Download Section 2: ß-Cell Genes: Functional Aspects

Section 2: ␤-Cell Genes: Functional Aspects
Regulation of pdx-1 Gene Expression
Danielle Melloul, Sonya Marshak, and Erol Cerasi
The homeodomain-containing transcription factor pancreatic duodenal homeobox 1 (PDX-1) plays a key role
in pancreas development and in ␤-cell function. Upstream sequences of the gene up to about ⴚ6 kb show
islet-specific activity in transgenic mice. Attempts to
identify functional regulatory elements involved in the
controlled expression of the pdx-1 gene led to the
identification of distinct distal ␤-cell–specific enhancers in human and rat genes. Three additional sequences,
conserved between the mouse and the human 5ⴕ-flanking regions, two of which are also found in the chicken
gene, conferred ␤-cell–specific expression on a reporter
gene, albeit to different extents. A number of transcription factors binding to and modulating the transcriptional activity of the regulatory elements were
identified, such as hepatocyte nuclear factor (HNF)-3␤,
HNF-1␣, SP1/3, and, interestingly, PDX-1 itself. A fourth
conserved region was localized to the proximal promoter around an E-box motif and was found to bind
members of the upstream stimulatory factor (USF)
family of transcription factors. We postulate that disruption of pdx-1 cis-acting regulatory sequences and/or
mutations or functional impairment of transcription
factors controlling the expression of the gene can lead
to diabetes. Diabetes 51 (Suppl. 3):S320 –S325, 2002
P
ancreatic duodenal homeobox 1 (PDX-1) is an
orphan homeodomain protein that plays an important role in pancreas development. It is initially detected on embryonic day 8.5 in the part of
the dorsal and ventral primitive gut epithelium that later
develops into the pancreas. A high expression is maintained in most epithelial cells of the pancreatic bud until
day embryonic day 10.5 and then decreases to later
reappear predominantly in the differentiated ␤-cell. Targeted inactivation of this gene in the mouse (1,2) as well as
its mutation in humans (3) result in agenesis of the
pancreas. In the mouse model, malformations in areas
within the duodenum and absence of Brunner’s glands
were observed (1,2,4,5). Although pdx-1 gene expression
does not appear to be required for pancreatic determina-
From the Department of Endocrinology and Metabolism, Hadassah University
Hospital, Jerusalem, Israel.
Address correspondence and reprint requests to Dr. Danielle Melloul,
Department of Endocrinology, Hadassah University Hospital, P.O. Box 12 000,
Jerusalem 91120. E-mail: [email protected].
Received for publication 16 April 2002 and accepted in revised form 8 May
2002.
HNF, hepatocyte nuclear factor; MODY, maturity-onset diabetes of the
young; PDX-1, pancreatic duodenal homeobox 1.
The symposium and the publication of this article have been made possible
by an unrestricted educational grant from Servier, Paris.
S320
tion of the endoderm, it is crucial for the development
of endocrine and exocrine cell types (2,6). Differentiation
and maintenance of the ␤-cell phenotype also require
PDX-1. In mice, ␤-cell–selective disruption of pdx-1 led to
the development of diabetes with increasing age and was
associated with reduced insulin and GLUT2 expression
(7). Indeed, mice heterozygous for pdx-1 were found to be
glucose intolerant (7,8). In transgenic mice expressing
an antisense ribozyme specific for mouse pdx-1 in the
␤-cells, the expression of the endogenous gene was decreased and followed by impaired glucose tolerance and
elevated glycated hemoglobin levels (9). Impaired expression of PDX-1 as a consequence of hyperglycemia or
increased lipid concentrations (10) is associated with
diabetes.
In humans, a subpopulation of type 2 diabetes is monogenic and carries mutations in genes important for normal
␤-cell function. Heterozygous individuals carrying one of
the mutant genes develop a form of maturity-onset diabetes of the young (MODY). MODY4 has been linked to
heterozygosity for mutations in pdx-1 (11,12). Other monogenic forms of MODY have been associated with mutations in genes coding for transcription factors hepatocyte
nuclear factor (HNF)-1␣, HNF-1␤, HNF-4␣, and Beta2 (13),
most of which will be described below as regulators of
pdx-1 gene transcription. Together, these data indicate
that PDX-1 has a dosage-dependent regulatory effect on
the expression of ␤-cell–specific genes and therefore assists in the maintenance of euglycemia. As a consequence,
mutations or functional impairment of other transcription
factors that control the expression of the pdx-1 gene in the
␤-cell could result in additional subtypes of MODY or be
candidates for susceptibility to diabetes. Because PDX-1
appears to play such a central role in ␤-cell differentiation
and function, as well as in pancreatic regeneration (14),
understanding the molecular basis of its regulation and its
maintained expression in the ␤-cell will enable the identification of factors that govern these processes.
STRUCTURE OF THE pdx-1 GENE
The coding region of the pdx-1 gene comprises two exons.
The first exon encodes for the NH2-terminal region of
PDX-1, and the second encodes for the homeodomain and
COOH-terminal domain. The human, mouse, and rat genes
are localized on chromosomes 13 (15,16), 5 (17), and 12
(18), respectively. Although the activation domain of
PDX-1 is contained within the NH2-terminal domain, its
homeodomain is involved in DNA binding; both are involved in protein–protein interactions (19 –25). The pdx-1
gene is TATA-less; thus, it utilizes three principal tranDIABETES, VOL. 51, SUPPLEMENT 3, DECEMBER 2002
D. MELLOUL, S. MARSHAK, AND E. CERASI
scription initiation sites (17), followed by a short 5⬘
untranslated sequence of ⬃100 nucleotides.
REGULATION OF pdx-1 EXPRESSION
Distal rat and human pdx-1 enhancer elements. To
understand the mechanisms that control the expression of
PDX-1 during pancreas development and in the adult
␤-cell, the pdx-1 gene from different species was mapped.
Regulatory regions lying upstream from the transcription
start sites are under characterization in transgenic mice as
well as in cultured cells in several laboratories. A genomic
fragment containing ⬃6.5 kb of the 5⬘-flanking rat pdx-1
sequence was sufficient to target ␤-galactosidase expression to pancreatic islets and duodenal cells in transgenic
mice (17). A longer fragment containing the coding region
and the 3⬘-flanking sequences of the gene restored the
development of all pancreatic lineages and corrected
glucose intolerance in pdx-1⫺/⫺ animals (24). In transiently transfected ␤-cells, the fragment extending from
⫺6.2 to ⫹68 linked to a reporter gene showed 20- to
100-fold higher activity than that in non-islet cells. Using
deletion analyses, the ␤-cell–specific regulated expression
of the rat sequence appeared to require a distal enhancer
element located between the ⫺6.2- and ⫺5.67-kb region of
the gene. This element was shown to bind the endodermal
factors HNF-3␤ and Beta2, which act cooperatively to
induce PDX-1 expression. Furthermore, glucocorticoids
reduced pdx-1 gene expression by interfering with
HNF-3␤ activity on the islet enhancer (26).
To characterize the regulatory elements and potential
transcription factors necessary for the expression of human pdx-1 in ␤-cells, a series of 5⬘ and 3⬘ deletion
fragments of a 7-kb sequence of the 5⬘-flanking region of
the gene, fused to a reporter gene, was tested. By transient
transfections in ␤-cells and non–␤-cells, a ␤-cell–specific
distal enhancer element located between ⫺3.7 and ⫺3.45
kb was delineated. This enhancer fragment strongly stimulated reporter gene activity in all ␤-cell lines tested and
was much less active in non–␤-cells, including glucagonproducing cells, and in acinar and hepatic cells. No sequence similarity was revealed between the enhancer
sequence and the available mouse or rat pdx-1 genomic
sequences. DNase I footprinting analysis revealed two
protected regions: one binding the transcription factors
SP1 and SP3 and another binding HNF-3␤ and HNF-1␣.
Similar to the rat distal enhancer, these factors act in
concert to regulate the transcriptional activity of the pdx-1
gene (27).
Human and mouse pdx-1 conserved regulatory elements. In vivo characterization of the mouse pdx-1 sequences was independently initiated using a fragment of
⬃4.5 kb upstream of the initiation start site (28 –30). The
transgene driven by these sequences was shown to approximately recapitulate the endogenous expression pattern. An extended fragment containing the coding region,
3 kb of the 3⬘-flanking region, and 6.2 kb of upstream
sequences was able to completely rescue the apancreatic
pdx-1 null phenotype and ultimately restore glucose homeostasis. Moreover, the sequences sufficient for appropriate developmental and islet-specific expression were
located within ⬃4.5 kb of 5⬘-flanking DNA (30). The
␤-galactosidase was detected in Brunner’s glands of the
DIABETES, VOL. 51, SUPPLEMENT 3, DECEMBER 2002
proximal duodenum and pyloric glands of the distal stomach and coincided with the expression of pdx-1 mRNA.
Occasionally, ectopic activity was observed in exocrine
tissue of the adult pancreas, submucosal layer of the
duodenum, and even in the spleen (28).
Failure of the pancreas to develop in both humans and
mice lacking PDX-1, as well as the dosage-dependent
effect of PDX-1 on the expression of ␤-cell–specific genes
(and on the maintenance of euglycemia), led to the assumption that sequences conserved between the two
species could be essential for its transcriptional control. A
striking divergence at the nucleotide level was observed
between the two species with the exception of four
regions that showed significant (94, 81, 73, and 78%)
similarity. In addition to the conserved proximal promoter
sequence (20), three short highly homologous regions
were found between ⫺2.81 and ⫺1.67 kb of the human and
between ⫺2.7 and ⫺1.8 kb of the mouse pdx-1 gene (Fig.
1). These regions were designated PH1, PH2, and PH3 for
PDX-1 homologous regions 1–3 (22) or areas I, II, and III,
as determined by Gerrish et al. (31). In transient transfection experiments, each of the conserved sequences was
able to confer ␤-cell–specific activity on a heterologous
promoter; however, it was done to different extents.
PH1/areaI and PH2/areaII showed the highest preferential
induction in ␤-cell versus non–␤-cell activity (22,31). An
interesting observation was the absence of the PH2/areaII
domain in the chicken 5⬘-flanking region (31), suggesting
that the regulation of pdx-1 expression in birds may differ
from that in rodents and humans.
Attempting to identify factors that regulate the transcriptional activity of the conserved domains, DNase I
footprinting analyses, gel electrophoretic mobility shift
assays and mutational studies led to the identification of
several transcription factors (Fig. 1). PH1/areaI and PH2/
areaII sequences bind and are transactivated by HNF-3␤.
Although mutations in the HNF-3␤ binding site within the
PH2/areaII sequence did not modify its transcriptional
activity, in PH1/areaI, it had a profound effect. Interestingly, the PH1/areaI enhancer element was reported to
bind the PDX-1 transcription factor itself both in vitro (22)
and in vivo (32), suggesting a possible autoregulatory loop
as a mechanism for PDX-1 to control its own expression.
The involvement of HNF-1␣ in regulating PH1/areaI was
also determined (32). It was further shown that the PDX-1
protein binds HNF-3␤, and all three transcription factors
appear to act cooperatively to regulate transcription. Identification of factors binding and regulating conserved
sequence PH3 is the focus of ongoing studies.
Thus, from the studies on the binding and the cooperativity between the different factors acting in concert to
control the transcriptional activity of pdx-1 regulatory
elements, it emerges that at least some aspects of the
expression of the gene rely on the transcription factor
HNF-3␤. Indeed, its absence in mouse embryonic stem
cells had a profound effect on pdx-1 gene expression (29).
HNF-3␤ is a member of the forkhead/winged helix family
of transcription factors and is essential for endodermal
cell lineages (33,34). It is structurally related to histone H5,
which can alter the nucleosomal structure and thus prime
target genes for expression by opening the chromatin
structure and providing promoter access to other tranS321
pdx-1 GENE EXPRESSION
FIG. 1. Organization of mouse, human, and rat pdx-1 enhancer/promoter regions. cis-acting regulatory elements of the pdx-1 5ⴕ-flanking sequences
are boxed. Conserved sequences between human, mouse, and rat pdx-1 genes are indicated as dark boxes and nonconserved enhancer elements
as empty boxes. A: DNA fragments of the mouse pdx-1 gene used in transgenic mice experiments in their expression patterns are depicted
(28 –30). B: Transcription factors binding to the regulatory elements are indicated above each box. PH1, PH2, and PH3 equals PDX-1 homology
regions 1, 2, and 3, respectively.
scription factors (35,36). Because HNF-3␤ is not restricted
to ␤-cells, the selective transcription of PDX-1 is likely to
rely on the combination of additional factors, among them
HNF-1␣, SP1, PDX-1 itself, and possibly other unidentified
factors.
In addition, the distal human enhancer element and the
PH1/areaI domain bind the HNF-1 members of transcription factors. HNF-1␣ and HNF-1␤ homeoproteins are capable of binding the HNF-1 site as homodimers or
heterodimers (37,38). Transient transfection experiments
in fibroblasts demonstrated that both HNF-3␤ and HNF-1␣
independently activate the distal human enhancer and,
when cotransfected, act in a synergistic manner. However,
HNF-1␤ did not affect enhancer-driven transcription separately or in combination with HNF-3␤. Although high
levels of HNF-1␤ mRNA are observed at 6 –7.5 days of
gestation (39), HNF-1␣ is expressed at a later developmental stage (40) and appears to be the predominant form
present in adult ␤-cells (27). We found that the relative
abundance of HNF-1␣ and HNF-␤ proteins differ in various
pancreatic cell lines, suggesting that differences in HNF-1
subtype ratios may be one of the factors contributing to
tissue-specific expression of the pdx-1 gene. The relative
abundance of the two major HNF-1 species was recently
suggested to be a mechanism for expression pattern
S322
determination for the glut-2 gene (41). The in vivo studies
on HNF-1␣ knockout mice did not give an unequivocal
answer regarding the importance of this transcription
factor for pdx-1 gene expression. Shih et al. (42) reported
that in HNF-1␣ null animals’ pdx-1 mRNA levels were
significantly decreased (42); however, little or no effect on
its expression was obtained by Parrizas et al. (43). Furthermore, no reduction in PDX-1 protein was observed in
transgenic mice with selective expression of a dominantnegative form of HNF-1␣ in pancreatic ␤-cells (44).
Mutations in the HNF-1␣ gene represent the most frequent form of MODY (45). The observed diabetic phenotype was first suggested to be the result of impaired
binding of HNF-1␣ to the insulin promoter, thus causing
decreased transcriptional activity of the gene. However,
binding of HNF-1␣ to the flanking AT sequences (FLAT)-F
element of the insulin gene appears to be unique to the rat
insulin I gene because this AT-rich motif is not conserved
among insulin promoter sequences. In fact, HNF-1␣ is a
weak transactivator of the human insulin gene; we therefore favor the explanation that the impact on insulin gene
expression is indirect, via its effect on pdx-1 gene transcription. Impaired pancreatic function in MODY3 is also
the result of defective transcription of genes involved in
␤-cell glucose sensing and glucose metabolism (42).
DIABETES, VOL. 51, SUPPLEMENT 3, DECEMBER 2002
D. MELLOUL, S. MARSHAK, AND E. CERASI
FIG. 2. The proximal promoter of the mouse, human, and rat pdx-1 gene. Sequence homology between the proximal region of the TATA-less rat,
mouse, and human pdx-1 genes. The sequences corresponding to the transcription start sites (S1–S3) are highly conserved and indicated as
determined for the rat gene (17).
The in vivo importance of the conserved domains was
addressed in transgenic mice (28 –30). Fragment XbaI–
XhoI, spanning the sequence between ⫺4.3 and ⫺1.88 kb
of the mouse pdx-1 gene, which includes both PH1/areaI
and PH2/areaII domains, directed the transgene expres‘sion to pancreatic islets but not to any other cell population in which PDX-1 is normally expressed. However, a
smaller region (PstI–BstEII) that still contained PH1/areaI
and PH2/areaII sequences was active in the islets as well
as in the pyloric sphincter and the common bile duct.
Furthermore, within the pancreas, expression of the reporter gene driven by the PstI–BstEII fragment was found
in the majority of insulin-, glucagon-, and somatostatinproducing cells; therefore, this fragment was considered
to be an endocrine-specific enhancer. Furthermore, the
fragment (XhoI–BglII) containing PH3/areaIII drove the
reporter expression to clusters of insulin-producing cells,
whereas it was almost inactive in glucagon- and somatostatin-positive cells of the neonatal pancreas. However,
this ␤-cell–specific activity was transient and lost in adult
pancreases (30). Although the reason for the silencing of
this transgene is not clear, it may be proposed that the
XhoI–BglII region plays a specific role in immature ␤-cells
or provides critical developmental cues for the initiation of
the mature ␤-cell lineage. Altogether, these data suggest
that the conserved domains confer islet-specific, and to a
certain extent ␤-cell–specific, transcription both in vivo
and in vitro. However, separately or in combination, these
elements cannot faithfully recapitulate the expression of
endogenous pdx-1, thus implicating that additional regulatory elements must be involved in this process. Mutations or deletions of important regulatory sequences in the
context of the endogenous pdx-1 gene will help assess
their critical importance in the regulated expression of the
gene.
THE PROXIMAL PROMOTER OF THE pdx-1 GENE
As mentioned earlier, the pdx-1 gene is TATA-less, and
although the sequences corresponding to the transcription
start sites among the rat, mouse, and human genes are
highly conserved (17) (Fig. 2), great heterogeneity is
revealed further upstream. The ␤-cell–specific transcripDIABETES, VOL. 51, SUPPLEMENT 3, DECEMBER 2002
tional regulation of the rat pdx-1 gene was reported to rely
in part on a proximal promoter sequence containing an
E-box motif located at ⫺104. Pursuing our search for
functional regulatory elements in the human pdx-1 5⬘flanking region, deletion analysis of the proximal promoter
was performed, and a ␤-cell–specific regulatory sequence
between ⫺160 and ⫺100 bp was identified. Comparison of
this region between the promoters of insulin genes from
different origins showed a relatively high degree of homology (78%), with greater heterogeneity further upstream.
DNAse I footprinting, using the conserved proximal promoter of the human gene and ␤-cell extracts, revealed a
specific protected region around the conserved E-box
motif (CACGTG) (17). This site predominantly binds a
complex containing the transcription factor USF (17).
Mutations abolishing its binding impaired the activity of
the pdx-1 promoter. Expression of a dominant-negative
form of USF-2 in ␤-cells reduced both the pdx-1 promoter
activity as well as PDX-1 mRNA and protein levels. This
led to a reduction of PDX-1 binding to the insulin promoter
and consequently a dramatic decrease in insulin gene
expression (46). Thus, USF1 and USF2 interactions with
the E-box sequence in the pdx-1 promoter appear to
contribute to the preferential expression of the gene in
␤-cells.
DISCUSSION
Taken together, these results suggest that the transcriptional stimulation of pdx-1 in ␤-cells is mediated by a
unique combination of protein–protein interactions and
that separate modules in the gene can be active at a given
stage by binding a specific set of transcription factors.
Indeed, the transcription factors HNF-3␤, HNF-1␣, HNF1␤, SP1/3, USF1/2, and PDX-1 itself regulate the expression of the pdx-1 gene. Most of these factors have been
previously shown, mainly by knockout experiments in
mice, to be important developmental regulators. PDX-1 is
a key factor with multiple functions both during development and in the differentiated ␤-cell. In the adult, its role in
regulating islet-specific genes and, most importantly, in
mediating the glucose effect on insulin gene transcription
S323
pdx-1 GENE EXPRESSION
emphasizes its particular role in normal and diabetic
states.
ACKNOWLEDGMENTS
The study was supported by the Juvenile Diabetes Research Foundation International (1-2001-325).
The authors wish to express their gratitude to Tamara
Gurevich, Michal Shoshkes, and Etti BenShushan for their
contributions.
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