Endocrinology and Hormonal Biochemistry
units, Hospital Clínic, Institut d'Investigacions
Biomèdiques August Pi i Sunyer, Barcelona, Spain 08036
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INTRODUCTION |
Hepatic nuclear factor 1-
(HNF1-) is an atypical homeodomain-containing protein which was
first identified based on its ability to bind to critical regulatory
cis elements present in the 5'-flanking region of
liver-specific genes, such as the albumin, 
-fibrinogen, and
1-antitripsin genes (9, 12, 18). It was initially
regarded as a hepatic-specific transcriptional regulator but was later
found to be expressed in other tissues, such as kidney, gut, and
pancreas (4). More recently, heterozygous mutations in the
gene encoding HNF1- were identified as the most common cause of
monogenic diabetes mellitus in humans (44). The discovery
resulted from a positional cloning strategy with no clear a priori
evidence for a role of HNF1- in regulating glucose homeostasis
(39). Interestingly, although HNF1- is expressed
in multiple tissues, the sole phenotypic abnormality identified to date
in humans with mutations in the gene encoding HNF1- is pancreatic
-cell dysfunction (4, 8). This suggests that HNF1-
must perform an essential cell-restricted role in regulating
transcription of key genes in pancreatic insulin-producing cells.
Targeted disruption of the hnf1- gene has been attained
by two laboratories (23, 28). Although young heterozygous
mutant mice do not have a distinct phenotype, hnf1-
homozygous null animals are small and have abnormal liver function,
developing phenylketonuria due to the lack of hepatic transcription of
phenylalanine hydroxylase (28, 29). These mice display
glycosuria secondary to renal tubular dysfunction, and like humans with
heterozygous mutations in the gene encoding HNF1-, they develop
overt diabetes (23, 30). Also in analogy to the human
phenotype, nullizygous mice have severely blunted glucose-induced
insulin secretion, resulting at least in part from abnormal aerobic
glucose metabolism in pancreatic -cells (14, 30).
Nevertheless, the molecular defects which underlie this -cell
glucose sensing abnormality are not known. In fact, to date no distinct
gene has been reported to be abnormally regulated in -cells from
mice lacking HNF1-. However, in studies carried out with a clonal
-cell line, overexpression of HNF1 dominant-negative mutants
(41, 42) has been shown to lead to a marked decrease in
the mRNAs encoding insulin, the type 2 glucose transporter isoform,
liver-type pyruvate kinase, aldolase B, 3-hydroxymethylglutaryl
coenzyme A reductase, and mitochondrial 2-oxoglutarate. These genes
represent strong candidates to explain -cell dysfunction in mice and
humans with mutations in the gene encoding HNF1-. It is reasonable
to assume that understanding the intimate molecular mechanisms involved
in the transcriptional control of -cell genes by HNF1- could
provide a rational basis to manipulate -cell function in humans with
mutations in the gene encoding HNF1- or other forms of diabetes.
The molecular mechanisms involved in how HNF1- controls
transcription are still poorly understood. However, there is evidence which suggests that HNF1- may be responsible for regulating the chromatin dynamics of its target genes (29, 31, 32). In hnf1-
/ mice transcription of the
phenylalanine hydroxylase gene (pah) is abolished in liver
tissue, and this is associated with an abnormal methylation pattern of
CpG islands and disappearance of nuclease hypersensitivity sites in the
pah gene 5'-flanking region in hepatic cells
(29). Furthermore, HNF1- is necessary to elicit
chromatin opening and transcription of the -antitrypsin gene cluster
in a hepatoma cell line in vitro (31). Although the
molecular basis for these HNF1--dependent phenomena is still not
understood, more recent work has shown that HNF1- can interact with
coactivator proteins which possess histone acetyltransferase (HAT)
activity, such as CREB binding protein and P/CAF, and that this HAT
activity is important for the potentiation of the effects of HNF1-
on a reporter minigene containing multimerized HNF1- binding sites (32). An emerging notion derived from these studies is
that HNF1- could bind its cognate DNA element in target genes and then physically interact with HAT coactivator proteins and hence induce
local conformational changes which ultimately lead to gene activation
in keeping with the prevailing view, whereby histone acetylation is
likely to be instrumental in transcriptional activity (for reviews, see
references 34 and 43). It should be pointed out nevertheless that evidence that HNF1- does, in fact, play a role
in the acetylation of chromosomal histones is currently lacking.
In this study we have investigated the mechanisms involved in the
control of -cell-specific transcription by HNF1-. This was done
by using mice lacking HNF1- (23) rather than by relying on cultured cell systems based on overexpression of transcription factors and episomal reporter plasmids. We first identified genetic targets which are dependent on HNF1- exclusively in pancreatic -cells, indicating that HNF1- plays a -cell-specific
transcriptional regulatory role in vivo. Using chromatin
immunoprecipitations, transcription factor occupancy and histone
acetylation of the promoters of these -cell genes and a known
hepatic HNF1- target were analyzed in the different tissues. The
results revealed that promoter occupancy by HNF1- in islets and
liver tissue does not correlate with its requirement for gene
expression. However, a close linkage was observed between
tissue-specific HNF1--dependent gene activity and localized histone
hyperacetylation. These findings provide for the first time evidence
based on a live mammalian system to support the physiological relevance
of the notion that a single activator can be essential to target HAT
activity to promoters as a mechanism to induce transcription.
Furthermore, they provide in vivo evidence to indicate that
gene-specific regulation by HNF1- is likely mediated by its ability
to acetylate nucleosomal histone tails.
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MATERIALS AND METHODS |
Animal breeding and genotyping.
A colony of mice segregating
a null hnf1- allele on a C57BL/6J background was
established locally. hnf1- null mice were generated in
the laboratory of Frank Gonzalez (National Institutes of Health,
Bethesda, Md.) by Cre-loxP recombination and deletion of the first exon
and have been previously described (23). Animals were kept
in a controlled environment and genotyped by PCR coamplification of
mouse -globin and hnf1- exon 1 sequences.
Pancreatic islet and hepatocyte isolation.
Four-week-old
hnf1-/ mice and their age-matched
heterozygote (+/
) and wild-type (+/+) siblings were anesthetized with
urethane (15% solution, 1 ml/kg) and decapitated. Pancreatic islets
were isolated by a modification of procedures previously described for
rats (17). Briefly, a cannula was inserted in the main
pancreatic duct and the pancreas was inflated with Hanks' balanced
salt solution (HBSS) buffer containing 3 U of collagenase P (Roche) per
ml. The pancreas was then removed and digested for 11 min at 37°C in
a water bath with continuous agitation. The cell suspension was washed
several times in cold HBSS-0.5% bovine serum albumin (BSA), and
islets were purified by using a discontinuous gradient in which the
lower phase was formed by a Histopaque mixture containing a 7:3 ratio
of Histopaque 1077 and Histopaque 1119 (Sigma), and the upper phase was
HBSS-0.5% BSA. The islet-enriched fraction was aspirated from the
interface, washed three times in HBSS-0.5% BSA, and further purified
by handpicking under a stereomicroscope.
Mouse hepatocytes were isolated by in situ collagenase digestion of the
liver (2). A cannula was inserted in the portal vein and
the liver was perfused sequentially with phosphate-buffered saline
(PBS) supplemented with 0.5 mM EGTA, HBSS-0.5% BSA, and at least 20 ml of HBSS buffer containing 5 mM CaCl2 and 0.25 U of
collagenase A (Roche) per ml at 37°C. The liver was then excised and
transferred to a petri dish cooled on ice. Digestion was stopped by the
addition of ice-cold HBSS-1% BSA. The liver was minced and filtered
through a 100-µm-pore-size cell strainer (Falcon). The cell
suspension was centrifuged at 50 × g for 1 min, and
the supernatant was discarded. The pellet was resuspended in complete RPMI medium, and centrifugation was repeated two more times. The final
pellet was resuspended in fresh RPMI and transferred to a petri dish.
RNA extraction and reverse transcription (RT)-PCR analysis.
Total RNA was extracted from 80 to 100 mg of liver, kidney, or duodenum
tissue, or 25 to 100 isolated islets of individual wild-type and
knockout mice, using TRIzol (Life Technologies, Inc.) according to the
instructions of the manufacturer. Total RNA (120 ng for liver, kidney,
and duodenum, 24 ng for islets) was heated at 70°C for 10 min in the
presence of 10 ng of random primers (Promega) per µl and
reverse-transcribed into cDNA in a 20-µl solution containing 3 mM
MgCl2, 1 mM deoxynucleoside triphosphates, 10 mM
dithiothreitol, and 200 U of SuperScript II reverse
transcriptase (Life Technologies, Inc.). Reaction mixtures were
incubated for 10 min at 25°C, 1 h at 42°C, and 10 min at
97°C. PCR was carried out with oligonucleotides designed to span an
intron to detect genomic DNA contamination using Primerselect 4.0 (DNASTAR) software. Sequences and specific PCR amplification conditions
are available upon request. Gene products of interest were coamplified
with an internal control gene (-actin or tbp).
The cycling and reaction conditions were adjusted by performing test
reactions at multiple cycles to ensure that the two products were in
the exponential phase of amplification and showed similar amplification
efficiencies. Amplification products were resolved on ethidium
bromide-stained acrylamide gels, and low-exposure images were analyzed
using ScionImage (Scion) software. To coamplify insulin I and II cDNAs,
oligonucleotide primers with full homology to both mouse genes were
designed, and the two amplification products were discriminated by
selective restriction of the insulin II gene using BstEII.
All reactions included a blank control (without cDNA) in addition to
controls lacking reverse transcriptase.
Immunohistochemistry.
Mouse tissues were dissected, fixed in
fresh 4% paraformaldehyde overnight, and then embedded in paraffin.
Three-micrometer sections of paraffin-embedded tissue were
deparaffinized in xylene, rehydrated through a serially diluted ethanol
sequence, and heated in a microwave for 5 min in 0.01 M citrate buffer
(this step was omitted when using glut2 antiserum). Sections were
incubated for 30 min at room temperature in antibody diluent (DAKO
Corporation) with 3% normal serum from the same species as the
secondary antibody. The primary antibody (dilutions: insulin, 1:1,000;
glucagon, 1:200; glut2, 1:200; and pdx1/idx1, 1:1,000) was added in
DAKO antibody diluent and incubated overnight at 4°C. Slides were
washed in PBS and incubated with the fluorescent-conjugated secondary
antibodies (Alexa Fluor 488 and 546 from Molecular Probes and Cy3 from
Jackson Immunoresearch) at a final dilution of 1:500 in DAKO antibody diluent for 45 min at room temperature. After being washed in PBS, the
sections were mounted and coverslipped using ProLong Antifade mounting
media (Molecular Probes). Images were collected by using a Leica TCS 4D
confocal microscope and processed using Adobe Photoshop 5.0 (Adobe
Systems). Guinea pig anti-insulin, rabbit anti-glut2, and rabbit
anti-idx1 were kind gifts from Chris Van Schravendijk (Vrije
Universiteit Brussel, Brussels, Belgium), Bernard Thorens (University
of Lausanne, Lausanne, Switzerland), and Dr. Joel Habener (Howard
Hughes Medical Institute, Massachusetts General Hospital, Boston,
Mass.), respectively. Rabbit anti-glucagon was obtained from DAKO.
Formaldehyde cross-linking and immunoprecipitation of
chromatin.
The formaldehyde cross-linking and immunoprecipitation
of chromatin procedure was adapted from existing protocols for use in
small amounts of cells isolated from mammalian tissues (5, 21,
26). Formaldehyde (37% HCHO-10% methanol stock solution; Merck) was diluted to 11% in a buffer containing 0.1 M NaCl, 1 mM
EDTA, 0.5 mM EGTA, and 50 mM HEPES (pH 8.0) and added directly to
freshly isolated cells maintained in culture medium (150 to 500 pancreatic islets or 2 × 106 hepatocytes), providing
a final concentration of 1%. Fixation was allowed to proceed for 6 min
at 22°C for acetylated histone immunoprecipitations or 10 min at
22°C followed by 20 min at 4°C for HNF1- immunoprecipitations.
Fixation was stopped by the addition of glycine to a final
concentration of 0.125 M. Cells were collected by centrifugation,
washed once in 1 ml of ice-cold PBS supplemented with 1 mM
phenylmethylsulfonyl fluoride (PMSF) and protease inhibitors (Complete
protease inhibitor cocktail; Roche), and swelled in 0.5 ml of 5 mM
piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) (pH 8.0),
85 mM KCl, 0.5% NP-40, 1 mM PMSF, and protease inhibitors for 15 min
on ice. Approximately one volume of 400- to 625-µm glass beads
(Sigma) was then added, and samples were incubated for 10 min on a
vortexer at 4°C. Samples were separated from the glass beads by
covering the upper end of the tube with a nylon mesh and centrifuging
it inverted inside a 15-ml tube at 1,000 × g for 2 min. Pellets were resuspended in the same supernatant, transferred to a
fresh 1.5-ml centrifuge tube, and pelleted again by microcentrifugation
at 20,000 × g. Pellets were resuspended in 150 to 400 µl of sonication buffer (1% sodium dodecyl sulfate [SDS], 10 mM
EDTA, 50 mM Tris-HCl [pH 8.1], 1 mM PMSF, and protease inhibitors)
and incubated on ice for 15 min. Samples were then sonicated to an
average length of 1,000 to 2,000 bp with a Bandelin sonifier for six
30-s pulses at microtip maximum output and microcentrifuged at
20,000 × g for 1 h. The efficiency of the
fragmentation was always verified by purification of DNA and analysis
on a 1% agarose gel stained with ethidium bromide, which typically
yielded fragments spanning 500 to 2,000 bp (see Fig. 4A). The resulting
supernatants were diluted 10 times in IP buffer (0.01% SDS, 1.1%
Triton X-100, 1.2 mM EDTA, 167 mM NaCl, 16.7 mM Tris-HCl [pH 8.1], 1 mM PMSF, and protease inhibitors). This chromatin solution was
precleared with 50% protein A Sepharose (Amersham Pharmacia Biotech)
containing 0.1% BSA and 200 µg of sonicated salmon sperm DNA per ml
for 30 min at 4°C. Precleared chromatin (500 µl, corresponding to
75 to 150 pancreatic islets or 300,000 hepatocytes) was incubated with
2 µg of affinity-purified rabbit polyclonal anti-acetylated histone
H3 or H4 antibodies (Upstate Biotechnology, Inc.), 1 µg of mouse
monoclonal anti-HNF1- antibody (Transduction Laboratories), or 2 µl of preimmune rabbit serum (Jackson Immunoresearch) and was rotated
at 4°C for 16 h. Two micrograms of rabbit anti-mouse immunoglobulin G (Jackson Immunoresearch) was added to the samples incubated with the anti-HNF1- monoclonal antibody at least 1 h
prior to ending the incubation. Samples were microcentrifuged at
20,000 × g for 5 min and transferred to fresh tubes
prior to addition of 25 µl of protein A Sepharose containing BSA and
sonicated salmon sperm DNA. Incubation was allowed to proceed for
2 h at 4°C, and the samples were then washed sequentially in
low-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl
[pH 8.1], 150 mM NaCl), high-salt buffer (0.1% SDS, 1% Triton
X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 500 mM NaCl), LiCl buffer (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl [pH 8.1]), and three times in TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA). SDS was omitted in the wash buffers for the HNF1- immunoprecipitated samples. Immune complexes were eluted by adding 400 µl of elution buffer (0.1 M NaHCO3, 1% SDS) and being
rotated for 15 min at room temperature. Cross-links were reversed by
the addition of NaCl to a final concentration of 200 mM, followed by
incubation at 65°C for at least 4 h. Samples were then treated with 1 µg of proteinase K for 1 h at 45°C, extracted with
phenol-chloroform, and ethanol-precipitated at 20°C overnight. The
final pellets were resuspended in 25 µl of TE buffer (10 mM Tris-HCl
[pH 7.5], 0.1 mM EDTA) and analyzed by PCR. Precipitated samples were
left undiluted (islets) or diluted 1:3 (hepatocytes). Total input
samples were diluted 1:20, 1:100, and 1:300 before PCR.
PCRs were performed in a 15-µl reaction volume containing 2 µl of
immunoprecipitate or diluted input samples and primer mixtures designed
to coamplify segments located in the transcription initiation site
region of selected promoters. PCR conditions were adjusted to ensure
nonsaturation kinetics and similar amplification efficiencies for all
amplicons within a reaction mixture. Primer mix 1 consisted of
glut2 promoter primers at 1 µM, pah promoter
primers at 0.3 µM, and ins2 promoter primers at 0.6 µM.
Mix 2 consisted of pah promoter primers at 0.3 µM and
pklr exon 1 and ins2 promoter primers at 0.6 µM. Mix 3 consisted of glut2 promoter primers at 0.5 µM and sur1 promoter primers at 1 µM. Mix 4 consisted of
glut2 promoter primers at 0.75 µM and myoD1
promoter primers at 1 µM. Mix 5 consisted of pah promoter
primers at 0.2 µM and myoD1 promoter primers at 1 µM.
Mix 6 consisted of pklr exon 1 primers at 0.5 µM and
myoD1 promoter primers at 1 µM. Amplification conditions
for multiplex PCR were 95°C for 30 s, 60°C for 30 s, and
68°C for 1 min for 27 cycles. PCR products were run on a 9%
acrylamide gel, stained with ethidium bromide, and analyzed using
ScionImage software (Scion). Oligonucleotide sequences of the primer
pairs used are detailed in Table 1.
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TABLE 1.
Primer sequences and locations relative to reported major
transcription start sites corresponding to the PCR products used in the
chromatin immunoprecipitation experiments
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RESULTS |
HNF1- is not essential for insulin mRNA transcription.
In
keeping with previously reported data (23, 30),
hnf1-/ mice develop mild diabetes at an
early stage of postnatal life (data not shown). Immunofluorescence
analysis of islet hormones in the pancreas of 2-week-old
hnf1-/ mice showed normal insulin,
glucagon, and somatostatin staining intensity, a normal density of
insulin-positive and glucagon-positive cells per field, and a conserved
distribution of glucagon-positive cells in the periphery and insulin
cells in the core of islet structures (Fig.
1A and data not shown). Although
immunofluorescence analysis did not suggest a major decrease in insulin
content, previous studies performed with cultured cells suggested that HNF1- may regulate insulin gene transcription (16, 42).
We therefore sought to determine if a lack of HNF1- resulted in a
selective decrease of either insulin I or II mRNA in young animals which have still not developed overt diabetes. A single pair of oligonucleotides with 100% homology to the two nonallelic genes was
designed to simultaneously amplify both sequences, and the gene
products were distinguished by selective BstEII restriction of insulin II. Amplification at different cycle numbers revealed that
the two sequences were being amplified at similar efficiencies. Under
these conditions no major differences were encountered for insulin I
and II mRNAs between purified islets from 4-week-old wild-type and null
mice (Fig. 1B). This suggests that HNF1- is not a major regulator of
insulin gene transcription.
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FIG. 1.
Microscopic islet structure and insulin mRNA content are
not altered in pancreas tissue of hnf1-/
mice. (A) Representative paraffin-embedded pancreatic sections from
2-week-old control (a) and hnf1-/ (b)
mice, immunostained with anti-insulin (red) and anti-glucagon (green)
antisera. (B) Semiquantitative RT-PCR analysis of insulin mRNA levels
in freshly isolated islets of 4-week-old control (+/+ and +/) and
hnf1-/ mice. Primers with full homology
to the two mouse insulin genes were used for PCR. The two amplification
products were distinguished by selective restriction of the insulin II
gene with BstEII (upper panel). Insulin I and II mRNA
content of hnf1-/ islets was not
different from that of control islets. The same samples were
simultaneously analyzed by RT-PCR for -actin as a control
for RNA loading (lower panel). PCRs were carried out at different
numbers of cycles to ensure lineal amplification rates (not shown).
These results are from a representative experiment from the analysis of
four HNF1- null mice, which yielded analogous results.
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HNF1- controls tissue-specific gene expression.
We next
assessed the expression of the glut2 glucose transporter
gene in hnf1-/ mice. glut2
mRNA has a very similar tissue distribution to that of
hnf1- (36). Immunofluorescence costaining
with glut2 and insulin antisera revealed that -cells from 2-week-old
hnf1-/ mice have undetectable amounts of
the sugar carrier (Fig. 2A). Similarly,
RT-PCR from freshly isolated islets detected only trace amounts of the
normally abundant glut2 RNA (Fig. 2B and C). This result was
not due to differences in the purity of pancreatic islets between
control and null mice, as shown by the analysis of islet amylin
polypeptide, an islet-restricted transcript. To ensure that the
reduction is not a secondary phenomenon resulting from diabetes, which
is known to cause reduced expression of glut2 in islets
(37), we analyzed pancreata from embryonic day 20 (E20)
fetal hnf1-/ mice obtained from
normoglycemic heterozygous mothers. These animals also displayed absent
glut2 staining in islets (Fig. 2A).
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FIG. 2.
HNF1- is essential for glut2 expression in
differentiated -cells but not other tissues. (A) Paraffin-embedded
sections were immunostained with glut2 antiserum (green) and costained
with insulin to identify -cells (not shown). Pancreatic
glut2 expression was readily apparent and restricted to
-cells in 2-week-old wild-type mice (a) but was absent in
hnf1-/ mice (b). HNF1- dependence of
glut2 expression is already apparent in prenatal pancreas at
E20 (c and d). However, no significant decrease in glut2
expression was detected in null mouse liver (f), duodenum (h), and
kidney (j) tissues as compared with their respective wild-type controls
(e, g, and i). (B) glut2 mRNA content was analyzed by
semiquantitative multiplex RT-PCR from tissues of 4-week-old mice.
Coamplification of actin and glut2 shows a marked
decrease in glut2 mRNA content in
hnf1-/ mouse purified islets but not in
liver, duodenum, and kidney tissues (upper panel). The bar chart below
shows a densitometric analysis comprising the results of two
independent experiments. The experiment was performed at two different
cycle numbers (23 and 26, or 30 and 36, as indicated under the bar
graph) to ensure similar amplification rates for both products. (C) The
fall of glut2 expression in islets parallels that of
pklr and mirrors the loss of pah expression in
liver tissue. Semiquantitative multiplex RT-PCR analysis was performed
as described above for isolated islets and liver tissue from wild-type
and hnf1--null mice. Coamplification with tbp
as an internal control shows that both glut2 and
pklr mRNA levels were decreased in
hnf1-/ islets but were unaffected in
liver tissue. pah showed a marked reduction of expression in
liver tissue but no major decrease in islets from mutant mice. Islet
amyloid polypeptide (iapp) mRNA levels were unaffected in
hnf1-/ islets, indicating a comparable
islet purity of the different samples.
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As expected, glut2 staining was also readily observed in wild-type
liver, kidney, and duodenum tissue. Surprisingly, hnf1- mutants showed no clear reduction of glut2 expression in
these organs (Fig. 2A). Likewise, glut2 mRNA levels were not
decreased in liver, duodenum, and kidney tissue of
hnf1-/ mice (Fig. 2B). Thus, HNF1- is
required for expression of the glut2 gene in
insulin-producing cells but not in other tissues which coexpress
HNF1- and glut2.
We next assessed whether tissue-specific dependence on HNF1- could
be extended to other HNF1- targets. The L-type pyruvate kinase gene
(pklr) is a key glycolytic enzyme whose tissue distribution also partially overlaps that of hnf1- and
glut2 (13, 25). Furthermore, it has been shown
to be regulated by HNF1- in cell lines (13, 25, 41),
thus constituting a suitable in vivo HNF1- candidate target gene. In
analogy to the results obtained for glut2 gene expression,
pklr mRNA levels were dramatically decreased in pancreatic
islets but not in liver tissue of hnf1-/
mice (Fig. 2C). We also analyzed tissue-specific, HNF1--dependent expression of a gene which has already been previously shown to be
entirely dependent on HNF1- for its expression in liver tissue, phenylalanine hydroxylase (pah). Our experiments confirmed
previous findings that the gene is nearly extinguished in liver tissue in the absence of HNF1- (Fig. 2C) (28) and extend these
in showing that pah, normally expressed at substantially
lower levels in pancreas tissue (24), is not significantly
reduced in islets from hnf1-/ mice (Fig.
2C). In summary, these results show that HNF1- is essential for
glut2 and pklr expression only in islet cells,
whereas it is required for pah expression specifically in
the liver. This indicates that different HNF1- target genes
establish distinct tissue-specific requirements for HNF1-.
Lack of glut2 expression in -cells of
hnf1-/ mice is not a consequence of
reduced expression of pdx1.
In light of the
tissue-specific dependence of glut2 expression on HNF1-,
we hypothesized that cell-specific factors which regulate
glut2 could act as downstream -cell targets of HNF1-. pdx1/idx1 (also known as IPF1 or stf1), the product of a gene which is
also capable of causing early-onset monogenic diabetes (33), is a potential candidate intermediary target, as
this protein has been shown to regulate glut2 in different
experimental models (1, 40). However, immunofluorescence
analysis indicated that -cells from young hnf1- null
mice who have not yet developed full-blown diabetes have intense
pdx1/idx1 nuclear staining (Fig. 3A) and do not display significant
changes in pancreatic islet pdx1/idx1 mRNA content relative
to control littermates (Fig. 3B).
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FIG. 3.
Loss of glut2 is not associated with decreased
expression of pdx1. (A) Paraffin-embedded pancreatic
sections immunostained with anti-pdx1 (green color, a and b) antiserum
do not display differences among hnf1-/
(b) and control (a) 2-week-old mice. Samples were coimmunostained for
insulin to detect -cells (shown in red). (B) Semiquantitative
multiplex RT-PCR analysis was performed for freshly isolated islets to
compare the mRNA levels of pdx1 in three wild-type and three
null mice. Results were analyzed by densitometry, yielding
pdx1/-actin ratio values of 0.60 ± 0.1 versus
051 ± 0.07 arbitrary units (not significant).
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HNF1- directly interacts with glut2 and
pklr promoter chromatin templates in both liver and islet
cells.
We next sought to determine whether tissue-specific
dependence on HNF1- for gene activity correlated with differences in the ability of HNF1- to effectively bind to the endogenous
glut2 promoter chromatin in pancreatic islets and liver.
Occupation of the glut2 promoter by HNF1- was assessed by
in vivo cross-linking and sonication of chromatin from freshly isolated
islets and hepatocytes (Fig. 4A),
followed by immunoprecipitation of HNF1--bound chromatin. Immunoprecipitates were then analyzed by PCR for enrichment of promoter
segments cross-linked to HNF1-. To ensure that results correspond to
promoter-selective enrichment as opposed to random fluctuations in the
efficiency of the PCR or variability in the amount of unselected DNA,
semiquantitation of specific DNA fragments was carried out by PCR
coamplification of a control promoter segment and nonsaturation cycling
conditions. A threefold dilution of input DNA was amplified in parallel
to assess the expected amplification pattern when coamplified gene
segments are present in equimolar amounts as well as to ascertain
nonsaturation amplification kinetics. Results were also compared to an
immunoprecipitation with preimmune serum. These precautions are
critical given that the small amount of tissue material used to analyze
chromatin from isolated mouse pancreatic islets is orders-of-magnitude
lower than that commonly used in chromatin immunoprecipitation
procedures (5, 21, 26). As shown in Fig. 4,
immunoprecipitations with anti-HNF1- from pancreatic islet and
hepatocyte chromatin resulted in a selective enrichment of
glut2 promoter DNA relative to the results obtained with
preimmune serum. Furthermore, as compared to the internal control
amplification product in each reaction mixture, the relative amount of
glut2 DNA in the immune serum sample markedly differs from
that observed when amplifying input DNA (Fig. 4B and C). Thus, in
contrast to what was observed for glut2 DNA, neither the
sulfonylurea receptor promoter (sur1), which regulates a
-cell gene which is not modified in HNF1--deficient mice and does
not have a consensus HNF1- binding site (data not shown), nor the myoD1 promoter (myoD1), which regulates a gene which is not
expressed in islets or liver tissue, was enriched in the precipitated
sample in either tissue (Fig. 4B and C). As a further control to assess the specificity of the experiment, hnf1-/
islets were also analyzed to confirm that the observation of glut2 DNA immunoprecipitated with anti-HNF1- is indeed
specifically dependent on the presence of HNF1- (Fig. 4B). Likewise,
a pklr 5' genomic sequence was enriched in the HNF1-
immunoprecipitates from pancreatic islets and hepatocytes compared with
the myoD1 promoter (Fig. 4B and C). These data indicate that
HNF1- occupies the 5'-flanking region of the glut2 and
pklr genes in both islets and liver. The phenylalanine
hydroxylase promoter was also clearly enriched in liver chromatin
immunoprecipitated with anti-HNF1- immunoglubulins (Fig. 4C), while
only weak association with HNF1- could be seen in pancreatic islet
chromatin (data not shown).
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FIG. 4.
HNF1- contacts the endogenous glut2 and
pklr promoters in both pancreatic islets and hepatocytes in
vivo. (A) Ethidium bromide-stained agarose gel indicating a
representative example of sonicated DNA. M, molecular weight markers;
I, sonicated islet DNA. (B and C) One hundred freshly isolated
wild-type pancreatic islets or 3 × 105 hepatocytes
were fixed with formaldehyde and immunoprecipitated using either
anti-HNF1- antibody or nonimmune serum. The precipitated samples
were analyzed by duplex PCR using primers for the glut2,
pklr, or pah promoter together with either the
sur1 or myoD1 promoter as negative control. The
HNF1- immunoprecipitated sample is selectively enriched in
glut2 promoter chromatin in wild-type but not
hnf1-/ islets (lanes 1 and 5 and 9 and
13, panel B). HNF1- also contacts glut2 promoter
chromatin in hepatocytes (lane 1, panel C). The pklr exon 1 sequence is similarly enriched in HNF1- immunoprecipitated from both
islets and liver cells (lane 17, panel B, and lane 5, panel C). The
pah promoter is occupied by HNF1- in hepatocytes (lane
13, panel C). Only trace amounts of these promoter fragments were
precipitated with preimmune serum (PI). Diluted input DNA (In) samples
were assayed in parallel to illustrate the amplification profile when
all promoter fragments are present in equimolar amounts. The data shown
here represent at least two PCR amplification assays from two
independent immunoprecipitations for islets and three for hepatocytes,
yielding essentially identical results.
|
|
Therefore, HNF1- is not acting exclusively through intermediary
transcriptional regulators forming part of a transcriptional cascade.
Furthermore, the observed differences in tissue-specific requirements for HNF1- for the expression of glut2 and
pklr cannot be explained by the existence of
hnf1- promoter occupancy in only certain cell types.
Tissue-specific HNF1--dependent gene expression correlates with
the effects of HNF1- on localized nucleosomal hyperacetylation.
We then initiated studies to assess the mechanisms involved in
HNF1--dependent transcription of tissue-specific target genes. Experiments were designed to address the possible role of
gene-selective nucleosomal histone acetylation in transcriptional
activation by HNF1- in vivo. We hypothesized that while HNF1-
interacts with target promoters in diverse tissues, its presence may be required to recruit HAT activity-containing coactivators exclusively to
promoters that display HNF1--dependent transcription. Chromatin immunoprecipitations from mouse tissues were carried out with antisera
recognizing histone H3 acetylated at lysines 9 and 14 (AcH3) and
tetraacetylated histone H4 (AcH4). Selective enrichment of specific
promoters in the hyperacetylated chromatin fraction was tested by
multiplex PCR. In particular, 5' genomic segments corresponding to
ins2, pah, glut2, and pklr were tested, four genes for which we have established distinct tissue-specific expression patterns in wild-type and hnf1--deficient mice (see Fig.
1 and 2).
Analysis of hepatocytes from wild-type mice revealed enrichment of
glut2 and pah promoter DNA in the AcH3 and AcH4
immunoprecipitates but not of insulin 5'-flanking DNA (Fig.
5A, lanes 9 and 10). Amplification of
multiple dilutions of input DNA confirmed that all three amplicons were
being amplified at comparable efficiencies when the target sequences
were present at equimolar amounts (Fig. 5A, lanes 12 and 13). These
findings indicate that insulin gene promoter chromatin is
hypoacetylated in liver tissue, whereas transcriptionally active
glut2 and pah promoter chromatin is
hyperacetylated. In hnf1-/ hepatocytes
glut2 promoter nucleosomes appeared to be hyperacetylated to
an extent similar to that in wild-type cells (Fig. 5A, lanes 14 and
15). Similarly, pklr exon 1 showed high levels of histone H4
hyperacetylation in normal mice (Fig. 5B, lane 9) as well as in
hnf1- mutants (Fig. 5B, lane 13). However, histone
acetylation of the pah 5'-flanking region was drastically
reduced (Fig. 5A, lanes 14 and 15). Thus, HNF1--dependent histone
acetylation results closely paralleled the observation that in liver
tissue, pah but not glut2 and pklr
expression is altered in the absence of HNF1-.
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|
FIG. 5.
HNF1- is essential for nucleosomal hyperacetylation
of its tissue-specific transcriptional targets. Chromatin
immunoprecipitation assays using anti-acetylated histone H3 and H4
antibodies were performed with wild-type (+/+) and
hnf1-/ pancreatic islets and hepatocytes.
The precipitated samples were analyzed by multiplex PCR using primers
for the insulin (ins2), pah, pklr, and glut2
promoters. (A) Anti-acetylated histone H4 selectively precipitated
ins2 and glut2 promoter DNA but not
pah DNA in islets from wild-type mice (lane 1). In
hnf1-/ islets anti-histone H4
immunoprecipitate was depleted of glut2 DNA with no major
changes in ins2 and pah (lane 5). Wild-type
hepatocytes showed high levels of histone H3 and H4 acetylation of
pah and glut2 promoter chromatin but not of the
insulin II promoter (lanes 9 and 10). In liver from mice lacking
hnf1-, pah chromatin was hypoacetylated while
glut2 acetylation was unaffected (lanes 14 and 15). PI,
preimmune serum; In, input DNA. The bar chart shows the densitometric
analysis of the chromatin immunoprecipitation experiments described.
Values for each promoter are densitometry results of PCR products
expressed as the following equation: (immune serum PI)/input
DNA. (B) Anti-acetylated histone H4 selectively precipitated
pklr chromatin in islets of wild-type but not
hnf1-/ mice (lane 1 versus lane 5). In
contrast, pklr chromatin remained acetylated in liver tissue
in the absence of HNF1- (lane 9 versus lane 13). The data represent
two to three independent immunoprecipitations with at least two PCR
experiments from each, yielding essentially identical results, except
for anti-AcH3 and pklr in islets, which represent two PCRs
from a single immunoprecipitation.
|
|
In wild-type pancreatic islets, immunoprecipitation with anti-AcH4 and
anti-AcH3 showed enrichment of glut2 and insulin promoter sequences relative to preimmune serum (Fig. 5A, lane 1 versus lane 2).
pklr exon 1 was also clearly enriched in the AcH4
immunoprecipitated samples (Fig. 5B, lane 1 versus lane 2). In
hnf1-/ mice glut2 promoter DNA
and pklr exon 1 were selectively depleted from the
acetylated histone immunoprecipitates, whereas the insulin II gene
promoter was not significantly modified (Fig. 5A and B, lane 5). These
results indicate that HNF1- is necessary for histone hyperacetylation of glut2 and pklr promoter
chromatin in islets. Thus, HNF1- is required for the acetylation of
nucleosomes of target promoters exclusively in those tissues in which
it is indispensable for gene activity.
| |
DISCUSSION |
HNF1- plays an essential role in tissue-specific gene
expression.
Human genetic studies have revealed that heterozygous
mutations in the gene encoding HNF1- cause pancreatic -cell
dysfunction, with no conspicuous phenotype in other tissues where
HNF1- is expressed (8, 44). This finding strongly
suggests that HNF1- plays a previously unsuspected but crucial role
in -cell-specific transcription. We intend to understand how this is
accomplished in the context of whole animal models. Our first step was
thus to search for pancreatic islet-specific genetic targets of
HNF1- by analyzing the expression of candidate genes in
hnf1- null mice. We initiated this analysis with genes
for which studies performed in cultured cell lines indicated that
HNF1- is able to regulate its expression (16, 42). Our
studies indicated that in mice, glut2 glucose transporter
gene expression is fully dependent on HNF1- in pancreatic -cells
but not in other tissues which normally coexpress both genes, including
liver, gut, and kidney tissue. Furthermore, glut2 expression
already requires HNF1- in insulin-producing -cells immediately
prior to birth, thus suggesting that glut2 silencing is not
secondary to the development of diabetes in the adult mouse. Studies
performed with E12.5 embryonic pancreas cells suggest that HNF1-
dependence for glut2 expression is not present in early
pancreatic epithelial cells (M. A. Maestro and J. Ferrer,
unpublished results), suggesting that it may represent a feature of
mature insulin-producing cells. Tissue-specific dependence on HNF1-
is not restricted to the glut2 gene. Thus, HNF1- is required for the expression of the liver-type pyruvate kinase gene
(pklr) in pancreatic islets but not in liver cells, and this is mirrored by the liver-specific dependence on HNF1- for the expression of pah. Thus, HNF1- plays tissue-specific
transcriptional regulatory roles in both pancreatic islets and liver tissue.
This study has focused on the glut2 and pklr
promoters as tools to study -cell-specific transcriptional
regulation by HNF1-. Therefore, the role that the loss of
glut2 and pklr gene expression can play in the
-cell secretory defects present in
hnf1-/ mice has not been specifically
addressed here. Inactivation of glut2 in mice results in a
severe diabetic phenotype ascribed to loss of glucose-sensing
capabilities secondary to defective -cell glucose transport
(19), although the defect can be rescued by reexpression
of very small amounts of this carrier (35). The glucose
transport capacity of islets of hnf1-/
animals was not quantified in our study to assess if it is likely to be
rate limiting for glycolytic flux, but detailed analysis of
stimulus-secretion coupling in islet cells of
hnf1-/ mice suggests the existence of
defective glycolytic metabolism distal to glyceraldehyde-3-phosphate
(14), while analysis in clonal -cells overexpressing
dominant-negative HNF1 revealed both glycolytic and mitochondrial
defects (41, 42). Both types of evidence argue against
glucose uptake being the sole cause of abnormal glucose sensing. On the
other hand, while pyruvate kinase plays a key role in aerobic glucose
metabolism, null mutations of the L-type pyruvate kinase gene in humans
result in hemolytic anemia but not diabetes (3). The data
presented here, along with recently reported studies, clearly point to
the existence of pleiotropic effects of inhibition of HNF1- function
(14, 30, 41, 42). Thus, defective glucose-stimulated
insulin release in HNF1--deficient mice is likely to result from an
aggregate -cell gene expression defect rather than from a single
target abnormality. If the primary role of HNF1- is to regulate a
genetic program involved in glucose sensing, which represents a highly specialized function of differentiated -cells, the glut2
and pklr genes can be regarded as paradigms to dissect the
intimate mechanisms involved. The observation that HNF1- targets
have tissue-specific requirements for HNF1- is conceptually relevant to the development of diabetes in humans with heterozygous mutations in
the gene encoding HNF1- (8). Thus, selective pancreatic -cell dysfunction could indicate that a genetic program required for
glucose sensing in human -cells is vulnerable to HNF1- haploinsufficiency.
We were unable to elicit an analogous dependence on HNF1- for the
expression of insulin I and II mRNA. This suggests that HNF1- is not
essential for insulin gene expression in mice, and therefore lack of
insulin gene transcription is unlikely to be a major factor in the
pathophysiology of HNF1--deficient diabetes in humans. Previous
studies based on the overexpression of HNF1- in cultured cells
showed transactivation of rat insulin I promoter reporter minigenes,
while expression of high concentrations of dominant-negative HNF1
mutants results in decreased insulin mRNA in cultured tumor -cell
lines (16, 41, 42). One potential explanation to reconcile
these experimental results with the data presented here is that at the
endogenous concentrations present in mice, HNF1- plays no role at
all in the regulation of chromatinized insulin gene templates from
mature native -cells. However, taken together, the data are also
consistent with other explanations, such as the existence of
islet-specific redundant mechanisms which are inhibited by the
dominant-negative mutant experiments but which remain intact in the
HNF1- null mice, the possibility that HNF1--dependent insulin
transcription represents a minor fraction of the total gene activity
which is not reflected in our assays of mRNA abundance and histone
acetylation, or that it is involved in physiological regulatory
conditions not examined here. This scenario is not entirely unlike that
encountered previously with liver HNF1- targets. The mRNA content of
some genes previously thought to be regulated by HNF1-, such as the
phosphoenolpyruvate carboxy kinase (PEPCK) and albumin genes, was
described as either normal (for PEPCK) or only slightly reduced (for
albumin) in hnf1-/ mice
(28). This consideration emphasizes the value of
recognizing glut2 and pklr as major -cell
HNF1- targets in a null mutant animal model as opposed to studies
based exclusively on cultured cell systems.
HNF1- regulates glut2 expression in pancreatic
-cells by a direct mechanism.
One model to explain the cellular
specificity of HNF1- in regulating gene expression in -cells is
that it is achieved indirectly through the regulation of cell-specific
factors acting downstream of HNF1-. Studies performed in hepatoma
cell lines suggest that the transcription of HNF1- in hepatocytes
may be controlled by HNF4- (20), while both appear to
be positioned downstream of HNF3- and HNF3- in an endodermal
regulatory cascade (15). Mutations in the gene encoding
HNF4- in humans result in a very similar -cell phenotype to those
with mutations in the gene encoding HNF1- (7),
supporting the possibility that both are indeed involved in a common
regulatory circuit which is likely operative in pancreatic -cells.
It is conceivable that such a transcriptional regulatory circuit may
incorporate as-yet unidentified cell-specific factors in different cell
types. We hypothesized that pdx1/idx1 could represent such a factor
given that pdx1/idx1 has been shown to transactivate glut2
promoter minigene constructs (40), -cell-specific inactivation of this gene results in -cells which express insulin but not glut2 (1), and humans with heterozygous mutations
in this gene also have a monogenic diabetic phenotype
(33). Moreover, the murine pdx1 5'-flanking
region reported in the GenBank database contains a perfect HNF1-
binding consensus site (data not shown). Our studies, however, show
that there is not a major decrease in pdx1/idx1 content in mice with
disruption of the hnf1- gene. Although functional
interactions between pdx1/idx1 and HNF1- in regulating the
glut2 gene need to be investigated, these results argue
against a simple transcriptional hierarchy model, in which pdx1 is located dowstream of hnf1-, to explain
the cell-specific effects of HNF1- on the glut2 gene.
An alternate model is that HNF1- regulates glut2
transcription at least in part through direct interactions with the
glut2 promoter. HNF1- is able to bind in vitro to
conserved AT-rich elements present on the rat and murine
glut2 promoters and to stimulate transcription
(10; M. Parizas, M. A. Maestro, and J. Ferrer,
unpublished data). However, this does not necessarily predict the
situation observed in the in vivo chromatin template context. A related
issue is whether in live cells HNF1- could effectively establish
interactions with target promoters selectively in tissues where it is
required for gene activity. We have been able to address these types of
questions for the first time in a whole-animal model by implementing a
DNA cross-linking and chromatin immunoprecipitation procedure to study
transcription factor occupancy in different mouse tissues. The data
presented here indicate that HNF1- occupies the glut2 and
pklr promoters in pancreatic islets, where it acts as an
indispensable transactivator for these two genes, as well as in the
liver, where it is not required. This suggests that different
tissue-specific promoter contexts determine that HNF1- is essential
for glut2 and pklr transcription only in
pancreatic -cells.
Differences in HNF1--dependent nucleosome hyperacetylation,
rather than in vivo binding, explain the tissue-specific requirements
for HNF1- to regulate glut2 and pklr.
Gene activity has been correlated with hyperacetylation of lysine
residues at the N-terminal tail of histones H3 and H4 located in
nucleosomes spanning transcriptionally active loci (for a review, see
reference 34). The studies presented here reveal that in mice, HNF1- is required to maintain hyperacetylation of histones H3
and H4 of the glut2, pklr, and pah
promoters exclusively in those tissues in which it is essential for
activity of these genes. This effect of HNF1- on histone acetylation
is locus specific, as opposed to a global effect on histone acetylation
in either tissue. Taken together, the results show that HNF1- binds
the glut2 and pklr promoters independently of its
requirement for transcription, whereas the tissue-specific dependence
on HNF1- for transcription of distinct genes is likely to be at
least in part mediated by the role of HNF1- in inducing localized
hyperacetylation of chromatin.
The results are therefore consistent with a model whereby HNF1- is a
functionally obligate component of the set of activator-coactivators forming complexes in vivo at the glut2 and pklr
promoters in islets. In contrast, in liver tissue it is present as a
dispensable component, presumably due to the presence of redundant
positive factors or the lack of tissue-specific repressive elements. In
either tissue, the activating complex is necessary to target histone
acetyltransferase complexes which result in the hyperacetylation of
nucleosomal histones and subsequent conformational changes which are
instrumental in transcriptional activation. This is, in fact,
consistent with prevailing models which indicate that one of the
mechanisms whereby transcriptional activators can induce gene activity
consists in the recruitment of histone acetylase activity to its target
sites. However, existing evidence for this concept stems primarily from yeast genetics, reconstituted in vitro systems, and more recently from
cultured mammalian cell systems analyzing hormonal and viral induction
of transcription (see, for example, references 6, 11, 22, 27 and
38). Thus, the bearing on the diversity of eukaryotic promoters
in live mammals of the notion that transactivators target localized
nucleosomal hyperacetylation in order to activate transcription remains
untested. The results presented here fortify its physiological
relevance, inasmuch as they represent a demonstration in mammals of the
essential role of a single activator in inducing gene-selective histone
hyperacetylation and provide evidence that this hyperacetylation
process is linked to gene expression of distinct promoters.
This work was supported by SAF98-0005 (CICYT), Fundació
Marató TV3, Eli Lilly/European Association for the Study of
Diabetes, and QLRT1999-546 (European Commission) (J.F.). M.P. is the
recipient of a postdoctoral fellowship from the CICYT.
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Directs Nucleosomal
Hyperacetylation to Its Tissue-Specific Transcriptional
Targets
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Abstract
Introduction
