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Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, and Hypertension-Endocrine Branch, National Heart, Lung, and Blood Institute (H.C., M.J.Q.), National Institutes of Health, Bethesda, Maryland 20892-1770
Address all correspondence and requests for reprints to: Dr. Marc Reitman, Diabetes Branch, Building 10, Room 8N-250, 10 Center Drive, MSC 1770, Bethesda, Maryland 20892-1770. E-mail: mlr{at}helix.nih.gov
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and
mediated trans-activation by C/EBP, -ß, and -
.
Mutation of a consensus Sp1 site reduced promoter activity 2.5-fold and
abolished binding of Sp1. Mutation of a fourth factor-binding site,
denoted LP1, abolished protein binding and reduced promoter activity
2-fold. Factor binding to the LP1 motif was observed with adipocyte,
but not with nonadipocyte extracts. Adipocytes from
fa/fa Zucker rats transcribed the reporter plasmids more
efficiently than did control adipocytes. No effect on the transient
expression of leptin was noted upon treatment with a thiazolidinedione,
BRL49653, or upon cotransfection with peroxisome proliferator-activated
receptor-
/retinoid X receptor- or sterol response element-binding
protein-1. Mutations of the Sp1, LP1, and C/EBP sites in pairwise
combinations diminished promoter activity to the extent predicted
assuming these motifs contribute independently to leptin promoter
function. Our identification of motifs regulating leptin transcription
is an important step in the elucidation of the mechanisms underlying
hormonal and metabolic regulation of this gene. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice homozygous for a nonsense mutation in the leptin gene (lepob/lepob) show a 20-fold increase in leptin RNA levels (1), suggesting that the leptin gene is subject to transcriptional regulation. Similarly, mutations in the leptin receptor (leprdb/leprdb mice and fa/fa rats) cause increased leptin RNA. Leptin levels are regulated by factors in addition to adiposity. Protein and RNA levels decrease in response to ß-adrenergic agonists or starvation and are increased by glucocorticoids or insulin (9, 10, 11). To understand leptin’s transcriptional regulation, we isolated the leptin promoter (12) and report here its detailed characterization.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 4 for base changes) are named using the base number of the
3'-most base in the mutation, with the sequence and cap site determined
previously (12); there are slight numbering differences between these
and those previously reported (15, 16). Our laboratory designations for
these plasmids are: m7, p1761; m16, p1760; m21, p1757; m27, p1581; m47,
p1649; m52, p1594; m53, p1645; m59, p1647; m67, p1579; m85, p1797; m95,
p1578; m109, p1799; m135, p1803; m47,59, p1651; m52,85, p1834; m52,95,
p1801; and m85,95, p1848. Plasmids p(-135)lep-luc (p1809),
p(-109)lep-luc (p1807), p(-95)lep-luc (p1583), p(-85)lep-luc
(p1805), p(-67)lep-luc (p1585), p(-52)lep-luc (p1589), and
p(-27)lep-luc (p1587) were constructed by digestion with
HindIII and religation of plasmids m135, m109, m95, m85,
m67, m52, and m27, respectively. PCR-generated regions were
confirmed by sequencing.
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expression plasmid was provided by C. Vinson (17).
Expression plasmids for C/EBPß (p1607) and C/EBP (p1608) were
constructed by insertion of an 850-bp EcoRI fragment of
pMEX-CRP2 (C/EBPß) or a 850-bp BamHI fragment of pMEX-CRP3
(C/EBP) with HindIII linkers (18) into
HindIII-digested pRc/cytomegalovirus (CMV; Invitrogen, San
Diego, CA).
Transient expression
Transient expression in primary rat adipocytes (CD strain,
Charles River Laboratories, Wilmington, MA) (12, 19) and luciferase
(Promega Luciferase Assay System) and chloramphenicol acetyltransferase
(CAT) assays (20) were performed as previously described. Two
independent clones were assayed for each construct. Rous sarcoma virus
(RSV)-CAT was used as an internal control. Results are expressed as a
percentage of the activity of p(-762)lep-luc in the same experiment
(e.g. 100 x
(luciferaseexp/CATexp)/(luciferasep(-762)lep-luc/CATp(-762)lep-luc)
and and are the mean ± SEM of the indicated number of
experiments performed in duplicate or triplicate. Results have been
normalized to the number of moles of plasmid transfected. To avoid cell
breakage, manipulations were performed more gently with adipocytes from
fa/fa Zucker rats. For example, only gentle shaking every 15
min was used during the collagenase digestion. The electroporation
protocol uses a constant volume of cells; thus, the cell number of
fa/fa Zucker adipocytes transfected was smaller.
HeLa cells were transiently transfected using Lipofectamine (Life Technologies, Grand Island, NY) as described by the manufacturer. The internal control was pRL-CMV (Promega), and the dual luciferase assay system (Promega) was used.
Electrophoretic mobility shift analysis
Electrophoretic mobility shift assays were performed as
previously described (21, 22) except for the following. Adipose cells
were lysed (23) (without the Polytron), and nuclei and nuclear extracts
were prepared (24). Protein concentrations were determined (Bio-Rad
Protein Assay, Hercules, CA), and binding reactions were performed in
25 mM HEPES (pH 7.5), 16 mM KCl, 50
mM NaCl, 1 mM MgCl2, 2
µM ZnCl2, 1 mM dithiothreitol, 40
µg/ml BSA, 0.01% Nonidet P-40, and 8% glycerol. Electrophoresis in
4% or 6% polyacrylamide gels used 0.5 x TBE. Polyclonal
antisera to Sp1, Sp3, and Sp4 (1 µg; Santa Cruz Biotechnology, Santa
Cruz, CA) was added after the DNA and then incubated for 60 min (4 C)
before electrophoresis. Figure 4
describes the wild-type
oligonucleotide sequence and mutations used in the C/EBP binding
assays. Other oligonucleotides are (only one strand is shown):
aP2 (25) (x312/x313), 5'-AACCAAAGTTGAGAAATTTCTATTAAAAAC; wt95
(x314/x315), 5'-GCCCGCTGGGTGGGGCGGGAGTTGGCGCTC; m95 (x267/x271),
5'-GCCCGCTGGGTGaaGCttGAGTTGGCGCTC; wt85 (x414/x415),
5'-AGTTGGCGCTCGCAGGGACTGGGGCTGGCC; wt85a (x490/x491)
5'-GGGGCGGGAGTTGGCGCTCGCAGGGACTGG; and m85 (x408/x409),
5'-GGGGCGGGAGTTaagctTCGCAGGGACTGG. Methylation interference analysis
was performed essentially as previously described (13).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Seven plasmids containing between
about 6700 and 109 bp of 5'-sequence showed a 2.2-fold range in
reporter activity. More dramatic decreases in promoter activity were
observed on deletion from -109 to -95, from -85 to -67, and from
-67 to -52. Deletion from -52 to -27 may also decrease expression,
but this could not be assessed due to the already low activity of the
-52 deletion. These data are consistent with small, but significant,
effects on expression from regions upstream of -109 and identify three
promoter regions between -109 and the TATA motif that contribute
strongly to promoter activity.
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Leptin promoter point mutants
To define more accurately the specific bases contributing to
proximal promoter function, a series of clustered point mutants was
tested for promoter activity. In the regions implicated by the deletion
mutants, sequences conserved between mouse and human were chosen for
mutation (Fig. 2). The region between the
TATA and cap sites was strikingly conserved (more so than exon 1 or
other promoter regions), but mutations in this region (m7, m16, and
m21) did not have a large effect on promoter activity. Presumably our
transient expression assay is insensitive to the conserved function(s)
of this region. Mutations in two nonconserved regions (m67 and m135)
served as controls and had little effect on promoter activity.
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Transient expression in fa/fa Zucker adipocytes
To look for adiposity-mediated regulation of leptin expression, we
transfected the leptin promoter constructs into adipose cells from
fa/fa Zucker rats. These rats have a mutated leptin receptor
(26), greatly increased adipose stores, and increased leptin RNA
levels. Due to their larger size, fewer fa/fa cells are
contained in the volume used for transfection. Consistent with the
fewer number of cells, luciferase and CAT activities were
proportionately lower in the fa/fa cells. To allow
comparison between these two cell types, each assay included samples
transfected with RSV-luc and CMV-luc. However, the RSV-luc and CMV-luc
reporters were expressed at different levels in the fa/fa
and CD cells. In Table 1, we present the
leptin promoter activity normalized to RSV-luc and CMV-luc, as it is
not clear which is the appropriate choice. Leptin promoter activity was
2- or 7-fold higher in the fa/fa cells (depending on whether
normalization was to RSV-luc or CMV-luc).
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). The numbers at the
top of Fig. 3 are the ratio of mutant promoter activity in
fa/fa cells relative to that in CD cells, normalized so that
the ratio of p(-762)lep-luc is 1, thus removing any assumptions about
transfection efficiency or relative strength of the control reporter.
The shorter promoters were expressed relatively better in
fa/fa cells. This suggests that shorter promoters are
sufficient for maximal activity in the fa/fa cells, whereas
the CD cells need a longer promoter. To examine the role of the C/EBP
and Sp1 motifs, the promoter activity of point mutations in these
motifs was tested. Expression from plasmids m52 and m95 was reduced to
11.5 ± 1.8% (n = 5) and 31.9 ± 1.1% (n = 4)
that of p(-762)lep-luc, respectively. Thus, individual C/EBP or Sp1
mutations in the context of the 762-bp promoter had similar effects in
fa/fa Zucker and CD adipocytes. Taken together, these data
suggest that the leptin promoter is transcribed more efficiently in
fa/fa cells, with a disproportionate contribution from the
proximal promoter (although neither the C/EBP nor the Sp1 motif
contributes disproportionately).
Analysis of the C/EBP-binding region
We previously identified the leptin promoter C/EBP motif and
reported that C/EBP coexpression increased leptin promoter activity
(12). We have now undertaken a detailed analysis of this motif. Binding
of recombinant C/EBP (17) to this region was studied using
electrophoretic mobility shift experiments. The C/EBP protein bound
the leptin C/EBP motif with high avidity, comparable to that for the
C/EBP site in the aP2 promoter (25) (Fig. 4a and data not shown). Mutations within
the leptin promoter C/EBP motif reduced (m53) or abolished (m52)
C/EBP binding (Fig. 4a). Competition experiments confirmed these
results and were consistent with a 10- to 100-fold reduction in
C/EBP binding by the m53 site (data not shown). Mutations abutting
the C/EBP motif (m47, m59, and m47,59) had no effect on C/EBP
binding (Fig. 4a).
Transient expression experiments showed a correlation between C/EBP
binding and promoter activity (Fig. 4b), suggesting that C/EBP factors
function at this site in cells. Two E box motifs (CAnnTG), similar to
sites used to regulate genes important in metabolism (27), overlapped
the C/EBP site. However, mutations of these E boxes (m47, m59, and
m47,59) did not affect promoter activity.
We next examined the ability of two other C/EBP family members to
trans-activate the leptin promoter. Cotransfection with
C/EBPß or C/EBP also stimulated transcription (Fig. 4c).
Obliteration of the C/EBP site (m52) abolished
trans-activation, whereas the mutant with a less severely
mutated site (m53) could still be trans-activated, albeit at
a reduced level and with a shifted dose-response curve (Fig. 4c and
data not shown). Taken together, these data demonstrate that the C/EBP
site is of fundamental importance for leptin promoter activity.
We also tested C/EBP trans-activation of
leptin promoter deletion constructs. Remarkably,
p(-67)lep-luc (the minimal C/EBP and TATA promoter) was
trans-activated about 800-fold, compared with approximately
25-fold for p(-762)lep-luc (12). Thus, with cotransfected C/EBP,
these two plasmids showed a similar absolute level of luciferase
expression. The p(-67)lep-luc plasmid is one of the most
C/EBP-responsive constructs known. The strong
trans-activation by C/EBP of p(-67)lep-luc suggests that
upstream elements may modify C/EBP action in the intact
promoter.
Analysis of the Sp1-binding region
The site centered at -97 is an exact match to the Sp1 core motif
sequence. To test for protein binding to this region, the binding of
recombinant Sp1 and that of rat adipocyte nuclear extracts was
examined. The recombinant Sp1 bound well to the -97 region, but less
avidly than to the highest affinity Sp1 sites from the simian virus 40
promoter (Fig. 5, lanes 1–6), probably
due to bases -93A (G binds better) and -92G (C or T bind better)
(28). Electrophoretic mobility shift assays using adipocyte nuclear
extracts showed a complex of the expected mobility for Sp1-DNA and
faster migrating complexes (Fig. 5, lanes 7–14). Three independent
nuclear extracts gave similar results. Mutation m95 abolished all
binding to this region (Fig. 5, lane 15), as did competition with
unlabeled wt95 DNA (not shown). To confirm that Sp1 was responsible for
the slowest complex, antibody to Sp1 was used to specifically retard
the mobility of Sp1-DNA complexes. Most of the putative Sp1-DNA
complexes were indeed reactive with anti-Sp1 (Fig. 5, lanes 7–14) and
not with other antibodies (to Sp3, Sp4, or chicken globin; data not
shown). Taken together, these data suggest that Sp1 is the predominant
protein binding to the -97 region of the leptin promoter and that the
2.5-fold reduction in expression in m95 is due to the loss of this
factor’s contribution.
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, lanes 16–21). Similar patterns were obtained with three
independent nuclear extracts and with the corresponding region of the
human leptin promoter. Complex formation was inhibited by unlabeled
oligonucleotide, but not by an oligonucleotide containing an Sp1 motif.
Mutation m85 abolished protein binding (Fig. 5, lane 21), and anti-Sp1
antibodies did not affect complex formation (data not shown). Weak
binding was observed with nuclear extracts from undifferentiated 3T3-L1
preadipocytes, which was unchanged in differentiated 3T3-L1 adipocytes
(data not shown). A protein-DNA complex was not seen when nuclear
extracts from HeLa and K562 cells was used (Fig. 5, lanes 23 and 25).
These data are consistent with an adipose specificity for binding to
this site.
Binding at the LP1 site was examined further using methylation
interference (Fig. 6). Methylation of
residues at positions -81, -82, -83, -85, -86, -88, -89, and
-90 relative to the cap site inhibited factor binding. This binding
site (GGCGCTCGC) is not an obvious match to known consensus
sequences.
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). These data suggest that the Sp1, LP1,
and C/EBP sites contribute independently to promoter activity; one does
not require another for function.
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(PPAR) and sterol response element-binding
protein-1 (SREBP) is a steroid superfamily transcription factor that
promotes differentiation into adipocytes (29). The thiazolidinedione
class of antidiabetic drugs is made up of activating ligands for
PPAR (30). There are a number of reports that thiazolidinediones
decrease leptin expression (31, 32, 33, 34, 35). We investigated the roles of
PPAR and thiazolidinediones in leptin transcription using transient
expression in rat adipocytes. Luciferase activity of p(-762)lep-luc
and m52 was not significantly changed in the presence of the
thiazolidinedione ligand BRL49653 at 10 µM (127% and
115%, respectively, of the untreated control value; mean of two
experiments). Cotransfection with PPAR and retinoid X receptor-
(RXR), its dimerization partner, either with or without BRL49653 was
also performed (Table 3). Two
PPAR-responsive reporters showed high basal levels of expression.
These data suggest that the adipocytes have high levels of endogenous
factors acting via the PPAR (DR+1) motif. Inclusion of PPAR/RXR
(with or without BRL49653) did not alter expression of the leptin
reporters, but, as expected, did increase expression of thymidine
kinase-PPREx3-Luc control. Similar results were obtained in HeLa cells
(Table 3). In these transient expression systems, we did not find a
major role for a thiazolidinedione or for exogenous PPAR in the
regulation of leptin.
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). Expression of the activated
transcription factor protein, SREBP-(1–410), increased leptin
expression about 2-fold, but also increased RSV-CAT expression slightly
while massively increasing expression of the positive control. Thus, in
this system, SREBP is not a major regulator of leptin expression.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To examine these mechanisms, it is necessary to understand the leptin promoter. We show that a 109-bp promoter is as effective as longer promoters in directing leptin transcription in transient expression assays. Four elements in the proximal 109 bp contribute to leptin promoter activity: the TATA box at -30, a C/EBP motif at -53, the LP1 region at -87, and an Sp1 motif at -97. The data are consistent with a small effect on adipose expression of more distant regions. No distant elements with a large effect on adipose expression have been identified, although a placental enhancer is found upstream of the human leptin promoter (39). It seems plausible that a distant element(s) with a large effect on adipose expression also exists.
PPAR ligands have been shown to have a small negative influence on
endogenous leptin expression. In transient expression assays, a slight
decrease in leptin expression by PPAR ligands has been observed (34, 35). Our inability to see this effect could be due to the small
magnitude of the effect, subtle differences between the reporter
plasmids, or other differences between the model systems.
C/EBP regulation of the leptin promoter
C/EBP is a basic region/leucine zipper transcription factor
important for the transcription of most adipocyte genes and of other
genes involved in energy metabolism (40). Before adipocyte
differentiation, C/EBP, -ß, and - levels are low. During
differentiation, first C/EBPß and - rise transiently, and then
C/EBP levels rise and remain high in the mature adipocyte (41).
Forced expression of C/EBP promotes adipogenesis (42, 43), and mice
with a nonfunctional C/EBP gene do not deposit lipid in their
adipose tissue (40).
Since the suggestion that C/EBP stimulated leptin expression via the
-53 motif (12), supporting evidence has come from a number of studies
(15, 16, 34, 44). Here we have expanded these observations by showing a
correlation between C/EBP binding affinity to the -53 site and the
degree of trans-activation. Furthermore, we demonstrate that
mutation of adjacent nucleotides has no effect, and that C/EBPß and
-, in addition to , can trans-activate via this motif.
These results suggest that the -53 C/EBP motif contributes to the
tissue-specific expression of the leptin gene. As C/EBP is the
predominant C/EBP family factor in mature adipocytes, it is likely that
in vivo this factor acts at this C/EBP site. However, there
are at least eight C/EBP-related proteins (45), so it is possible that
other family members also function at this site in vivo.
Sp1 regulation of the leptin promoter
The site at -97 of the leptin promoter is conserved in evolution,
binds Sp1 present in adipocyte nuclear extracts, and contributes to
promoter activity. Although these data cannot rule out the possibility
that other C2H2 transcription factors might also act at this site, the
simplest interpretation is that Sp1 is trans-activating the
leptin promoter via this motif.
In a hepatocyte cell line cotransfected with C/EBP, de la Brousse
et al. (16) did not observe a decrease in promoter activity
upon deletion of the Sp1 site. When we cotransfected C/EBP in
adipocytes, we obtained similar results. However, in our experiments
without C/EBP cotransfection, we saw a decrease in activity upon
either deletion of this region or point mutation of the Sp1 motif.
These data suggest that overexpression of C/EBP obscures the
contribution of the Sp1 element to leptin transcription.
Other promoters [e.g. GLUT4 (46), CYP2D5 (47), and C/EBP
(48, 49)], like leptin, are regulated via both C/EBP and Sp1-like
motifs. However, none of these appears similar enough to the leptin
promoter to allow inferences about its regulation.
Regulation of the leptin promoter by the LP1 region
The sequence of the -87 region of the leptin promoter is
conserved between mouse and human, suggesting that this site is
functional. Indeed, mutation of the region caused a decrease in
expression, and this site bound a factor present in preadipocytes and
adipocytes but not in other cell types. The binding motif does not
match that of other known transcription factors. Thus, the data suggest
that the LP1 region binds a novel trans-activating factor
that is present in adipose cells but not in the other cells
examined.
Regulation of leptin expression by adiposity, metabolites, and
hormones
Transiently expressed leptin reporters showed increased activity
in fa/fa Zucker adipocytes. These data are consistent with
cell autonomous regulation of leptin expression by increased adiposity.
Higher levels of promoter activity in fa/fa adipocytes have
been observed for other genes [GLUT4 (50) and
GAPDH (51)]. The increased fatty acid synthetase expression
in fa/fa adipocytes is due to inhibition of expression in
lean cells by a factor binding to a Sp1 site (52). Our data are not
consistent with such a mechanism for regulation of the leptin promoter.
Indeed, other than an increased contribution to leptin expression by
the proximal promoter, we have not been able to identify specific
sequence motifs involved in the increased expression in
fa/fa adipocytes.
The identification of three functional motifs in the leptin promoter
raises an obvious question. Does regulation of leptin expression by
hormones and metabolites occur via modification, in amount or activity,
of the factors that bind to these sites? One hint that it may is the
observation that expression and phosphorylation of C/EBP family members
is regulated by both glucocorticoids and insulin (53, 54). We do not
yet know whether the increase in leptin expression by these hormones is
accomplished by modification of C/EBP expression or activity.
Sp1, another potential target for regulatory cascades, is also a phosphoprotein. Increased Sp1 phosphorylation has been reported to facilitate transcription (55), whereas Sp1 dephosphorylation has been reported to increase its binding affinity for DNA (56). The factor(s) binding to the LP1 motif may also be a target of the regulatory signals converging on the leptin promoter.
The elucidation of the functional DNA elements of the leptin promoter and their cognate transcription factors presented here is a significant step toward a detailed understanding of the transcriptional regulation of the leptin gene.
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Acknowledgments |
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protein and
plasmids, and Drs. S. Bi, O. Gavrilova, D.-W. Gong, D. LeRoith, and S.
Taylor for comments on the manuscript. |
Footnotes |
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2 Scholar of the Lucille P. Markey Charitable Trust.
Received July 18, 1997.
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as are those reduced more than 85% (•).