMT, and to
ultimately characterize human genetic variants at the BHMT locus, we
have characterized the organization of the human BHMT gene. Here, we
report the intron-exon splice junctions of the gene, report the major
transcriptional start site, and provide 3.2 kilobases of DNA sequence
5' to this transcriptional start site.
Materials--
Human liver samples and human hepatoma cells (Hep
G2) were obtained from the Anatomic Gift Foundation (Laurel, MO) and
the American Type Culture Collection (Manassas, VA), respectively. [ Diets and Animal Protocol--
Three animal studies were
performed to investigate the interaction between Met restriction and
methyl donor intake on hepatic BHMT gene expression. These studies were
approved by the University of Illinois' Laboratory Animal Care
Advisory Committee.
The components of the amino acid-defined diets used in these studies
have been previously described in detail (13) and are based on the
American Society for Nutritional Sciences' nutrient recommendations
for growing rats (14). The dietary treatments only varied in Met (1.0, 1.5, 2.0, and 3.0 g/kg of diet) and methyl donor content. The methyl
donors employed were choline, betaine, DMAT, and DMPT, and these
compounds were present at levels ranging from 0 to 37.5 mmol per kg of
diet, as indicated in Table I. DMAT and DMPT are sulfonium analogs of
betaine; the latter is a plant metabolite found at high concentrations
in marine algae (15) and is present in some terrestrial plants as well
(16). These compounds were used only in study 1 and were added to the diet at levels that were isomethyl to the level of betaine used in the
same study. All diets contained 3 g of cystine and 10 g of
succinylsulfathiozole per kg of diet. Succinylsulfathiozole is an
antibiotic that was added to inhibit the microbial metabolism of methyl
donors in the gastrointestinal tract, but because of its use, all diets
were supplemented with menadione sodium bisulfite (50 mg/kg of diet).
The feeding trials were conducted using 3-week-old Sprague-Dawley rats
(Harlan, Indianapolis, IN), which were housed as described previously
(13). All rats were initially fed a control amino acid-defined diet for
3 days. The control diet contained adequate Met (3 g/kg) and choline
bitartrate (5 mmol/kg) and was devoid of other methyl donors. Following
the adaptation period, rats were randomly divided into experimental
groups such that mean body weights among groups were not significantly
different. When rats are fed diets severely restricted in any essential
amino acid, they voluntarily decrease their food intake (grams/d) by 40-50% (17). Therefore, where indicated in Table I, food intake among
some treatment groups was restricted to the average food intake of
groups fed the diets severely deficient in Met (1 g/kg of diet), which
did not significantly differ from each other. Each group was given free
access to water throughout the feeding trial, which varied from 10 to
16 days as indicated in Table I. Rats were killed and their livers
stored at Assay Procedures--
Northern analysis was performed as
described previously (13) except that the oligonucleotide probes for
BHMT were made using a rat cDNA as template, rather than a porcine
cDNA. mRNA content was quantified by phosphorimaging, and BHMT
gene expression was normalized against rat glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) (EC 1.2.1.12) expression.
Hepatic BHMT protein was visualized by Western analysis. Fifteen
micrograms of crude liver extract were subjected to SDS polyacrylamide gel electrophoresis using a 5% stacking gel and a 12% separation gel
using a Tris-glycine discontinuous buffer system. Duplicate gels were
run, and protein from one gel was blotted onto nitrocellulose using a
Tris-glycine-methanol transfer buffer and a semidry blotting apparatus.
The second gel was Coomassie-stained for protein and densitometrically
analyzed to confirm equal protein loading. Blotted protein was probed
for BHMT using rabbit polyclonal antibodies prepared against highly
purified recombinant human BHMT (3). The antigen and primary antibody
interaction was detected using a peroxidase-coupled antirabbit IgG
(Vector Laboratories, Burlingame, CA) and tetramethylbenzidine as the
peroxidase substrate.
BHMT activity in crude liver extracts were measured as described
previously (4). Protein in crude liver extracts were measured by a
Coomassie dye binding assay (Bio-Rad) using bovine serum albumin as
standard. A unit of BHMT activity is defined as 1 nmol of Met formed
per hour, but activities are expressed here relative to the mean BHMT
activity of the appropriate control group for each study. Treatments 1, 7, and 14 were the control groups for Studies 1, 2, and 3, respectively, and the mean BHMT activities of these groups were given
the relative value of 1.
Statistics--
The data obtained from each nutrition study were
analyzed by one-way analysis of variance. When analysis gave a
significant F value (p < 0.05), treatment differences
were evaluated using Fisher's least significant difference procedure.
Isolation of Genomic Clones Encoding the BHMT Gene--
A human
lung fibroblast cell line W138 genomic library in the Lambda FIX II
vector (Stratagene, La Jolla, CA) was screened (106
plaques) with [32P]dCTP-labeled probes generated using
the Rediprime labeling system (Amersham Pharmacia Biotech) and a human
BHMT cDNA as the template. The human cDNA was originally
isolated in pBluescript II SK (Stratagene), and this construct was
named pTG9 (4). Four positive clones (
PAC clones (G2, D8, A9, D11, M13, G19, and B23) were isolated using
probes synthesized from the entire human cDNA as template. The PAC
DNA from these clones was purified and characterized by restriction
mapping and Southern hybridization. Radiolabeled probes for Southern
analysis were made using an EcoRI 835-bp fragment of the 5'
region and a 1615-bp EcoRI-XhoI 3' region
fragment of the human cDNA. A 4.5-kb SacI fragment of
PAC clone D11 was purified and cloned into pBluescript II KS phagemid.
This clone was designated pEP-D11-4.5. A region of genomic DNA 3' to
pEP-D11-4.5 was isolated by PCR using the following primers:
5'-GGCATAGTGACTCACACCTGTAATC-3', 5'-GCATGACCTCTTTTCTAATACACTG-3', and
the Expand long template PCR system (Boehringer Mannheim). The primers
correspond to the 3' region of pEP-D11-4.5 and a region of DNA very
near the 3' terminus of the human cDNA, respectively. The
approximate 2.3-kb PCR product was cloned into a plasmid vector
producing plasmid pEP-D11-2.3. Clones pEP-D11-4.5 and pEP-D11-2.3 were
analyzed by DNA sequencing.
DNA Sequencing and Intron Size Determination--
DNA was
sequenced using an Applied Biosystems 373A automated DNA sequencer at
the University of Illinois' Biotechnology Center (Urbana, IL).
Exon-intron junctions in the pEP clones were determined by direct
sequencing across the junctions using oligonucleotide primers designed
from the cDNA sequence. Intron sizes were determined by sequencing
through the regions, PCR amplification using oligonucleotide primers
based on exon sequence, or by the combination of DNA sequence data in
conjunction with estimates of DNA fragment sizes by their mobilities in
agarose gels. The exon-intron junction sequences and 3.2 kilobases of
DNA 5' to the translational start site (from pEP8) were determined by
sequencing both DNA strands. The 5' region of the gene was analyzed
using the Transcription Element Search Software with the Transfac
version 3.3 data base (http://agave.humgen.upenn.edu/tess/index.html).
5'-RACE Analysis of BHMT cDNA Ends--
Total RNA was
isolated from human liver and Hep G2 cells using the Ultra-SpecII RNA
isolation system (Biotecx, Houston, TX). cDNA corresponding to the
5'-end of human liver mRNA was synthesized and amplified with the
5' RACE system (Life Technologies, Inc.) using the manufacturer's
instructions. The primers used were 5'-GCAGTCAGGAGTGTGGTAAGC-3' (gene-specific primer 1), 5'-GCAGCTAAGGTATGAAGGTGT-3' (gene-specific primer 2), 5'-CTCTTCTCCAGTGCAAAGACAAACC-3' (nested gene-specific primer), and the universal primers provided with the kit. The products
were cloned into plasmid vectors and analyzed by DNA sequencing.
The Influence of Diet on Rat Growth and BHMT Expression--
The
relative degree of Met deficiency was monitored by growth. Weight gain
and feed efficiency (weight gain/feed intake) are shown in Table
I. Rats given free access to the diets
severely deficient in Met (1 g/kg) consumed significantly less food
than those given free access to the diets containing adequate Met (3 g/kg). As expected, in Studies 1 and 2, the feed efficiencies of rats
that were restricted-fed the control diets (treatments 2 and 8) were
between those of rats given free access to the control diets
(treatments 1 and 7) and those fed the Met-deficient diets (treatments
3 and 12). Overall, rats fed diets containing 1.0 or 1.5 g of
Met/kg of diet (treatments 4-6, 11-13, and 14-17) had lower feed
efficiencies than rats fed the diets containing 2.0 or 3.0 g of
Met/kg of diet (treatments 2 and 8-10). These data indicate that the
Met content of the former diets cannot support maximum growth and so in
this regard are deficient in this amino acid. However, all rats gained
weight, indicating that 1.0 g of Met/kg of diet is slightly above
the maintenance requirement of the weanling rat. The feed efficiency of
rats fed the diet containing 2.0 g of Met/kg of diet (treatment
10) was approaching the feed efficiency of rats fed the control diet
containing 3.0 g of Met/kg of diet (treatment 9). This indicates
that 2.0 g of Met/kg of diet is approaching the minimum
requirement for the weanling rat. These observations are consistent
with Funk et al. (18), who showed that 2.5-3.0 g of Met/kg
of diet was the minimum level of dietary Met that could support the
maximum growth rate of weanling rats consuming an amino acid-defined
diet and that rats consuming 2.0 g of Met/kg of diet were
approaching that maximal growth rate. Furthermore, they observed linear
growth responses between 1.0 and 2.0 g of Met/kg of diet.
The addition of various methyl donors to diets severely deficient in
Met (1 g/kg) produced mixed results. In Study 1, the addition of 25 mmol of betaine/kg of diet slightly reduced feed efficiency, whereas
the addition of an isomethyl level of DMAT clearly inhibited growth
(treatments 4 and 5 versus treatment 3). The addition of
DMPT resulted in a slight increase in feed efficiency (treatment 6 versus treatment 3). In Studies 2 and 3, the addition of
choline to diets severely deficient in Met (1 g/kg) tended to decrease
feed efficiency. Most of these data support the idea proposed by Storch
et al. (9), who suggested that excess methyl donor
consumption may increase Met requirements due to an enhanced oxidation
rate of this amino acid.
Study 1 was performed to determine whether sulfonium analogs of betaine
can induce hepatic BHMT expression when fed concomitant with a diet
deficient in Met, as had been previously observed for betaine
consumption (13). Enzyme activity levels and mRNA and protein
contents were measured. The data for BHMT activity and mRNA content
can be seen in Fig. 1A. Based
on our previous reports (13, 19), we expected that rats consuming the
diet containing 1 g of Met/kg of diet and supplemental betaine
(treatment 4) would have significantly higher BHMT mRNA content and
enzyme activity compared with rats fed the Met-adequate (3 g/kg of
diet) control diet (treatment 1). As shown in Fig. 1A, about
8-9-fold changes were observed. Furthermore, feeding the sulfonium
analogs of betaine, DMAT (treatment 5), and DMPT (treatment 6),
concomitant with Met deficiency, also induced BHMT gene expression to a
similar extent. In general, the relative level of activity mirrored
mRNA content. Fig. 1B shows an autoradiograph of a
representative Northern blot in which rat liver total RNA was probed
for BHMT and GAPDH mRNA transcripts. The autoradiograph shows that
the relative changes in BHMT gene expression were dramatic and easily
quantified by phosphorimaging, as depicted in Fig. 1A. Fig.
2 shows a Western blot of total liver
protein probed for BHMT. Three liver samples were chosen at random from
rats fed either the control Met diet (treatment 1) or the Met-deficient
diet containing supplemental betaine (treatment 4). The blot shows that
the relative levels of BHMT protein were markedly affected by these
dietary treatments, and this result qualitatively mirrored mRNA
content and activity levels.
Study 1 also was designed to determine whether the large changes in
BHMT expression were due to the significant reduction of food intake
that accompanies the dietary restriction of Met. As shown in Fig.
1A, although the rats restricted-fed the control diet
(treatment 2) had levels of BHMT expression about 50% higher than
those of rats given free access to the same diet (treatment 1), this
change in expression is small compared with those changes observed when
rats were fed diets containing 1 g of Met/kg of diet and
supplemental levels of methyl donor (treatments 4-6), in which
6-9-fold increases were observed.
In Study 1, the rats fed the diet solely deficient in Met (treatment 3)
had BHMT activities that were approximately 2-fold higher than the rats
fed the diet containing adequate Met (treatment 1); however, changes in
mRNA content were not detected. In our previous study (13), we
reported a 4-fold change in BHMT mRNA content and activity when
rats were fed a diet solely deficient in Met. However, in that study,
the level of dietary choline was twice that used in this follow-up
study, i.e. 5 versus 2.5 mmol of choline/kg of
diet. Whether coincident or not, these observations lead us to
hypothesize that Met restriction per se does not induce BHMT
expression, but that some level dietary methyl donor is also required,
concomitant with Met restriction, before an induction of BHMT
expression will be observed. Furthermore, as we previously suggested
(19), there may be a methyl donor dose-dependent
relationship to its induction level. Study 2 was designed to test these hypotheses.
Study 2 dietary treatments used choline as the sole methyl donor. The
results of Study 2 (Fig. 3A)
showed that when dietary Met was adequate (treatment 8), a moderate
level of choline intake (5 mmol/kg) had no effect on BHMT expression
compared with rats consuming the same diet devoid of choline (treatment
9). Furthermore, when rats were fed diets devoid of choline, Met intake
ranging from adequate (3 g/kg) to severely deficient (1 g/kg) had no
effect on BHMT expression (treatments 9-11). However, when choline was added to diets severely deficient in Met, a significant induction of
BHMT expression was observed, and the effect appeared to be dose-dependent (treatments 11-13). In this study, the
changes observed in BHMT activity were also mediated by changes in
mRNA content and immunodetectable protein (not shown), as observed with betaine and the sulfonium analogs of betaine in Study 1.
As in Study 1, in Study 2, the feed-restricted rats consuming the
control diet (treatment 8) had about 70% higher levels of BHMT
activity compared with rats given free access to the same diet
(treatment 7). Although feed restriction caused a 50% (Study 1) and
70% (Study 2) increase in BHMT expression in our studies, it is clear
that the high induction of BHMT gene expression observed in these
studies was primarily due to the levels of nutrients in the diets and
was nearly independent of food intake. The relatively smaller increases
in BHMT expression caused by food restriction could be due to increases
in blood glucocorticoid concentrations. Previous studies have shown
that the intraperitoneal injection of hydrocortisone significantly
increases hepatic BHMT activity (20) and mRNA levels (21). However,
we did not measure plasma hormone concentrations in any of the rat
studies reported here.
The results of Study 3 confirm that the induction of BHMT activity is
dependent upon the methyl donor content of the diet when added to a
diet also severely deficient in Met (treatments 14-16; Fig. 3). This
effect was clearly dose-dependent. Although Study 2 showed
that severe Met restriction alone is not sufficient for BHMT induction,
Study 3 showed that the severity of Met restriction can influence the
magnitude of the methyl donor-induced increase in BHMT expression
(treatments 14, 16, and 17). Namely, the magnitude of the induction was
proportional to the severity of the deficiency.
The maximum relative BHMT induction levels observed in Study 1 (treatment 4) was approaching twice that observed in Study 3 (treatment
16), although the dietary treatments were similar. Treatment 4 (Study
1) was limiting in Met (1 g/kg) and contained excess betaine (25 mmol/kg), and treatment 16 (Study 3) was limiting in Met (1 g/kg) and
contained excess choline (20 mmol/kg). Therefore, although high
induction of BHMT expression was consistently observed when rats were
fed diets severely deficient in Met containing supplemental methyl
donor, we observed an unexplained variation in the magnitude of
induction between studies.
Organization of the Human BHMT Gene and 5' Region--
A Lambda
Fix II human genomic library was screened as described under
"Experimental Procedures." Four clones (
To obtain the remaining 3' region of the gene, PAC clones were isolated
using probes made from the entire human cDNA. Seven clones (G2, D8,
A9, D11, M13, G19, and B23) were isolated. Using SacI
digestion patterns (not shown), PAC clones were divided into those that
did (G2 and D11) and those that did not (D8, A9, M13, G19, and B23)
contain the diagnostic 9.5-kb fragment characterized in pEP10 (Fig. 4).
PAC clones D8 and D11 were chosen as representatives of each group and
analyzed by Southern blot using probes synthesized from the
EcoRI (835 bp) and EcoRI-XhoI (1615 bp) fragments of the human cDNA, which encoded a 5' and a 3' region
of the cDNA, respectively. The 5' probe encoded exons 1-5 but only
a portion of the 5' region of exon 6, whereas the 3' probe encoded the
3' region of exon 6 and the remainder of the cDNA. A 4.5-kb
SacI fragment of D11 hybridized to both probes and was
analyzed further by DNA sequencing. As hoped, sequence analysis of this
clone overlapped pEP10-1.5 and encoded exon 7 and the 5' portion of
intron 7. Exon 8, the last exon of the human gene, was PCR-amplified
using a primer designed from the known 5' sequence of intron 7 and
another primer designed from the end of the 3' untranslated region of the human cDNA. The resulting 2.3-kb PCR product was sequenced and
found to encode the remaining 3' region of intron 7, and all of exon 8. Exon 8 encodes the last 59 amino acids of the open reading frame and
the entire 3' untranslated region of the human cDNA. In summary,
the PAC D11 clone was used to isolate a DNA fragment (pEP-D11-4.5)
which overlapped the 3'-end of the
A diagram of the human BHMT gene can be seen in Fig. 4. It consists of
8 exons and 7 introns, and all intron-exon splice junctions follow the
GT-AG rule (Table II). Both strands of
the 5'-flanking region of the gene and the intronic sequence flanking
each exon have been sequenced, and these sequences have been deposited
in the GenBankTM data base. In general, the exon sequence
of the gene was in good agreement with our previously published
cDNA sequence (4); however, two nucleotide differences have been
detected. Using the numbering used for the original cDNA,
nucleotides 23 and 742 are both A in the human cDNA but are both G
in the genomic sequence reported here. The first difference is in the
5' untranslated region of the cDNA, and the second difference is
found in exon 6. The A to G change in exon 6 corresponds to a
Gln-to-Arg amino acid change. At this juncture, it is unknown whether
these two differences represent polymorphisms or mutations.
5'-RACE analysis of RNA isolated from either human liver or human
hepatoma cells (Hep G2) indicated only one major mRNA species in
liver. A total of seven PCR amplification products were sequenced, and
each ended 77 bases 5' to the first nucleotide of the start codon in
the original cDNA. These 5'-RACE products had a purine at the
terminal 5' position, and this extended the 5' untranslated region of our previously published cDNA sequence by 51 nucleotides. mRNAs generally begin with a purine, and therefore this A at
position
The 5' genomic region of human BHMT is shown in Fig.
5. This sequence was analyzed for
consensus transcription factor sequences, and many putative
transcription factor binding sites were found. Centered 26 bases 5' to
the major transcriptional start site is a putative TATA-binding protein
site. The location of this TATA-binding protein site in relation to the
5'-end of the mRNA is consistent with this being the primary
promoter region in liver. Analysis of this entire region of DNA
indicates the presence of four consensus TATA boxes, but only the one
noted above was identified by the TSSG and TSSW human polymerase II
promoter region and start-of-transcription analysis programs as a
potential promoter. Just 5' to this TATA box are four putative Sp1
sites and one putative activator protein-2 site. Just 3' to this TATA
box are two putative Sp1 sites. Regions 5' to this TATA box contain
putative binding sites for HNF-1, HNF-3, and CAAT enhancer-binding
protein, all of which have been identified as liver-specific or
liver-enriched transcription factors. It is interesting to note that
there are putative transcription factor binding sites for homeobox 4c,
4d, and 4e (22). These sites are centered 794 bases 5' to the
transcriptional start site. Homeobox transcription factors are known to
be important in early embryonic and fetal development, and the BHMT
mRNA has been detected in fetal tissue (GenBank accession number
W97296). Also shown in Fig. 5 are several consensus sites for steroid
hormone receptors, including glucocorticoids, progesterone, estrogen,
and androgen binding sites. Hydrocortisone has been reported to
increase hepatic BHMT gene expression (21), whereas there have been no
reports on the effects progesterone or androgens have on BHMT
expression.
Choline may be a significant source of one carbon units in
metabolism because it is abundant in human diets and its oxidation interfaces with Met and methylenetetrahydrofolate synthesis. Choline oxidation takes place primarily in the liver and kidney with enzymes found in the mitochondria and cytoplasm. The complete oxidation of
choline results in four of its five carbons entering the one-carbon pool. In the cytosol, one carbon directly becomes the labile methyl carbon of Met via the BHMT-catalyzed reaction. In the mitochondrion, three other one carbon units enter the folate pool at the oxidation state of formaldehyde by reactions catalyzed by dimethylglycine dehydrogenase, sarcosine dehydrogenase, and the glycine cleavage system, respectively. It has been established that folate metabolism in
the mitochondrion functions in part to produce formate, which leaves
this organelle to become activated into the folate one-carbon pool of
the cytoplasm (23, 24). In the cytoplasm these one carbon units are
used for purine and thymidylate biosynthesis and the folate- and
vitamin B12-dependent methylation of Hcy. Because choline is abundant in the diet of humans, primarily as phosphatidylcholine, its oxidation may contribute substantially to the
total one carbon needs of the liver and kidney.
The regulation of hepatic choline oxidation is not completely
understood. Free choline resides at a metabolic branch point; it can be
incorporated into phospholipids, converted to acetylcholine, or
oxidized to glycine. Quantitatively, the use of choline for acetylcholine synthesis is negligible. The competition for choline to
be incorporated into phospholipids or proceed through the oxidation pathway is between cytosolic choline kinase and transport into the
mitochondrion; the latter is reportedly the rate-limiting step in the
irreversible oxidation of choline to betaine (25). The
Km of rat liver choline kinase for choline is about 0.03 mM (26), and that for choline transport into rat liver mitochondrion has been estimated to be about 0.22 mM (27).
Choline concentrations in rat liver range from 0.05 to 0.25 mM (28, 29), and presumably, higher levels are attained
when dietary choline is supplemented in the diet. Taken together, these
data suggest that free choline in liver is preferentially incorporated into phospholipids and that choline oxidation functions as a spillover pathway that is sensitive to dietary choline intake and subsequent tissue levels. In fact, metabolic tracer studies using rat liver slices
have estimated that about 90% of the free choline proceeds through the
oxidation pathway (30). We are interested in BHMT because it resides at
an interface between choline oxidation and sulfur amino acid and
one-carbon metabolism, and it is possible that these pathways are
coordinately regulated in part by the BHMT-catalyzed reaction. We have
chosen to focus our initial efforts on the influence nutrition has on
BHMT gene expression because previous work indicated that activity
levels of hepatic BHMT are affected by diet.
There have been numerous studies investigating the effects of nutrition
on hepatic BHMT activity (13, 19, 31-34). The earliest studies used
rats and showed that the specific activity of BHMT varies with the
dietary intake of sulfur amino acids, choline, and betaine (31-33).
The greatest changes were observed with Met deficiency and Met
deficiency in combination with excess dietary choline where up to
3-fold increases of BHMT activity were observed. We subsequently
observed similar responses of BHMT activity in chicken liver when
chicks were fed varying levels of sulfur amino acids, choline, and
betaine (19). Although supplemental levels of Met, either with or
without excess methyl donor, slightly stimulated chick liver BHMT
activity, as was observed in rats (31), we confirmed that BHMT activity
is most dramatically up-regulated when the diet is deficient in Met.
Furthermore, the magnitude of induction was greater in our chicken
study than that previously reported for rats, reaching 6-fold induction
levels. A more recent study from our laboratory showed that 8-10-fold
increases in BHMT activity can be achieved by feeding rats amino
acid-defined diets deficient in Met containing excess betaine (13). In
this study, the diet-induced changes were shown to be mediated by
changes in the steady-state levels of mRNA. Finally, we recently
reported a preliminary investigation using pigs (34), and the results suggest that in this species the liver enzyme is refractory to changes
in diet, but the kidney enzyme is inducible under conditions of Met
deficiency with supplemental choline or betaine, although to a much
lower extent than what we observed with the chicken and rat liver
enzymes. Taken together, the data are consistent across species and
indicate that BHMT activity is influenced by Met, choline, and betaine
intakes. In all of the previous studies, it was concluded that Met
restriction induces BHMT activity. However, none of the studies to date
have controlled for the dramatic changes in food intake that accompany
the consumption of diets deficient in Met, and all of the studies in
which a major induction was observed used choline-containing diets.
The data we report here show that changes in BHMT activity are
primarily due to the changes in the nutrient content of the diet rather
than a physiological response to reduced food intake. These changes in
activity are due to changes in mRNA content and immunodetectable
protein. Furthermore, our data indicate that although high levels of
BHMT induction require Met restriction, Met restriction alone is not
sufficient; there must also be some methyl donor in the diet. As
speculated in our previous report (19), there is a
dose-dependent relationship between the level of BHMT
induction and the amount of methyl donor in a diet limiting in Met. We
also show that the magnitude of induction is related to the severity of
Met deficiency. Although the methyl donor-dependent induction of BHMT expression requires Met restriction, this induction occurs at a level of Met intake that is above the maintenance requirement for the weanling rat. Taken together, these data support the idea proposed by Finkelstein et al. (31) that BHMT
functions to conserve the backbone of Hcy under conditions of Met
deficiency; however, BHMT primarily does so only when there is also a
source of methyl donor in the diet, shown here to include choline,
betaine, or sulfonium analogs of betaine.
The requirement for both low dietary Met and adequate methyl donor
levels for the high induction of BHMT expression may be a regulatory
control to prevent the futile cycling of methyl groups. Because a diet
devoid or low in choline necessitates the synthesis of this compound
using S-adenosylmethionine, increasing BHMT expression when
both dietary Met and choline are deficient might enhance the oxidation
of choline, a compound being synthesized using methyl groups derived
from scarce Met supplies. Synthesizing choline from methyl groups
derived from Met only to in turn oxidize it would be a futile cycle. In
contrast, the induction of BHMT when dietary Met levels are low and
choline is adequate or high allows the liver to spare the labile methyl
carbon of Met two ways. First, choline directly spares the methyl group
of Met because the cell no longer needs to synthesize as much
phosphatidylcholine, as it can activate choline by phosphorylation and
incorporate it first into cytidine diphosphocholine and then into
phosphatidylcholine. Second, by increasing the expression of BHMT under
these nutritional conditions, the liver cell increases the probability
of Hcy being remethylated to Met, rather than have Hcy proceed through
the transsulfuration pathway.
Further studies will be required to determine what mechanisms mediate
the diet-induced changes in steady-state levels of hepatic BHMT
mRNA. Earlier reports indicate that actinomycin D could partially block BHMT induction in rat liver (20) or cultured cells (35), provided
varying levels of dietary or medium Met, respectively. It can be
inferred from these earlier studies that the diet-induced increases we
observed in BHMT mRNA are due in part to changes in transcription.
The influence of diet or physiological state on BHMT mRNA turnover
has not been studied. In order to begin studies on the transcriptional
regulation of BHMT expression, we have cloned the 5'-flanking region of
the human BHMT gene into a luciferase reporter vector and have
confirmed that changes in medium sulfur amino acid and betaine
concentrations cause significant changes in BHMT promoter activity in
Hep G2 transfectants.2 The
molecular signals that initiate these putative changes in BHMT
transcription remain to be identified.
In addition to the 5'-flanking region of the human BHMT gene, we have
isolated several overlapping clones encoding the human BHMT gene (Fig.
4). The intron-exon junctions were mapped and sequenced, and the intron
sizes were determined. The intronic sequences flanking each exon are
reported and all intron-exon junctions follow the GT-AG rule. The major
transcriptional start site in liver has been determined using 5'-RACE,
and computer analysis of the 5'-flanking region has identified many
putative transcription factor binding sites (Fig. 5). Centered 26 nucleotides upstream from the transcriptional start site is a TATA box.
This TATA box was the only one identified as a putative promoter using TSSG and TSSW programs. The 5'-flanking region also contains a significant number of potential steroid transcription factor binding sites. Some of the glucocorticoid response elements are presumably functional because glucocorticoids have been shown to increase rat
liver BHMT activity (20) and mRNA levels (21). Several transcription factor binding sites known to be important in
liver-specific expression were also identified, including HNF-1, HNF-3,
and CAAT enhancer-binding protein.
Although a genetic deficiency of BHMT activity has not been described,
it is possible that mutations or polymorphisms in the gene exist that
reduce BHMT activity and increase plasma Hcy levels and thus increase
vascular disease risk. We are unaware of any attempts to identify
individuals deficient in BHMT activity, most likely because the organs
that express this enzyme, the liver and kidney, are not routinely
biopsied and have not been specifically sampled for this purpose. The
sequence of the human BHMT gene reported here will permit investigators
to screen for gene variants using genomic DNA isolated from blood or
any other easily isolated cell type. The availability of the
5'-flanking region of the gene will allow further studies on the
nutrient-, hormone-, and tissue-specific regulation of BHMT gene transcription.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
EXPERIMENTAL PROCEDURES
-32P]dCTP (3000 Ci/mmol) and nitrocellulose filters
were obtained from Amersham Pharmacia Biotech. DNA restriction and
modifying enzymes were purchased from Promega (Madison, WI), Boehringer Mannheim, and New England Biolabs (Beverly, MA). Oligonucleotides were
synthesized at the University of Illinois' Biotechnology Center
(Urbana, IL). Phage 1 artificial chromosome (PAC) clones were obtained
from the Medical Resource Council of Canada Genome Resource Facility at
the Hospital for Sick Children (Toronto, Ontario, Canada). Betaine
hydrochloride was purchased from Sigma. Choline bitartrate,
L-Met, and other chemicals used for the L-amino acid-defined diets were purchased from Dyets (Bethlehem, PA). Dimethylacetothetin (DMAT) was a gift from NutriQuest (Chesterfield, MO), and dimethylpropiothetin (DMPT) was purchased from TCI America (Portland, OR). PCR products were cloned into pCRII (Invitrogen, San
Diego, CA) or pGEM-T (Promega) plasmids. All other reagents were of the
highest purity available from commercial vendors.
80 °C until analyzed for BHMT activity, mRNA, and
protein content.
6,
8,
10, and
13)
were identified following the tertiary screen. Phage from each plaque
were amplified by the plate lysate method, and their respective DNAs
were purified and characterized by restriction mapping and Southern
hybridization. Radiolabeled probes for Southern analysis were made
using a PstI 1531-bp fragment of the 5' region and a 925-bp
PstI-XhoI 3' region fragment of the human
cDNA as templates. The entire genomic DNAs, bordered by
NotI sites, were cloned into pBluescript II KS phagemid to generate pEP6, pEP8, pEP10, and pEP13 from the corresponding
clones. The approximate 1.5-, 4.5-, and 9.5-kb SacI
fragments of pEP10 were also cloned into pBluescript II KS phagemid.
These clones were designated pEP10-1.5, pEP10-4.5, and pEP10-9.5,
respectively. The 9.5-kb SacI fragment of pEP10 was further
digested into smaller fragments and cloned in pBluescript II KS
phagemid. The resulting clones, p10-9.5
XbaI-A,
p10-9.5
SmaI-A, p10-9.5
SalI-A,
p10-9.5
XbaI-B, p10-9.5
SmaI-B, and
p10-9.5
SalI-B, as well as the other clones derived from
pEP10, and 3.2 kilobases of DNA in the center region of pEP8 (promoter
and distal upstream region), were analyzed by DNA sequencing. The
sequencing for pEP8 began with primers designed from the most 5' region
of pEP10.
RESULTS
Dietary treatments and growth performance of rats fed diets varying in
Met, choline, betaine, DMAT, and DMPT content
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Fig. 1.
A, diet-induced changes of hepatic BHMT
activity and mRNA content (Study 1). Rats were fed the following
experimental diets for 16 days: 1, Met-adequate (0.3%)
diet; 2, Met-adequate diet; 3, severely
Met-deficient (0.1%) diet; 4, severely Met-deficient diet
containing 25 mmol of betaine/kg of diet; 5, severely
Met-deficient diet containing 37.5 mmol of DMAT/kg of diet; and
6, severely Met-deficient diet containing 37.5 mmol of
DMPT/kg of diet. All diets contained 2.5 mmol of choline/kg of diet.
Liver RNA was isolated and probed for BHMT and GAPDH mRNA levels by
Northern analysis and quantified by phosphorimaging. BHMT activity
and the BHMT:GAPDH ratio of the control group (treatment 1) were
assigned a value of 1, and other treatment groups are expressed
relative to this group. Values are means ± S.E. Means with unlike
superscript letters are significantly different
(p < 0.05). B, a representative Northern
blot of the diet-induced changes in hepatic BHMT mRNA content
(Study 1). One liver RNA sample was chosen at random from each dietary
treatment group of Study 1 and probed for BHMT and GAPDH mRNA
levels by Northern analysis as described under "Experimental
Procedures." BHMT and GAPDH mRNA were visualized by
autoradiography, and the autoradiographic image was captured using the
Foto/Analyst II Visionary system and Collage software (Fotodyne, New
Berlin, WI).
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Fig. 2.
Crude rat liver protein probed for BHMT
protein by Western analysis (Study 1). Crude liver extracts were
subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis,
blotted onto nitrocellulose, and probed with antihuman BHMT rabbit
polyclonal antibodies as described under "Experimental Procedures."
Three liver samples were chosen at random from rats fed either the
control Met diet (treatment 1) or the Met-deficient diet containing
supplemental betaine (treatment 4), shown in lanes 1-3 and
4-6, respectively.
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Fig. 3.
A, diet-induced changes of hepatic BHMT
activity (Study 2). Rats were fed the following experimental diets for
10 days: 7, Met-adequate (0.3%) diet containing 5 mmol of
choline/kg of diet (control); 8, Met-adequate control diet;
9, Met-adequate (0.3%) diet devoid of choline;
10, Met-deficient (0.2%) diet devoid of choline;
11, severely Met-deficient (0.1%) diet devoid of choline;
12, severely Met-deficient (0.1%) diet containing 5 mmol of
choline/kg of diet; and 13, severely Met-deficient (0.1%)
diet containing 10 mmol of choline/kg of diet. BHMT activity of
treatment 7 was assigned a value of 1, and other treatment groups are
expressed relative to this group. Values are means ± S.E. Means
with unlike superscript letters are significantly different
(p < 0.05). B, diet-induced changes of
hepatic BHMT activity (Study 3). Rats were fed the following
experimental diets for 14 days: 14, severely Met-deficient
(0.1%) diet devoid of choline; 15, severely Met-deficient
(0.1%) diet containing 5 mmol of choline/kg of diet; 16, severely Met-deficient (0.1%) diet containing 20 mmol of choline/kg of
diet; and 17, Met-deficient (0.15%) diet containing 20 mmol
of choline/kg of diet. BHMT activity of treatment 14 was assigned a
value of 1, and other treatment groups are expressed relative to this
group. Values are means ± S.E. Means with unlike
superscript letters are significantly (p < 0.05) different.
6,
8,
10, and
13) were obtained, and all were found to have some degree of
overlapping sequence based on restriction mapping and Southern analysis. The linear arrangement of two of these clones can be seen in
Fig. 4. The entire genomic inserts of the
clones, bordered by NotI sites, were inserted into
pBluescript II KS phagemids to generate pEP6, pEP8, pEP10, and pEP13
clones. The genomic inserts varied in size from 16 to 19 kilobases. The
restriction maps of pEP6, pEP8, and pEP13 indicated that these clones
were nearly identical, whereas pEP10 contained additional flanking
sequence. Therefore, pEP8 and pEP10 were chosen for further
characterization. The pEP10 clone was fragmented further as described
under "Experimental Procedures." The pEP8 and pEP10 clones, and
fragments thereof, were analyzed by DNA sequencing. Both pEP8 and pEP10
contained the 5' coding region of the gene, yet were missing varying
portions of the 3' region. The pEP8 clone was found to contain exons 1 and 2, but it encoded only part of intron 2. The pEP8 clone was estimated to have an additional 8 kb of DNA 5' to the start codon found
in exon 1, as originally identified in the human cDNA (4). The
pEP10 clone was found to encode exons 1-6 but only part of intron 6 (Fig. 4). In summary, pEP8 and pEP10, both of which were derived from
Lambda Fix II genomic inserts, together encoded exons 1-6 and about 8 kb of DNA 5' to exon 1. The sequence encoded by exons 1-6 correspond
to about two-thirds of the open reading frame, or about half of the
sequence that makes up the entire human cDNA.
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Fig. 4.
Organization of the human BHMT gene. The
positions of the overlapping genomic clones and fragments thereof, and
exon organization, are shown.
-derived pEP-10-1.5 clone and
encoded exon 7 and part of the 5' region of intron 7. The PAC D11 clone
was also used to PCR amplify the remaining 3'-end of intron 7 and exon
8, the latter being the last exon of the human BHMT gene.
Exon-intron, organization of the human BHMT gene
77 relative to the start ATG is the major transcriptional
start site used in liver.
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Fig. 5.
Nucleotide sequence of the 5'-flanking region
of the human BHMT gene and exon 1. The first exon is shown in
boldface. The 5' untranslated region is shown in
lowercase, and the major transcriptional start site is
identified with an asterisk. A putative TATA box centered 26 nucleotides upstream from the major transcriptional start site is
double-underlined. Other putative transcription factor
binding sites are underlined and include stimulating
protein-1 (Sp1) ( 11,
67,
121,
169,
179,
204,
347,
394,
610,
979,
1186,
1219,
1845,
2098,
2650,
2658, and
2887), glucocorticoid receptor (GR) (
250,
418,
429,
527,
539,
563,
714,
809,
966,
1088,
1188,
1212,
1249,
1273,
1300,
1324,
1401,
1405,
1494,
1607,
1994,
2140,
2304,
2307,
2428,
2450,
2465,
2476,
2550,
2554,
2631,
2681,
2818,
2906,
2921,
2941,
2944,
3005,
3044,
3111,
3119, and
3134), progesterone receptor (
966,
1273,
1300,
2476,
2941,
3005,
3111, and
3134), estrogen
receptor (
366,
626,
1300,
1616,
2476,
2825,
2941, and
2978), androgen receptor (
1300,
2476,
2941,
3111, and
3134), HNF-1 (
1726), HNF-3: (
2605), CAAT enhancer-binding protein
(
590,
606,
1217,
1585,
1692,
1893, and
2333), activator
protein-1 (
365,
458,
473,
625,
1047,
1280,
1456,
1513,
1616,
1643,
2266,
2824,
2960,
2978, and
3120), activator
protein-2 (AP-2) (
126 and
2071), homeobox 4c, 4d, and 4e (
871),
and ETS-related proteins 38, 49, and 55, (p38erg, p49erg, and p55erg)
(
2900).
DISCUSSION
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ACKNOWLEDGEMENTS |
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We thank Jacob D. Mulligan, Linda S. Garrow, Norman S. Millian, Margaret A. Griffiths, and Murty S. Renduchintala for technical assistance. We also thank Dr. Rima Rozen (McGill University, Montreal, Quebec, Canada) and Dr. Steve Scherer (Medical Resource Council of Canada Genome Resource Facility at the Hospital for Sick Children, Toronto, Ontario, Canada) for the P1 artificial chromosome clones and Dr. Marc Solioz (University of Berne, Switzerland) for the rat BHMT cDNA clone. We also thank Chris Perry for help in the preparation of the manuscript.
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FOOTNOTES |
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* This research was supported in part by the University of Illinois Agricultural Experiment Station Grant 60-0305; NIDDK, National Institutes of Health Grant 52501; and a predoctoral fellowship from the American Heart Association, Illinois Affiliate (to E. I. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF118371-AF118378.
To whom correspondence should be addressed: 463 Bevier Hall, 905 S. Goodwin Ave., University of Illinois, Urbana, IL 61801. Tel.:
217-333-8455; Fax: 217-333-9368; E-mail: t-garro{at}uiuc.edu.
2 A. P. Breksa and T. A. Garrow, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: BHMT, betaine-homocysteine methyltransferase; DMAT, dimethylacetothetin; DMPT, dimethylpropiothetin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Hcy, homocysteine; RACE, rapid amplification of cDNA ends; PAC, phage 1 artificial chromosome; PCR, polymerase chain reaction; HNF, hepatic nuclear factor; bp, base pair(s); kb, kilobase pair(s).
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REFERENCES |
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