From the Hormone Research Institute and the
¶ Department of Medicine, University of California San Francisco,
San Francisco, California 94143-0534
Received for publication, December 26, 2002, and in revised form, February 24, 2003
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ABSTRACT |
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Homeodomain transcription factor Nkx2.2 is
required for the final differentiation of the The development and differentiation of organs such as the pancreas
involve sequential modifications in gene expression controlled by a
cascade of transcription factors. Recently, several mouse strains with
mutations in genes encoding transcription factors that are expressed in
the pancreatic The onset of Nkx2.2 expression in mouse endoderm is coincident with the
onset of dorsal pancreatic bud evagination at embryonic day 9.5. Most
or all of the epithelial cells of the pancreas express Nkx2.2 from the
onset of bud formation until embryonic day 12.5; thereafter, Nkx2.2
expression becomes more restricted. During the peak period for To understand the mechanisms that regulate the expression of Nkx2.2, we
outline here the structure of the mouse nkx2.2 gene and
identify the regions that direct cell type-specific expression. The
nkx2.2 gene has three alternative first exons (exons 1a, 1b, and 1c). We found that the 5'-flanking region of exon 1a drives the
expression of Nkx2.2 predominantly in differentiated islet cells and is
activated by cooperative interactions between HNF3 Cloning of the Mouse nkx2.2 and Human NKX2B Gene
Promoters--
Plasmids containing mouse nkx2.2 genomic DNA
were kindly provided by L. Sussel (University of Colorado, Denver
(12)). The PI artificial chromosome clone containing the human
NKX2B gene and the plasmid containing human NKX2B
exons 1c and 2 were kindly provided by G. Bell and H. Furuta
(University of Chicago) (16). From the PI artificial chromosome clone,
the fragment containing exons 1a and 1b was isolated by Southern
blot analysis using a fragment of the mouse nkx2.2 gene
containing exon 1a and 5'-flanking sequences. The mouse and human
upstream regions were sequenced and are available from GenBank.
Oligoucleotide-capping Rapid Amplification of 5'-cDNA Ends
(5'-RACE)--
Total RNA was isolated from the mouse neural tube at
embryonic day 11.5, pancreas at day 2.5, and isolated adult islets of Langerhans, and from the mouse Reporter Gene Constructs--
To generate reporter plasmids,
fragments of the 5'-region of the mouse nkx2.2 gene obtained
by restriction digestion or PCR were ligated upstream from the
luciferase gene in the pFOXLuc1 plasmid or upstream from the thymidine
kinase minimal promoter in the pFOXLuc1TK (17). Mutagenesis of the
reporter gene constructs was performed using the QuikChange®
mutagenesis kit according to the manufacturer's instructions
(Stratagene). All constructs were confirmed by sequencing.
Cell Culture and Transient Transfections--
The Construction of Plasmids--
The HNF3 Electrophoretic Mobility Shift Assays (EMSAs)--
Nuclear
extracts from Immunoprecipitation Analyses--
The FLAG-fused HNF3 In Vitro Protein-Protein Interaction Assay--
GST fusion
proteins were produced in Escherichia coli BL21 competent
cells via the pPIG plasmid system (22). In vitro translated and [35S]methionine-labeled proteins were prepared using
the TNT-coupled reticulocyte lysate system (Promega). 25 µl of
35S-labeled protein was mixed with 10 µg of GST fusion
protein bound to glutathione-agarose beads in a total volume of 600 µl of interaction buffer (40 mM HEPES (pH 7.5), 50 mM KCl, 5 mM MgCl2, 0.2 mM EDTA, 1 mM dithiothreitol, 0.5% Nonidet
P-40). Samples were then incubated for 1 h at 4 °C with gentle
rocking, and the beads were washed three times with interaction buffer.
The bound protein was eluted with 2 × SDS sample buffer,
fractionated on SDS-polyacrylamide gels, and visualized by autoradiography.
Generation of Transgenic Mice and Detection of
The embryonic, neonatal, and adult tissues were harvested from the
established mouse lines. The pancreases from adult mice were harvested
after heart perfusion with 4% paraformaldehyde. Harvested tissues were
prefixed for 30 min at 4 °C in 4% paraformaldehyde. Tissues were
incubated overnight with 400 µg/ml X-gal substrate at room
temperature. Gross embryos and dissected pancreases were visualized
using a Leica dissecting microscope and imaged with a Spot RT digital
camera and Openlab software. The tissues were fixed again in 4%
paraformaldehyde, paraffin embedded, and sectioned at 5 µm.
Immunohistochemical Analyses--
Immunohistochemical and
immunofluorescence analyses were performed on paraffin sections as
described previously (8). The primary antibodies were used at the
following dilutions: guinea pig anti-insulin (Linco), 1:5,000; guinea
pig anti-glucagon (Linco), 1:10,000; rabbit anti-neurogenin-3 (14),
1:5,000; guinea pig anti-PDX-1 (14), 1:5,000; rabbit anti-HNF3
For immunohistochemistry, biotinylated anti-rabbit, anti-guinea pig or
anti-mouse antibodies were used at a 1:200 dilution (Vector) and
were detected with the ABC Elite immunoperoxidase system (Vector). The
secondary antibodies used for immunofluorescence were as follows:
FITC-conjugated anti-rabbit, anti-mouse or anti-guinea pig diluted
1:100 (Jackson Laboratory); Cy3-conjugated anti-rabbit diluted 1:800
(Jackson Laboratory). Fluorescence and brightfield images were
visualized with a Zeiss axioskop II and imaged with a Hamamatsu ORCA100
digital camera and Openlab software.
Structure of the Mouse nkx2.2 Gene--
As an initial step in
assessing its regulation, we identified the transcription initiation
sites in the mouse nkx2.2 gene using 5'-RACE. Using primers
complementary to the 5'-end of the known nkx2.2 cDNA
sequence (12), 5'-RACE was performed with cDNA from fetal mouse
pancreas at embryonic day 11.5, neural tube at day 10.5, adult
pancreatic islets, the
To identify sequences that might control transcription of the
nkx2.2 gene, we sequenced the regions flanking each major
transcription start site in both the mouse and human genes (Fig. 1,
C-E). As shown in Fig. 1, promoter 1a contains no TATA box
although it has a conserved GC-rich region that is frequently observed
in non-TATA box promoters (24). Promoters 1b and 1c each have a TATA
box sequence 30 bp upstream from the major transcription start site.
The proximal sequences of promoters 1a and both 1c are highly conserved
between mouse and human and contain multiple potential binding sites
for bHLH proteins (E boxes), homeodomain proteins, and nkx2 class
homeodomain proteins (25). Promoter 1a also contains a conserved
consensus binding site for HNF3 (26). The proximal region of promoter
1b is less well conserved and contains two conserved E boxes but no
other identifiable pancreatic transcription factor binding sites.
Promoter Function in Vitro--
To test for the ability to drive
transcription in cell lines, we constructed a series of plasmids with
upstream fragments of the mouse nkx2.2 gene linked to the
firefly luciferase gene. As shown in Fig.
2A, the relative activities of
the three promoters were compared in the
Focusing on the Nkx2.2 1a promoter, we mapped sequences within the
proximal 2,800 bp important for expression in islet cell lines. As
shown in Fig. 2B, a series of truncations of the promoter demonstrated that removal of the sequence between
Mutations introduced into the homeodomain binding site (Ho), or the two
5'-E boxes (E1 and E2) singly or together had modest effects on
promoter activity. In contrast, mutation of either the HNF3 (recently
renamed FoxA) binding site (H3) or the adjacent E box (E4) blocked
promoter activity almost completely. The fact that both mutations can
independently abolish promoter activity suggests that these two
elements may work synergistically.
Next, we generated reporter gene constructs containing three tandem
repeats of the H3/E4 region inserted upstream from the minimal
promoter. As shown in Fig. 3C, this small mini-enhancer is
capable of activating transcription in a cell type-specific and
orientation-independent manner. Together with the mutation data, these
results demonstrate that the H3 and E4 elements are both necessary and
sufficient for nkx2.2 1a promoter activity in the
transfected cell lines.
Transcription Factors Binding to the nkx2.2 E1a Promoter--
To
identify factors that bind to H3 and E4, we performed EMSAs using
double-stranded oligodeoxynucleotides corresponding to H3 and E4 as
probes. The H3 site conforms to an HNF3 binding consensus (26). The
three member of the HNF3 family of winged helix transcription factors
play key roles in development and gene expression in endoderm-derived tissues (27), and HNF3
E boxes contain the consensus sequence CANNTG, bind to dimers of the
bHLH class of transcription factors, and mediate cell-specific gene
expression. Among the bHLH proteins expressed in the pancreas, neurogenin-3 and NeuroD1 play critical roles in islet development and
gene expression (2, 7, 14, 31-33). As shown in Fig. 4B,
in vitro translated neurogenin-3 and NeuroD1 can bind to E4 when dimerized with the ubiquitous bHLH protein E47. A complex of
similar mobility detected in nuclear extracts from HNF3
In contrast, the related non-pancreatic bHLH factors MyoD and Tal1 did
not synergize with HNF3
To map the regions of the HNF3
In contrast to the 1a promoter, activity of the nkx2.2 1b
promoter was not enhanced by neurogenin-3, in islet or non-islet cell
lines.3
HNF3
To map the regions of HNF3 Promoter Function in Vivo--
To determine when and where the
individual nkx2.2 promoters function during fetal
development and islet cell differentiation, we generated transgenic
mice with each of the three promoters driving the bacterial
lacZ gene encoding
Although Nkx2.2 is expressed in all or most of the early epithelial
cells of the pancreatic bud (12), immunohistochemical analyses showed
that at embryonic days 10.5 (Fig. 9,
A-C) and 12.5 (data not
shown) the 1a promoter drove
A small subset of
To test whether the minimal nkx2.2 1a promoter is sufficient
to drive correct expression, we also generated mice carrying a
transgene with the
The nkx2.2 1b promoter gave a very different expression
pattern in vivo. We generated transgenic mice carrying a
transgene with
By embryonic day 15.5, much stronger
Finally, we also generated transgenic mice with a construct carrying
3.6 kb of the nkx2.2 exon 1c promoter ligated upstream from
the The transit of cells from a multipotent precursor state to a
specific differentiated fate involves progressive changes in their gene
expression program. In the developing pancreas, most of the cells that
form the initial buds have the potential to differentiate along several
different paths. When a subset of these cells commits to the pathway to
endocrine cells, they activate a set of islet transcription factor
genes that include neurogenin-3 and pax4 (14, 31,
36). These genes, however, are unique to the islet precursor cells and
are switched off as the cells differentiate further into mature
endocrine cells. In the differentiated, post-mitotic islet cells, a
different set of islet transcription factor genes is activated,
including NeuroD1, pax6, and isl1 (8, 14, 37).
Nkx2.2 is expressed in the early pancreatic progenitor cells, the
neurogenin-3-expressing islet precursor cells, and the differentiated islet cells (12, 14). The data presented here indicate that distinct
mechanisms direct the expression of Nkx2.2 in these three cell
populations. The transgenic animal studies demonstrate that sequences
5' of exon 1a direct expression in a few neurogenin-3-expressing islet
precursor cells, but primarily in mature islet cells ( The sequence of the exon 1b promoter is less well conserved between
mouse and human than the sequences upstream from exons 1a or 1c, but
two ideal bHLH binding sites (E boxes) are conserved. Despite the
presence of these binding sites and the observation that activity of
the exon 1b promoter closely parallels the expression of neurogenin-3
in vivo, several other lines of evidence suggest that
neurogenin-3 may not directly control the 1b promoter. First, in the
transgenic fetuses it should take some time for the 1b promoter to be
activated and for The close match of neurogenin-3 and On the other hand, our data provide good evidence that bHLH proteins
(neurogenin-3 and/or NeuroD1) regulate the nkx2.2 1a promoter. In the 1a promoter transgenic fetuses, activity of the promoter overlaps with neurogenin-3 expression, but only in a few
cells. Together with the evidence that neurogenin-3 can directly activate the promoter through the E4 site, these data suggest a model
in which neurogenin-3 initially activates the 1a promoter. In this
model, neurogenin-3 expression would be extinguished in most cells by
the time detectable levels of Neurogenin-3 does not act alone, however, in activating the
nkx2.2 1a promoter. Our data show that full activation by
neurogenin-3 requires the presence of the forkhead/winged helix factor
HNF3 Interestingly, HNF3 It should be noted that the expression patterns of Finally, it is interesting to speculate on the role of the Nkx2.2
binding sites found in the Nkx2.2 1a and 1c promoters. These sites fit
the ideal Nkx2.2 binding consensus (25) and are completely conserved
between mouse and human. It seems possible that, once Nkx2.2 expression
has been initiated from the 1b promoter, Nkx2.2 itself may feedback
through the 1a and 1c promoters to maintain its expression in mature
islet cells, in cooperation with other factors. In this model, a
cascade of signals in the form of transcription factors initiates
Nkx2.2 expression, but a network of interdependent signals maintains
Nkx2.2 expression and the differentiated phenotype of the mature islet cells.
-cells in the pancreas
and for the production of insulin. Nkx2.2 is expressed in islet cell
precursors during pancreatic development and persists in a subset of
mature islet cells including all
-cells. To understand the
mechanisms regulating the expression of Nkx2.2 in these different cell
populations, we outlined the structure of the mouse nkx2.2
gene and identified regions that direct cell type-specific expression.
The nkx2.2 gene has two noncoding alternative first exons
(exons 1a and 1b). In transgenic mice, sequences upstream from exon 1a
directed expression predominantly in mature islet cells. Within this
exon 1a promoter, cooperative interactions between HNF3 and
basic helix-loop-helix factors neurogenin-3 or NeuroD1 binding to
adjacent sites played key roles in its islet cell-specific expression.
In contrast, sequences upstream from exon 1b restricted expression
specifically to islet cell precursors. These studies reveal distinct
mechanisms for directing the expression of a key differentiation factor
in precursors versus mature islet cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-cells have been found to have severe abnormalities
in pancreatic development (1-12). Mice homozygous for a null mutation
of the homeodomain transcription factor Nkx2.2 develop severe
hyperglycemia and die shortly after birth (12). The mutant embryos lack
insulin-producing
-cells and have fewer
-cells and PP
cells. Remarkably, in these mutants there remains a large population of
islet cells that do not produce any of the four endocrine hormones.
These cells express some
-cell markers, such as islet amyloid
polypeptide and PDX-1, but lack other definitive
-cell
markers including GLUT2, glucokinase, and the
-cell-specific
homeodomain factor Nkx6.1. These mice demonstrate that Nkx2.2 is
necessary for the final differentiation of
-cells.
-cell
neogenesis, from embryonic day 13.5 to 18.5 (13), Nkx2.2 is expressed
in a subset of incompletely differentiated endocrine precursor cells
that coexpress the bHLH1
proendocrine transcription factor neurogenin-3 (12, 14). Unlike
neurogenin-3, which is expressed exclusively in precursor cells, Nkx2.2
is expressed also in differentiated endocrine cells. In the mature
pancreas, Nkx2.2 expression is limited to the differentiated endocrine
cells including
-,
-, and PP cells, but not
-cells. Therefore, Nkx2.2 is expressed at least three distinct stages in islet
cell differentiation: in the broad initial pancreatic precursor
population, in a subset of the neurogenin-3-expressing islet progenitor
cells, and in differentiated islet cells. In addition, Nkx2.2 is
expressed in the developing ventral neural tube and mature neurons in
the central nervous system (15). However, the mechanisms that control
Nkx2.2 expression in these different populations are unknown.
and either
neurogenin-3 or the related bHLH factor NeuroD1. On the other hand, the
5'-flanking region of exon 1b directs expression predominantly to islet
precursor cells. These data reveal distinct mechanisms regulating
Nkx2.2 expression in progenitor cells and in mature islet cells and
support a model in which HNF3
and neurogenin-3 lie upstream from
Nkx2.2 in the hierarchy of
-cell differentiation factors.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-cell tumor line
TC3. The 5'-end of the mouse nkx2.2 cDNA from each cell was identified
by the oligonucleotide-capping RACE method using the GeneRacer Kit
according to the manufacturer's instructions (Invitrogen). Briefly, 2 µg of total RNA was dephosphorylated, decapped, and ligated to
GeneRacer RNA oligonucleotides. Then reverse transcription was carried
out using an oligo(dT) primer. Using this cDNA pool as a template, we carried out 30 cycles of PCR using the GeneRacer 5'-primer and HW323
(5'-CACTTGGTCAATTCGTGG CTCTCC-3') as primers. For nested PCR, we used
the GeneRacer 5'-nested primer and HW324
(5'-CACGCAGAAATGTAGGCTGTGACTGG-3') as primers and performed 25 cycles
of PCR. The PCR products were subcloned into pCR4-TOPO
(Invitrogen) and sequenced.
TC3 cell
line and the
-cell tumor line
TC1.6 were grown in Dulbecco's
modified Eagle's medium supplemented with 2.5% fetal bovine serum and
15% horse serum. NIH3T3 cells were grown in Dulbecco's modified
Eagle's medium supplemented with 10% calf serum. The rat floor plate
cell-derived line Z13 (a generous gift from T. Jessell, Columbia
University, New York) was grown in OptiMEM with 10% fetal bovine
serum. For transient mammalian cell transfections, cells were plated in
six-well tissue culture plates 24 h before transfection. For
standard reporter gene analyses, 1.8 µg of each luciferase reporter
plasmid and 0.2 µg of the CMV
-Gal plasmid were cotransfected into
cells using the TRANSFAST cationic lipid reagent (Promega) according to
the manufacturer's instructions. For assessing the effect of each
transcription factor on the Nkx2.2 promoter, we cotransfected 10 ng of
the expression vector with 1.8 µg of the reporter gene vector.
Controls for transcription factor experiments always contained equal
amounts of the empty CMV expression vector (pBAT12). 48 h after
transfection, cells were harvested, and luciferase and
-galactosidase assays were performed as described previously (17).
All transfection experiments were performed in triplicate on at least
three separate occasions. Luciferase activity was corrected for
transfection efficiency by dividing by the
-galactosidase activity.
deletion
mutant constructs were generated by PCR starting with the CMV-HNF3
plasmid (a generous gift from M. Stoffel, Albert Einstein College of
Medicine, New York (18)) as a template and subcloned into either the
pBAT11 T7 in vitro transcription vector (17), the pBAT12 CMV
expression vector, or the pPIG11 glutathione S-transferase
(GST) fusion vector. The truncated neurogenin-3 constructs were
generated by PCR and subcloned into the pCITE4a T7 in vitro
transcription vector (Amersham Biosciences), pBAT12, or pPIG11.
HNF3
, E47, NeuroD1, and neurogenin-3 proteins were produced in
vitro using the TNT Quick Coupled Lysate System®
(Promega) and the in vitro expression vectors as templates.
TC1.6 cells,
TC3 cells, and NIH3T3 cells were
prepared following the procedure described by Sadowski and Gilman (19).
Single-stranded oligonucleotides corresponding to the sequences in the
mouse Nkx2.2 promoter were 5'-end labeled with
[
-32P]ATP using T4 polynucleotide kinase. The labeled
oligonucleotide was column purified and annealed to an excess of
complementary strand. EMSA buffers and electrophoresis conditions were
as described previously (17). One µl of in vitro reaction
mixture or 2 µg of nuclear extract was used for each 10-µl binding
reaction. For antibody experiments, 1 µl of antiserum was added to
each binding reaction, and the mix was incubated for 15 min at room
temperature before PAGE. The antisera directed against HNF3
,
,
and
were generous gifts from R. H. Costa (University of
Illinois at Chicago (20)). The following oligonucleotides along with
their complementary strands were used as binding probes: H31,
CGGGCTAGAAAAACAAACAGAGCGCTGCGC; E4, GATCCATTGGCCATATGTTCAGCGGTAATAAATTGA.
expression plasmids were generated using the pcDNA3FLAG plasmid (a
generous gift from S. Tomita, University of California, San Francisco
(21)). The FLAG-fused HNF3
and neurogenin-3 expression plasmids were
transfected into NIH3T3 cells, nuclear extracts were isolated, and 50 µg of each nuclear extract was used for immunoprecipitation. The
immunoprecipitation procedures were performed using FLAG-tagged protein
immunoprecipitation kit (Sigma) according to the manufacturer's instructions.
-Galactosidase--
We isolated 1.8 kb and 0.2 kb of the
nkx2.2 exon 1a promoter (from
1754 to +85 bp relative to
the 1a transcription start site and from
247 to +85 bp), 0.7 kb of
the exon 1b promoter (from
665 to +109 bp relative to the 1b
transcription start site), and 3.6 kb of the exon 1c promoter (from
approximately
3600 to +106 bp relative to the 1c transcription start
site) using suitable restriction enzymes or PCR. After cloning into the
p
gal-Enhancer vector (Clontech, CA), we obtained
p
gal.Nkx2.2E1a-1800, p
gal.Nkx2.2E1a-200, p
gal.Nkx2.2E1b-700, and p
gal.Nkx2.2E1c-3600. Each plasmid was purified using Endo Free Plasmid kit (Qiagen), linearized, and microinjected (1.5 ng/µl) into oocyte pronuclei from C3FeB6 mice. The
injected embryos were transferred to pseudopregnant BDF1 female mice.
After checking the genotype with PCR primers for the lacZ sequence, we established multiple mouse lines with each construct by
crossing each founder with C57B6 mice. For the Nkx2.2 1a
1754 bp
construct we characterized five independent lines, three gave detectable expression of
-galactosidase in identical patterns. For
the Nkx2.2 1a
247 bp construct we characterized six independent lines, two gave detectable expression in the same patterns. For the
Nkx2.2 1b construct we characterized 15 independent lines, four gave
detectable expression in the same pattern. For the Nkx2.2 1c construct,
we examined eight lines, and none gave detectable expression.
(23)
(gift of T. Jessell), 1:1,000; mouse monoclonal anti-Nkx2.2 (23)
(Developmental Studies Hybridoma Bank, University of Iowa), 1:10.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-cell tumor line
TC3, and the fibroblast
line NIH3T3. Sequencing of the PCR products revealed three major
transcription start sites (Fig.
1A), which produce three
different splice products (Fig. 1B). Two novel exons, 1a and
1b, each are spliced upstream from exon1c. Exon 1b is located ~8 kb
upstream from exon 1c and the translation initiation site, and exon 1a
is located ~0.7 kb farther upstream. Although the 5'-RACE results are
not quantitative, the adult islet and
TC3 RNA produced predominantly
exon 1a-containing products, whereas the pancreatic bud and neural tube
RNA produced predominantly exon 1b products, suggesting that
transcription initiating from these exons is regulated in a
tissue-specific manner. Products starting with exon1c were found at low
abundance in
TC3 cells and all four tissues but not in NIH3T3
cells.
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Fig. 1.
Structure of the nkx2.2
gene. A, map of the mouse nkx2.2 gene.
The coding sequence is shown in black. B, the
three identified splice variants of the nkx2.2 mRNA. In
C-E, comparisons of the proximal sequences of the human
NKX2B and mouse nkx2.2 1a (C), 1b
(D), and 1c (E) promoters are shown. Conserved
bases are marked by asterisks. The major transcription start
sites identified by oligonucleotide-capping RACE methods are shown as
boldface underlines, and potential transcription factor
binding sites are indicated in boldface letters.
-cell-derived line
TC3,
the
-cell-derived line
TC1.6, the rat fetal floor plate
cell-derived line Z13, and the fibroblast line NIH3T3. These cell lines
were chosen because Nkx2.2 protein was detected by Western blot
analysis in
TC3 cells,
TC1.6 cells, and Z13 cells, but not in
NIH3T3 cells (data not shown). In agreement with the 5'-RACE results,
the nkx2.2 1a promoter drove luciferase expression only in
islet cell lines, whereas the nkx2.2 1b promoter functioned
in Z13 cells. The nkx2.2 1c promoter showed minimal activity
in all cell lines. Although the longest construct, containing sequences
from promoter 1a through 1c, produced less absolute activity than the
shorter constructs, all transfections were performed with the same mass
of DNA, so that the molar concentration for this large plasmid was
2-3-fold lower than for the shorter promoters. Because, in addition,
the transfection efficiencies of such large plasmids may be decreased, relative activity of this very large construct can best be judged by
comparing the
TC3 cells and NIH3T3 cells infected with the same
construct. Using that comparison, the activity of the longest construct
was not significantly different from the construct with the isolated 1a
promoter in
TC3 cells.
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Fig. 2.
Identification of an islet cell-specific
enhancer element in the nkx2.2 promoter. A, the upper
panel shows a map of the mouse nkx2.2 gene. Reporter
plasmids were constructed with the nkx2.2 gene fragments
shown, inserted upstream from the luciferase gene in the pFOXLuc1
plasmid using appropriate restriction enzyme sites. Luciferase reporter
plasmids were cotransfected with a CMV promoter-driven
-galactosidase expression plasmid into a
-cell-derived line,
TC3 (hatched bars);
-cell-derived line,
TC1.6
(open bars); a neural tube derived line, Z13 (stippled
bars); and a fibroblast line, NIH3T3 (filled bars).
Relative luciferase activities were calculated with the activity of
cells transfected with the promoterless parent vector pFOXLuc1 set at
1. B, varying lengths of the 5'-flanking region of the mouse
nkx2.2 1a promoter were inserted upstream from the
luciferase gene in pFOXLuc1 as shown. These luciferase reporter
plasmids were then cotransfected with a CMV promoter-driven
-galactosidase expression plasmid into the cell lines
TC3
(hatched bars),
TC1.6 (open bars), and NIH3T3
(filled bars). Activity in
TC cells was not determined
(N.D.) for the
634 and
539 bp promoters. Relative
luciferase activities were calculated with the activity of cells
transfected with the promoterless parent vector pFOXLuc1 set at 1. All
data are shown as the mean ± S.E. The abbreviations for
restriction enzyme sites used in this figure are: B,
BamHI; K, KpnI; N,
NheI; Xb, XbaI; Xh,
XhoI; P, PstI; S,
SpeI; A, ApaI; Not,
NotI.
247 and
121 bp
completely disrupted the activity of the promoter in
TC3 cells. Within this region, we identified seven potentially important elements
based on their similarity to known transcription factor binding sites.
Mutations were introduced into each of these sites in the context of
the
247 bp reporter gene construct and tested in
TC3 cells (Fig.
3, A and
B).
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Fig. 3.
E box and HNF3 binding sites are necessary
and sufficient for nkx2.2 1a promoter activity. A, the
sequence of the domain mapped in Fig. 2 is shown. Potential binding
sites for pancreatic transcription factors are shown in
boldface. The locations of mutations introduced into the
promoter are underlined. B, reporter plasmids
were constructed with the indicated mutations in the minimal
nkx2.2 1a promoter upstream from the luciferase gene and
then were cotransfected with a CMV promoter-driven -galactosidase
expression plasmid into
TC3 (hatched bars),
TC1.6
(open bars), or NIH3T3 (filled bars) cells.
C, three tandem repeats of the nkx2.2 promoter
sequences from
187 to
105 bp were ligated bidirectionally upstream
from the THYMIDINE KINASE minimal promoter-driving luciferase gene and
then were cotransfected with a CMV promoter-driven
-galactosidase
expression plasmid into
TC3 (open bars) or NIH3T3
(filled bars) cells. Relative luciferase activities were
calculated with the activity of cells transfected with the promoterless
pFOXLuc1 plasmid set at 1. All data are shown as the mean ± S.E.
(FoxA2) is a key regulator of the
pancreatic/duodenal homeobox gene pdx-1 (28-30). As shown
in Fig. 4A, in vitro
translated HNF3
can bind to H3. A complex of similar mobility was
detected in nuclear extracts from
TC1.6 cells and
TC3 cells, but
not in NIH3T3 cell, and this complex was recognized by antiserum to HNF3
but not to HNF3
or HNF3
.
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Fig. 4.
HNF3 and
proendocrine bHLH proteins bind to the nkx2.2 1a
promoter. A, EMSAs using in vitro translated
HNF3
, or nuclear extracts from NIH3T3,
TC1.6, and
TC3 cells
are shown. 32P-Labeled oligonucleotides encoding the H3
enhancer element (sequences are shown under "Materials and
Methods") were incubated with 1 µl of in vitro
translation product or 2 µg of nuclear extract. In the four
right lanes, an additional 1 µl of non-immune serum or
the indicated immune serum was added to the binding mix.
B, EMSAs using in vitro translated E47, NeuroD1
(ND1), or neurogenin-3 (Ngn3), or nuclear
extracts from NIH3T3,
TC1.6, or
TC3 cells are shown.
32P-Labeled oligonucleotides encoding the E4 enhancer
element were incubated with 1 µl of each in vitro
translated protein or 2 µg of nuclear extracts. In the indicated
lanes, an additional 1 µl of non-immune serum or the
indicated immune serum was added to the binding mix. Binding mixes were
subjected to electrophoresis on a 5% polyacrylamide gel.
TC1.6 cells and
TC3 cells, but not NIH3T3 cells, was recognized by antisera to
NeuroD1 and E47.
and Neurogenin-3 Synergistically Activate the nkx2.2 1a
Promoter--
To test the ability of the bHLH proteins and HNF3
to
activate the islet-specific H3/E4 element in non-islet cells, we
expressed various bHLH factors in NIH3T3 cells along with a luciferase
construct driven by either the
247 bp nkx2.2 1a promoter
(Fig. 5A) or by three copies
of the H3/E4 element from the 1a promoter upstream from a minimal
THYMIDINE KINASE promoter (Fig. 5D). As shown in Fig.
5A, HNF3
or E47 alone activated the nkx2.2 1a
promoter modestly; and coexpression of HNF3
with E47 did not provide
any further activation. On the other hand, the addition of NeuroD1 or
neurogenin-3 to E47 and HNF3
synergistically activated the promoter.
Activity of the three factors transfected together was significantly
greater than the combined activities of the individual transcription
factors. To keep all transfections comparable, no attempt was made to
optimize relative synergistic activity by varying plasmid
concentrations.
View larger version (28K):
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Fig. 5.
HNF3 and
neurogenin-3 cooperatively activate the nkx2.2 1a promoter.
A and B, a reporter plasmid containing the
nkx2.2 1a proximal promoter driving luciferase and
expression plasmids expressing the transcription factor cDNAs
indicated under the control of the CMV promoter were cotransfected into
NIH3T3 cells. Ngn3
D indicates the neurogenin-3 cDNA with the DNA
binding domain deleted (Ngn3
76-131 is shown in Fig. 7C).
HNF3
D indicates the HNF3
cDNA with the DNA binding domain
deleted (HNF3
46-257 shown in Fig. 7B). C,
reporter plasmids containing the nkx2.2 1a proximal promoter
with or without the mutations indicated driving luciferase and
expression plasmids expressing the transcription factor cDNAs
indicated under the control of the CMV promoter were cotransfected into
NIH3T3 cells. D, reporter plasmids containing three tandem
copies of the nkx2.2 mini-enhancer element (from
187 to
105 bp) upstream from the THYMIDINE KINASE minimal promoter driving
luciferase and expression plasmids expressing the transcription factor
cDNAs indicated under the control of the CMV promoter were
cotransfected into NIH3T3 cells. Relative luciferase activities were
calculated with the activity of cells transfected with the expression
vector without cDNA insert (
) set at 1. All data are shown as the
mean ± S.E.
, although MyoD significantly activated the
promoter and mini-enhancer construct in the absence of HNF3
.
Synergistic activation of the nkx2.2 1a promoter requires the DNA binding domains of HNF3
and neurogenin-3 (Fig
5B), and intact H3 and E4 sites (Fig 5C). These
results demonstrate that neurogenin-3 and HNF3
synergistically
activate the nkx2.2 1a promoter when bound to the H3 and E4 sites.
and neurogenin-3 proteins outside of
the DNA binding domains that are necessary for this synergy, we
generated eukaryotic expression vector constructs expressing truncated
neurogenin-3 and HNF3
proteins and tested their ability to synergize
on the nkx2.2 1a minimal promoter. Interestingly, no single
domain outside the DNA binding domains was absolutely required by
neurogenin-3 or HNF3
for synergy (Fig.
6). Instead, neurogenin-3 requires either
the carboxyl- or the amino-terminal end of the molecule, both of which
contain a transcription activation domain.2 Similarly, HNF3
requires any one of its three activation domains (34).
View larger version (14K):
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Fig. 6.
Regions of HNF3 and
neurogenin-3 required for cooperative activation of the
nkx2.2 1a promoter. A, a reporter plasmid
containing the nkx2.2 1a proximal promoter (
247 bp)
driving luciferase was cotransfected into NIH3T3 cells along with
plasmids containing the CMV promoter driving the expression of HNF3
,
E47, and neurogenin-3 with the deletions indicated. B, a
reporter plasmid containing the nkx2.2 1a proximal promoter
(
247 bp) driving luciferase was cotransfected into NIH3T3 cells along
with plasmids containing the CMV promoter driving the expression of
neurogenin-3, E47, and HNF3
with the deletions indicated. Relative
luciferase activities were calculated with the activity of cells
transfected with the expression vector without cDNA insert
(control) set at 1. All data are shown as the mean ± S.E.
Physically Interacts with Neurogenin-3--
To determine
whether synergy between HNF3
and neurogenin-3 involves a physical
interaction, we tested for a direct interaction between the two
proteins. A FLAG-tagged HNF3
expression plasmid was transfected
along with a neurogenin-3 expression plasmid into NIH3T3 cells and
nuclear extracts were isolated. Immunoprecipitation was performed with
a FLAG antibody, followed by Western blotting with a neurogenin-3
antibody. As shown in Fig. 7A,
neurogenin-3 coprecipitated with FLAG-tagged HNF3
but not with the
FLAG peptide alone. These results demonstrate that HNF3
can directly
interact with neurogenin-3 in vivo.
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Fig. 7.
HNF3 and
neurogenin-3 interact directly through their DNA binding domains.
A, neurogenin-3 and the FLAG peptide or HNF3
fused to the
FLAG peptide were expressed in NIH3T3 cells. Nuclear extracts were
isolated, immunoprecipitated (IP) with a FLAG antibody,
separated by SDS-PAGE, and immunoblotted (IB) with the FLAG
antibody (upper panel) or a neurogenin-3 (Ngn3)
antibody (lower panel). Panels B-G show pulldown
assays of HNF3
-neurogenin-3 interactions. The various truncated
fragments of HNF3
and neurogenin-3 shown in B and
C were translated in vitro with
[35S]methionine and compared for translation efficiency
by separating on SDS-polyacrylamide gel and comparing 35S
incorporation (shown in D and F). In
vitro translated [35S]methionine-labeled HNF3
and
neurogenin-3 proteins were then incubated with GST or GST-fused
neurogenin-3 (amino acids 76-213) (E) or HNF3
(amino
acids 141-262) (G) and glutathione-Sepharose. After
washing, Sepharose-bound proteins were separated by SDS-PAGE, and
binding was gauged by retained 35S label (shown in
E and G).
and neurogenin-3 which are involved in
this interaction, we used a pulldown analysis with GST fused to
neurogenin-3 amino acids 76-213. As shown in Fig. 7E the
neurogenin-3-GST fusion protein bound in vitro translated HNF3
proteins containing the winged helix domain. It did not bind,
however, to HNF3
proteins lacking the winged helix domain, demonstrating that neurogenin-3 interacts with the winged helix domain
of HNF3
. Similar pulldown assays revealed that HNF3
bound to the
bHLH domain of neurogenin-3 (Fig. 7G). In contrast, the bHLH
domains of E47, MyoD, and NeuroD1 did not interact with HNF3
with
similar affinity, although a weaker interaction with NeuroD1 was
detectable. These results demonstrate a specific physical interaction
between the neurogenin-3 bHLH domain and the HNF3
winged helix domain.
-galactosidase. As shown in Fig.
8, the
1754 bp nkx2.2 1a
promoter produced obvious
-galactosidase activity in the fetal
pancreatic bud as early as embryonic day 10.5, along with expression in
the developing neural tube. By day 15.5, strong
-galactosidase
activity was observed in central epithelial cells of the developing
pancreas and scattered cells in the gut. At 1 and 3 weeks after birth,
-galactosidase activity was restricted to islets. Three independent
transgenic lines showed identical expression patterns.
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Fig. 8.
Gross expression of the 1754 bp
nkx2.2 1a promoter in vivo. In
A, a transgenic embryo at E10.5 with the
1754 bp
nkx2.2 1a promoter ligated upstream from the bacterial
lacZ gene is shown on the left with a
nontransgenic littermate on the right. Both embryos were
incubated with X-gal, yielding the blue product outlining
areas of
-galactosidase expression in the pancreas
(arrow) and neural tissues of the transgenic embryo. In
B, an Nkx2.2 1A1754-lacZ transgenic embryo at E12.5 is shown
after X-gal staining. The arrow indicates staining in the
pancreas. In C, viscera from an Nkx2.2 1A1754-lacZ
transgenic embryo at E15.5 is shown after X-gal staining. The
blue stain can be detected in the central areas of the
dorsal and ventral pancreas and in a speckled pattern in the
gut (distinct from the hazy staining in the lumen of the gut produced
by endogenous
-galactosidase activity). In D, the
pancreas from a 3-week-old Nkx2.2 1A1754-lacZ transgenic mouse is shown
after X-gal staining. The bar indicates 1 mm. St,
stomach; DP, dorsal pancreas; VP, ventral
pancreas; Du, duodenum.
-galactosidase expression predominantly
in differentiated hormone-expressing cells and not in the more abundant
PDX-1 expressing progenitor cells. At this stage, most of the
differentiated endocrine cells in the pancreatic buds express glucagon,
but these early glucagon-expressing cells are distinct from the
glucagon-expressing
-cells found in the mature islets after birth
(35). At embryonic day 15.5, the 1a promoter drove
-galactosidase
expression in all insulin-positive cells and a subset of
glucagon-positive cells (Fig. 9, D and E). Although most of the Nkx2.2-positive cells expressed
-galactosidase at day 15.5, some did not (data not shown), suggesting that the expression of Nkx2.2 in those cells may be regulated by other promoters. All
-galactosidase-expressing cells expressed HNF3
(Fig. 9G).
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Fig. 9.
Expression pattern of the 1754 bp nkx2.2 1a
promoter in vivo. In A-C, a single
section through the dorsal pancreatic bud at E10.5 from an
nkx2.2 1a1754-lacZ transgenic embryo is shown.
-Galactosidase activity was detected with X-gal and is shown in
blue; PDX-1 (red) and glucagon (green)
were detected by immunofluorescence. In D-G, serial
sections from the pancreas of an Nkx2.2 1a1754-lacZ transgenic embryo
at E15.5 are shown.
-Galactosidase activity was detected with X-gal
(blue); glucagon, insulin, neurogenin-3, and HNF3
were
detected by immunohistochemistry with peroxidase labeling
(brown). In H-J, a single section through the
pancreas of an adult Nkx2.2 1a1754-lacZ transgenic embryo is shown.
-Galactosidase activity was detected with X-gal and is shown in
blue; glucagon (red) and insulin
(green) were detected by immunofluorescence. The
bar indicates 25 µm.
-galactosidase-expressing cells also expressed
neurogenin-3, although most neurogenin-3-expressing cells did not
express
-galactosidase (Fig. 9F; and see also Fig. 11, A-C). If neurogenin-3 initiates
-galactosidase
expression, we assume that it would take some period of time for
-galactosidase protein accumulation to reach detectable levels, by
which time neurogenin-3 expression, which is brief, would already be
declining, resulting in only a few cells that express detectable levels
of both neurogenin-3 and
-galactosidase. Therefore, this expression pattern is consistent with the initiation of
-galactosidase
expression in neurogenin-3-expressing cells. In adult animals, the
expression of
-galactosidase was detected in islets in
insulin-expressing cells, but little or no
-galactosidase activity
could be detected in glucagon- or somatostatin-expressing cells (Fig.
9, H-J, and data not shown).
247 bp nkx2.2 1a promoter driving the
lacZ gene. Among six transgenic mouse lines carrying the
transgene, two transgenic lines expressed
-galactosidase. Although
the level of
-galactosidase activity was lower than in the
1754 bp
promoter transgenic lines, the
247 bp promoter produced the same
expression pattern (data not shown).
665 bp of the nkx2.2 1b promoter driving
the lacZ gene. Among 15 independent transgenic mouse lines
carrying the transgene, 4 lines expressed detectable levels of
-galactosidase in the same pattern. As shown in Fig.
10A,
-galactosidase activity was faintly detectable in a small region of the developing spinal cord at
embryonic day 12.5. Although not apparent grossly, very faint
-galactosidase activity was detectable in the dissected pancreas.
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Fig. 10.
Gross expression of the 665 bp nkx2.2 1b
promoter in vivo. In A, a transgenic
embryo at E12.5 with the
665 bp nkx2.2 1b promoter ligated
upstream from the bacterial lacZ gene is shown on the
left with a nontransgenic littermate on the
right. Both embryos were incubated with X-gal, yielding the
blue product outlining areas of
-galactosidase expression
in neural tissues (arrow) of the transgenic embryo. In
B, viscera from an nkx2.2 1b665-lacZ transgenic
embryo at E15.5 on the left and a nontransgenic littermate
on the right are shown after X-gal staining. The
blue stain can be detected in the central areas of the
pancreas. Hazy blue staining in the gut lumen of both embryos is
produced by endogenous
-galactosidase activity. St,
stomach; DP, dorsal pancreas; Du, duodenum, In
C, the pancreas from a 1-day-old nkx2.2
1b665-lacZ transgenic mouse is shown after X-gal staining. The
bar indicates 1 mm.
-galactosidase activity
appeared in central regions of the pancreas of the nkx2.2 1b promoter transgenic embryos (Fig. 10B); but by birth and in
the adult,
-galactosidase activity was undetectable in the pancreas. In the embryonic day 15.5 pancreas,
-galactosidase expression was
restricted to neurogenin-3-positive cells and was not detected in
mature islet cells (Fig. 11,
D-F). Careful examination reveals that most, but not all,
neurogenin-3-expressing cells had some detectable
-galactosidase
activity. There was little if any
-galactosidase expression in cells
that did not stain for neurogenin-3. These results revealed that the
nkx2.2 1b promoter was active in the cells of the islet
precursor population during the major phase of islet cell
neogenesis.
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Fig. 11.
Comparison of the activity of the 1754 bp
Nkx2.2 1a promoter and the
665 bp nkx2.2 1b promoter in islet
progenitor cells in vivo. Single sections through
the pancreas at E15.5 are shown from an nkx2.2 1a1754-lacZ
(A-E) and an nkx2.2 1b665-lacZ (F-I)
transgenic embryo.
-Galactosidase activity was detected with X-gal
and is shown in blue; PDX-1 (red) and
neurogenin-3 (green) were detected by immunofluorescence.
The bar indicates 25 µm.
-galactosidase gene. We established eight independent lines, but
-galactosidase expression was not detectable in any of these lines
(data not shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-,
-, and
PP cells), whereas sequences 5' of exon 1b direct expression to
neurogenin-3-expressing islet precursor cells. These distinct promoters
in turn are regulated by different sets of transcription factors.
-galactosidase protein to accumulate after
neurogenin-3 appears and then for
-galactosidase activity to decay
after neurogenin-3 is gone (38); but the restriction of detectable
-galactosidase activity to cells expressing neurogenin-3 provides no
evidence for such a lag. Second, in cell lines, the E boxes are not
required for full activity of the 1b promoter3; and
coexpression of neurogenin-3 has no effect on promoter
activity,3 together showing that neurogenin-3 does not
directly activate the 1b promoter.
-galactosidase expression in the
1b promoter transgenic fetuses suggests instead that the
neurogenin-3 gene promoter and nkx2.2 1b promoter
may be regulated in parallel in the developing pancreas. Although this
is an attractive hypothesis, there are no common sequence elements
between the two promoters and no binding sites in the 1b promoter for
the endoderm factors that regulate the neurogenin-3 promoter; in
addition, the 1b promoter is not inhibited by HES13 as is
the neurogenin-3 promoter. The tight connection between neurogenin-3
expression and nkx2.2 1b promoter activity may depend on
factors that have not yet been identified.
-galactosidase activity accumulate,
and NeuroD1 would maintain nkx2.2 1a promoter activity in
the mature cells.
(FoxA2) binding to the adjacent H3 site. This synergy requires DNA binding and transcriptional activation domains on both proteins and
is associated with a physical interaction between the two proteins
which maps to their DNA binding domains.
plays a wide ranging role in endoderm
development (27). In this study, we found that HNF3
is expressed broadly in pancreatic endoderm at embyro day 15.5, including ductal cells, exocrine cells, and all Nkx2.2-positive cells. Recently HNF3
has been implicated in the transcriptional regulation of several
pancreatic genes, including the pancreatic/duodenal homeobox gene
(pdx1) (28-30) and the neurogenin-3 gene itself
(39). Unfortunately, the role of HNF3
in pancreatic development and
gene expression cannot be determined by studying HNF3
homozygous
null mutant mice because they die early in embryogenesis well before
formation of the pancreas (40, 41); although a
-cell-specific
disruption of the HNF3
has been obtained, the expression of Nkx2.2
was not studied in these animals (42).
-galactosidase in
the transgenic fetuses using the nkx2.2 1a and 1b promoters do not recapitulate the broad expression of nkx2.2 protein seen prior
to embryonic day 13 in the pancreatic buds of normal mice. This
shortcoming could result from the absence of key sequences that lie
outside the regions of the nkx2.2 gene used for these transgenic animals. Furthermore, the use of individual, isolated promoters, although necessary to identify their distinct functions, could also limit expression if sequence elements from two or more of
the promoters cooperate in driving expression in some cell types. For
example, sequences in the 1a promoter could affect transcription from
the 1b promoter and broaden its activity to additional cell types.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank members of the German, Hebrok, and Vaisse laboratories for helpful comments and criticism and T. Jessell for providing Z13 cells, S. Tomita for providing the pcDNA3FLAG vector, L. Sussel for the mouse nkx2.2 genomic DNA, and G. Bell and H. Furuta for the human NKX2B DNA.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants DK553401 and DK21344.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY44657 (mouse) and AY44658 (human).
§ Recipient of a postdoctoral fellowship and an advanced postdoctoral fellowship from the Juvenile Diabetes Research Foundation International. Present address: Dept. of Medicine, Metabolism and Endocrinology, Juntendo University School of Medicine 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan.
To whom corrrespondence should be addressed: Hormone Research
Institute, University of California San Francisco, 513 Parnassus Ave.,
HSW Rm. 1090, San Francisco, CA 94143-0534. Tel.: 415-476-9262; Fax: 415-731-3612; E-mail: mgerman@biochem.ucsf.edu.
Published, JBC Papers in Press, February 25, 2003, DOI 10.1074/jbc.M213196200
2 S. Smith, H. Watada, and M. S. German, unpublished data.
3 H. Watada and M. S. German, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
bHLH, basic
helix-loop-helix;
CMV, cytomegalovirus;
EMSA(s), electrophoretic
mobility shift assays;
GST, glutathione S-transferase;
LUC, luciferase;
5'-RACE, rapid amplification of 5'-cDNA ends;
TK, thymidine kinase;
X-gal, 5-bromo-4-chloro- 3-indolyl--D-galactopyranoside.
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