From the Brookdale Center in the Department of
Biochemistry and Molecular Biology, Mount Sinai School of
Medicine-New York University, New York, New York 10029, the
¶ Medical Research Council, Clinical Sciences Center,
Imperial College School of Medicine, Hammersmith Campus, London W12
ONN, United Kingdom, the
Gene Targeting Group, Department of
Neuromuscular Diseases, Imperial College School of Medicine, Charing
Cross Campus, London W6 4RF, United Kingdom, and the ** Department
of Molecular Genetics, The University of Texas, M. D. Anderson
Cancer Center, Houston, Texas 77020
Received for publication, February 13, 2001
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ABSTRACT |
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We have examined the chromatin structure around
and upstream of the transcriptional start site of the human The extracellular matrix plays a critical role in morphogenesis
and growth, as well as in tissue homeostasis and repair (1). Type I
collagen is the most abundant extracellular component of the vertebrate
connective tissue and consists of two The The regulatory network of Col1a2 seems to adhere to the
functional domain model of gene expression, where functional domain defines the genomic region that contains cis-acting
sequences regulating a particular locus (14). Such DNA elements include promoter, enhancer, and insulator sequences, as well as the DNase I
hypersensitive site (HS) and the locus control region (LCR), (14). The
model is probably best exemplified by the organization of the
Cell transfection and DNA binding assays have located the transforming
growth factor- The present study was undertaken to delineate the overall organization
of the putative functional domain of COL1A2 and to begin defining its
structural-functional relationship to the mouse unit. Toward this end,
we analyzed the chromatin structure around and upstream of the start
site of transcription; identified previously unknown sites of
DNA-nuclear protein interaction; and assessed the activity of relevant
genomic regions in transgenic mouse embryos. Altogether, the results
indicate that the human and mouse genes share similar chromatin
organizations and structurally homologous regulatory sequences. They
also suggest that distinct enhancer/promoter interactions may underlie
species-specific differences in the tissue-specific expression of the
two genes.
Cells and DNA Constructs--
Human embryonic lung fibroblasts
(WI-38, ATCC CCL-75), Jurkat T cells (ATCC TIB-152), and umbilical
vascular endothelial cells (HUVEC, C-003-5C, Cascade Biologics, Inc.,
Portland, OR) were grown in Dulbecco's modified Eagle's medium, RPMI
1640, and M200 medium, respectively. Dulbecco's modified Eagle's
medium and RPMI 1640 were supplemented with 10% fetal bovine serum,
100 µg/ml streptomycin, and 100 units/ml penicillin; M200 was
supplemented with low serum growth LSGS (Cascade Biologics, Inc.) in
the absence of antibiotics. Construct Chromatin Analysis and DNase I Footprinting Assay--
Cells
were washed with ice-cold phosphate-buffered saline, scraped, pelleted,
and resuspended in buffer A (15 mM Tris-HCl (pH 7.6), 15 mM NaCl, 60 mM KCl, 1 mM EDTA, 0.5 mM EGTA, 0.3 mM sucrose, 0.1% Triton X-100,
0.15 mM spermine, 0.5 mM spermidine, 1 mM phenylmethylsulfonyl fluoride, 1 mM
dithiothreitol). Cells were mechanically disrupted, the resulting
homogenate was diluted with an equal volume of buffer B (buffer A
without Triton X-100), and the nuclei were sedimented by
centrifugation. Nuclear pellets were resuspended in 5 volumes of buffer
C (buffer A without Triton X-100, EDTA and EGTA) and DNA concentrations
were estimated by UV absorption at 260 nm; 15 OD units were used for
each DNase I digestion. These reactions were performed in 40 mM Tris-HCl (pH 7.6) and 6 mM MgCl2
using 10 µl of DNase I (Amersham Pharmacia Biotech, Piscataway, NJ)
at concentrations between 0 and 10 units/µl. After 15-min incubation
at room temperature, the reaction was halted by adding 2 volumes of 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 100 mM EDTA, 1% SDS, and 40 µl of proteinase K (20 mg/ml). DNA purification, restriction nuclease digestion, and Southern analysis
were performed according to the standard protocol (22). For DNase I
footprinting, plasmid DNA was digested with the appropriate restriction
enzyme and end-labeled by filling-in 3'-recessed ends with the Klenow
enzyme (22). Gel purification of labeled DNA fragments, preparation of
nuclear extracts from WI-38 cells, nuclear protein binding, and DNase I
footprinting reactions were performed as previously described (16).
Generation and Analysis of Transgenic Embryos--
Transgenic
embryos were produced by the standard pronuclear injection of DNA into
fertilized C57Bl/10 × CBA/J F1 eggs (23). Plasmid DNA was
digested with appropriate enzymes, purified from agarose gel, and
microinjected at a concentration of 2-4 ng/ml in 10 mM
Tris (pH 7.4) and 0.1 mM EDTA. Injected eggs were
transferred in pseudo-pregnant CD1 females. Embryos were collected from
the recipient females at 15.5 days post coitum (E15.5) for
whole-mount fixation and staining. Aside from corresponding to a time
of strong Col1a2 expression (24), stage E15.5 was chosen to
avoid the problem of diminished permeability due to skin
keratinization. Integration of the transgenes was assessed by Southern
blot hybridization to a LacZ (the gene coding for
COL1A2 Chromatin Structure--
DNaseI-hypersensitive sites in
chromatin are structural landmarks indicative of control regions
involved in constitutive and tissue and/or stage-specific transcription
(25). The mouse Col1a2 gene has been shown to contain five
major hypersensitive sites at discrete locations within the genomic
region that spans from
Probe Pa is a 0.2-kb fragment located in intron 1, which hybridizes to
a 1.75-kb EcoRI genomic fragment (Fig. 1, A and
B). A new 1.3-kb-long fragment was detected in DNA derived
from Jurkat nuclei treated with DNase I, in addition to a significantly
fainter band of similar size in DNase-treated WI-38 nuclei (Fig.
1B). Probe Pb is a 0.2-kb fragment located 3.6 kb upstream
of the start site of transcription, which recognizes a 3.9-kb
EcoRI genomic fragment (Fig. 1, A and
C). Southern analysis of DNA from DNase-treated WI-38 nuclei
revealed that probe Pb hybridizes faintly to a 3.5-kb species and more
strongly to a 1.3-kb band; of these new hybridizing bands, the 1.3-kb
species was not observed in DNase-treated Jurkat nuclei (Fig.
1C). Probe Pc is located 19 kb upstream of probe Pb and
hybridizes to a 6.2-kb BamHI genomic fragment (Fig. 1, A and D). Unlike DNase-treated nuclei from HUVEC
and Jurkat cells, probe Pc recognized three additional bands in
DNase-treated nuclei from WI-38 fibroblasts (Fig. 1D). The
relative positions of the six hypersensitive sites (designated as
HS(In) and HS1-5 in Fig. 1) were validated by
Southern analysis using probes corresponding to the opposite ends of
the Pa, Pb, and Pc genomic fragments (data not shown). Accordingly,
HS(In) was located at about +730 bp; HS1 at Comparison of COL1A2 and Col1a2 Sequences--
To relate the above
data at the DNA level, we sequenced ~3 kb encompassing mouse HS3-5
and compared it to the sequence generated by the Human Genome Project.
We also compared selected areas of the proximal promoters and first
introns where the other hypersensitive sites had been mapped.
Dot-matrix analysis of the relevant genomic regions revealed that the
cell type-specific hypersensitive sites common to COL1A2 and
Col1a2 (HS1-5) reside within stretches of highly homologous
sequences; by contrast, the unique HS in the first intron of COL1A2
[HS(In)] lies within a divergent sequence (Fig.
2A). A comparison of the
sequences around human HS(In), HS1, and HS2 is shown in Fig.
2B.
Computer-aided alignment of the sequences that contain human and mouse
HS3-5 revealed remarkably high homology (62%) between human
nucleotides Nuclear Protein Binding Sites in the Core Homology
Region--
Previous characterization of the mouse far-upstream region
has not included identification of binding sites for nuclear proteins, a prerequisite to deciphering enhancer function (11). The DNase I
footprinting assay was therefore employed in the present study to map
sites of nuclear protein interaction in the core homology region of
COL1A2. The analysis identified twelve distinct areas of nuclear
protein protection within identity islands IS1, IS2, IS3, and IS5 (Fig.
4). By contrast, no recognizable
footprint was identified in IS4 where HS4 is located (Fig. 3). The
twelve footprinted areas were designated FU1-12, in a 5' to 3'
direction (Fig. 3). The footprints are broadly distributed into three
separate clusters that reside within the upstream third of the core
homology region (FU1-7), and in the middle (FU8-9) and at the 3'-end
of it (FU10-12) (Fig. 5). Although the
identity of the cognate trans-acting factors remains to be
determined, the analysis nevertheless yielded the first indication of
the number and distribution of putative cis-acting elements
within the core homology region.
Functional Analyses--
Six LacZ reporter gene
constructs were engineered to determine the transcriptional
contribution of the COL1A2 far-upstream region in transgenic mouse
embryos. The first of them (
The activity of each construct was examined according to the overall
intensity of
The
Addition of the core homology region with (22.8/17.5pLAC) or without
(21.1/18.8pLAC) the divergent flanking sequences dramatically increased
LacZ staining, and broadened significantly the expression profile to closely resemble that of the endogenous gene (24). These
points are visually illustrated in the whole-mount and histological images of a positive 21.1/18.8pLAC transgenic embryos (Figs.
6B and 8). Among other mesenchymal tissues, LacZ
is transcriptionally active in smooth and skeletal muscles,
intramembranous bones, meninges, skin, liver, lung, and kidney (Fig.
8). Taken at face value, these data
equated the core homology of COL1A2 functionally to the far-upstream
enhancer of Col1a2 (11). An important difference was however
noted between the two last constructs.
Whereas five out of seven 22.8/17.5pLAC transgenes displayed strong
LacZ staining, a significantly lower percentage of
21.1/18.8pLAC (33%) were
The high percentage of 22.8/17.5pLAC and 20.8/17.5pLAC transgenes
expressing
Histological examination of tissues from 20.2/18.8pLAC transgenic
embryos documented an expression profile broader than The type I collagen genes are an instructive example of
coordinated regulation during embryogenesis in a variety of mesenchyme cells and with tightly controlled spatio-temporal patterns (2). Combinatorial interactions among restricted and ubiquitous
transcription factors are generally believed to direct gene expression
in individual cell types and at distinct developmental stages (28).
Identification of cell and/or stage-specific cis-acting
elements in type I collagen genes may therefore provide new insights
into the mechanisms that underlie the diversification of mesenchyme
cell specification. Finding evolutionarily retained DNA sequences in
different organisms provides strong indication of critically important
cis-acting elements and indirect evidence for the
identification of transcriptional regulatory networks (13). Using the
transgenic model and guided by the prior analysis of chromatin
structure, we have identified DNA elements that direct high and
tissue-specific expression of the human COL1A2 gene. The results have
revealed remarkably conserved chromatin structure and sequence
composition in the far-upstream region of the human and mouse Six distinct DNase I-hypersensitive sites were mapped around the start
site of transcription of COL1A2 and within 20 kb upstream of it; their
spatial arrangement, cell-type specificity and relative availability to
DNase I digestion are comparable to those of the mouse gene. There is
only one exception, namely a hypersensitive site in the first intron
(HS(In)) of COL1A2, which is absent in Col1a2. HS(In)
appears to be inaccessible to DNase I digestion in type I
collagen-producing fibroblasts, a finding consistent with a putative
silencing role of this unique hypersensitive site. Indeed, a previous
study has correlated the first intron of COL1A2 with transcriptional
repression of promoter constructs (4). Consistent with the uniqueness
of HS(In), the intronic sequences of the mouse and human genes are divergent.
Our chromatin survey identified three strong hypersensitive sites
(HS3-5) at about the same location as those of the mouse far-upstream
enhancer and within nearly identical sequences. Although limited to
three cell lines, the analysis nevertheless suggests that HS3-5 may
represent sites of open chromatin in type I collagen-producing cells.
DNase I footprinting assays correlated areas of sequence identity with
twelve distinct areas of nuclear protein interaction, which are
distributed in three different clusters within the core homology
region. Like previous transgenic work on the mouse gene, we
demonstrated that the far-upstream region of COL1A2 acts as a strong
tissue-specific enhancer in concert with the proximal promoter (11).
Our study has also extended the work on the mouse enhancer by revealing
two additional features of the far-upstream sequence of COL1A2.
The first feature pertains to the role of the 3'-flanking
sequence in augmenting position-independent expression of the
transgene. A variety of elements of different composition have been
described that protect expression of integrated transgenes from the
surrounding chromatin environment (13-15, 29-30). The 1-kb segment
that flanks the 3'-boundary of HS3-5 appears to fulfill such a
function, because deletion of this sequence leads to a substantial
decrease of The second feature pertains to the loss of skin expression
in the construct harboring the enhancer without putative
cis-acting elements FU1-7. This result contrasts with the
low but reproducible X-gal staining of skin fascia in Interestingly, reduced enhancer activity and loss of skin
expression were not observed in comparable mouse transgenes (11). The
discrepancy may be reconciled by arguing that these genomic segments,
albeit highly homologous, are bound by different transcriptional complexes. There is a precedence in support of our hypothesis. The
TGF In conclusion, the human 2(I)
collagen (COL1A2) gene. Four strong DNase I-hypersensitive sites
(HS2-5) were only detected in fibroblasts, and a weaker one (HS1) was identified in type I collagen-negative cells. Another hypersensitive site potentially involved in COL1A2 silencing was found in intron 1 (HS(In)). HS1 and HS2 were mapped within conserved promoter sequences
and at locations comparable to the mouse gene. HS3, HS4, and HS5 were
likewise mapped ~20 kilobases upstream of COL1A2 at about the same
position as the mouse far-upstream enhancer and within a remarkably
homologous genomic segment. DNase I footprinting identified twelve
areas of nuclease protection in the far-upstream region (FU1-12) and
within stretches nearly identical to the mouse sequence. The region
containing HS3-5 was found to confer high and tissue-specific
expression in transgenic mice to the otherwise minimally active COL1A2
promoter. Characterization of the human element documented functional
differences with the mouse counterpart. Enhancer activity substantially
decreased without the segment containing FU1-7 and HS5, and inclusion
of AluI repeats located 3' of HS3 augmented
position-independent expression of the transgene. Hence, subtle
differences may characterize the regulation of mammalian
2(I)
collagen genes by evolutionarily conserved sequences.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 chains and one
2 chain
(2). Type I collagen is produced at different levels by a large number
of tissues and organs, and in a tightly controlled spatio-temporal
manner (3). Transgenic studies, predominantly carried out on the rodent
1(I) and
2(I) collagen genes, have identified DNA sequences that
drive transcription in distinct mesenchyme lineages (4-12). Although
coordinated expression has been thought to underlie similar
transcriptional mechanisms, the transgenic studies failed to identify
comparable organization of regulatory sequences or common
cis-acting elements in the two collagen genes (4-12). In
contrast to the number of tissue-specific elements found within the 3.5 kb1 upstream sequence of the
rat and mouse
1(I) genes, regulated transcription of mouse
2(I)
collagen (Col1a2) involves yet to be identified DNA
sequences located in both the proximal promoter and a far-upstream
enhancer (6, 8-11).
350 promoter of gene Col1a2 is capable to direct
transcription in transgenic models, but expression is limited to a few tissues, such as skin fascia, tail tendon, and calvaria osteoblasts (6). Similar results were obtained when multiple copies of the
315 to
284 region were linked to the basal
40 Col1a2 promoter (6). Chromatin analysis of the upstream region of Col1a2
subsequently identified a cluster of three DNase I-hypersensitive sites
(HS3-5), located ~17 kb from the start site of transcription (11).
Inclusion of the 6-kb region containing the three HS sites upstream of
the
350 proximal promoter resulted in a high level of
-galactosidase expression in a larger number of type I
collagen-producing cells; this element was therefore termed the
far-upstream enhancer of the Col1a2 gene (11). There may be
functional redundancy among putative cis-acting elements in
the far-upstream enhancer, because deletion of the HS5-containing
segment had virtually no effect on the intensity or distribution of
-galactosidase activity (11).
-globin cluster (13-15). In this evolutionarily conserved gene cluster, the upstream LCR potentiates expression of downstream genes by competitively interacting with the respective promoters at
distinct stages of development (14, 15). Another important attribute of
the LCR is the ability to organize chromatin and thus, spatially
insulate the functional interactions between upstream enhancer and
proximal promoter (14, 15). Phylogenetic conservation of chromatin
organization and of related cis-acting elements are therefore strong and an indirect indication of a functional gene domain
(13). In the case of
2(I) collagen, current knowledge is limited to
the mouse gene; indeed, transcriptional analysis of the human
counterpart (COL1A2) has only focused on the proximal promoter.
(TGF
) and tumor necrosis factor-
(TNF
)
responsive elements of COL1A2 to the sequence lying between nucleotides
330 and
250, the human counterpart of the mouse
315 to
284
segment (16, 17). Recent studies have shown that the antagonistic
stimuli of TGF
and TNF
on COL1A2 transcription are integrated by
the binding of Sp1 in synergy with Smad and C/EBP proteins,
respectively (18-20). On the other hand, TGF
stimulation of
Col1a2 transcription has been reported to be mediated by the same sequence but through binding of CTF/NF-I (21). This last finding
has raised the intriguing possibility that species-specific differences
in seemingly identical cis-acting elements may result in
distinct mechanisms to regulate transcription of the mammalian
2(I)
collagen genes (16).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
378LAC in the p
gal-Basic
vector (CLONTECH, Palo Alto, CA) was derived from
the
378 COL1A2/LUC plasmid (17). Constructs were engineered by
subcloning various upstream sequences 5' of the
378 promoter. The
5.2-kb fragment that extends from
22.8 to
17.5 kb was derived by
double digestion with SpeI and XbaI of a larger
SpeI subclone of the BAC clone GS056H18 BAC clone (Genome
Systems, St. Louis, MO; GenBankTM accession number AC002074) to yield
construct 22.8/17.5pLAC. The 2.0- and 3.0-kb fragments that extend from
22.8 to
20.8 kb and from
20.8 to
17.8 kb of COL1A2 were derived
by internal HindIII deletion of the above clone to yield
constructs 22.8/20.8pLAC and 20.8/17.5pLAC, respectively. The 2.3-kb
fragment that extends from
21.1 to
18.8 kb was generated with
proofreading Pfu DNA polymerase (Stratagene, La Jolla, CA) on DNA template of the original BAC clone and using primers
5'-TTACCCCCAATTTACAGATGAAAG-3' and 5'-GCCTCAGCAAGCAACGTGG-3' to
yield construct 21.1/18.8pLAC. The 1.4-kb fragment that extends from
20.2 to
18.8 was generated by Swa1 digestion of
the above 2.3-kb fragment to yield construct 20.2/18.8pLAC. The
sequence of the mouse proximal promoter is in the GenBankTM under
accession number S48747, whereas the sequence of the far-upstream
enhancer has been deposited under accession number AF345994. Sequences
were compared using the MacVector package of programs.
-galactosidase) probe of DNA purified from embryonic
placentas. After cutting open thorax and abdomen to facilitate
substrate infiltration, embryos were placed in cold phosphate-buffered
saline and then fixed for 45-60 min in 0.2% glutaraldehyde, 2%
formalin in 0.1 M phosphate buffer, pH 7.3, containing 2 mM MgCl2 and EGTA. They were washed three times
for 1 h each in the same buffer with 0.1% sodium deoxycholate and 0.2% Nonidet P-40, and stained overnight at room temperature in 1 mg/ml 5-bromo-4-chloro-3-indolyo-
-D-galactoside solution
(X-gal) containing 5 mM potassium ferrocyanide and 5 mM ferricyanide. For histological analysis,
LacZ-expressing embryos were dehydrated and wax-embedded,
and 6-µm sections were prepared, dewaxed, and counterstained with eosin.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 kb to +1.5 kb, relative to the start site
of transcription; they have been termed HS1 to HS5, in a 3' to 5'
direction (Fig. 1A) (11). HS1
and HS2 are located in the proximal promoter to about
100 bp and
2.1 kb, respectively; whereas HS3-5 map at around
17 kb in the
far-upstream enhancer (Fig. 1A). We decided to search for
DNase I-hypersensitive sites within the human COL1A2 genomic segment
that extends from nucleotides
23 kb to +10 kb to assess whether the
chromatin organization may resemble the mouse counterpart. To this end,
three genomic fragments (probes Pa, Pb, and Pc in Fig. 1A)
were used to probe Southern blots containing DNA from fibroblasts,
which produce fibroblasts' high levels of type I collagen, and from
HUVEC and Jurkat cells, which are type I collagen-negative.
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Fig. 1.
DNase I-hypersensitive sites in the human
COL1A2 gene. A, partial restriction map of the human
gene with the locations of the Southern probes (Pa, Pb, and Pc) and
hypersensitive sites (HS) indicated in comparison to those of the
mouse. B-D, nuclei of the indicated cell lines were
digested with increasing amounts of DNase I and hybridized to probes Pa
(B), Pb (C), and Pc (D); the size of
the DNase I-resistant bands is indicated in each autoradiograph along
with the correlation of the bands and hypersensitive sites. The
asterisk identifies an unspecific digest.
130 bp; HS2 at
2.3 kb;
and HS3-5 at 19.1, 19.5, and 20.5 kb, respectively (Fig.
1A). Scanning of the remaining genomic region comprised
between HS2 and HS3 revealed no additional DNase I-hypersensitive sites
(data not shown). Altogether, the analysis demonstrated that COL1A2 and
Col1a2 share comparable chromatin organizations that are
characterized by cell type-specific hypersensitive sites clustered
immediately 5' of the start site of transcription and far-upstream of it.
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Fig. 2.
Conservation of sequences around
hypersensitive sites. A, dot-matrix analysis of the
proximal promoter (top) and far-upstream (bottom)
sequences of the mouse and human 2(I) collagen genes. B,
alignment of the human (top) and mouse (bottom)
sequences around HS(In), HS1, and HS2.
21.1 to
18.8 kb and mouse nucleotides
17.7 and
15.4
(Fig. 3); this highly conserved genomic
segment is herein referred to as the core homology region. The
alignment also identified five individual islands (IS) of sequence with
average identity of 80% or greater (Fig. 3). They include the
sequences around HS3 (
19.1 to
18.8 kb, IS5); HS4 (
19.6 to
19.4
kb, IS4); HS5 (
20.7 to
20.5 kb, IS2); between
20.1 and
19.8 kb
(IS3); and between
21.1 and
20.9 kb (IS1) (Fig. 3). IS5 displays
the lowest degree of sequence identity due to the presence of three
divergent segments within it (Fig. 3). The above observations thus
correlated comparable arrangements of open chromatin with
phylogenetically conserved DNA sequences.
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Fig. 3.
Comparison of the human and mouse
far-upstream sequences. The mouse sequence ( 17.7 to
15.4 kb)
is below the human sequence (
21.1 to 18.8 kb). Also shown are the
positions of the footprinted (FU) areas, identity islands
(IS), and hypersensitive sites (HS). The MAR
consensus sequence is highlighted by the shadowed
box, whereas the arrow indicates the 5'-end of
construct 20.2/18.8pLAC.
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Fig. 4.
DNase I footprinting analysis of the COL1A2
far-upstream region. The genomic region was analyzed using the
following probes: from 21 kb to
20.8 kb (labeled from 5'-end in
A, and 3'-end in B); from
20 kb to
20.4 kb
(labeled from 5'-end in C, and 3'-end in D); from
20.1 to
19.9 (labeled from 5'-end in E, and 3'-end in
F); and from
19.6 kb to
18.76 kb (labeled from 5' in
G and 3' in H). In each test, DNA was incubated
with increasing amounts of DNase I in the presence (+) or absence (
)
of WI-38 nuclear extracts. The numbers on the side of the
protected areas correspond to the footprinted sequences of Fig. 3,
whereas G/A indicates the Maxam and Gilbert reaction used as
a marker.
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Fig. 5.
Diagrams of the human transgenes. The
core homology region is shown in gray with the
arrowheads and black bars indicating the
positions of the hypersensitive sites and footprints, respectively. On
the right side are the statistics of the transgene analysis
with level of expression arbitrarily expressed as high and low based on
the uniformity of the whole-mount staining procedure (see Fig. 6). The
word none signifies undetectable expression under the same
experimental conditions. Indicated in parentheses are the total number
of transgenics found positive by Southern analysis. The copy number of
integrated transgenes ranged from 2 to 5.
378LAC) consists of the
378 to +54
proximal promoter fused to the reporter gene, whereas the others
contain additional combinations of far-upstream sequences subcloned 5'
of the proximal promoter (Fig. 5). To be precise, they include the core
homology region flanked by divergent 5' and 3' sequences
(22.8/17.5pLAC); only the core homology region (21.1/18.8pLAC); the 5'
divergent sequence and the segment of the core homology region
harboring FU1-4 (22.8/20.8pLAC); the core homology region and 3'
divergent sequence (20.8/17.5pLAC); and the core homology region
without the 5' third segment that contains HS5 and FU1-7
(20.2/18.8pLAC) (Fig. 5).
-galactosidase staining and to the percentage of
transgenic embryos expressing the reporter gene. The former parameter
was used to evaluate the transcriptional contribution of distinct
genomic fragments, and the latter was used to identify element(s) that
may contribute to position-independent expression of the transgenes.
All transgenic embryos were derived at the same developmental stage and
processed under the same experimental conditions to allow reliable
comparisons between embryos expressing different constructs. Within the
limits of the experimental approach, the comparisons revealed distinct
features of the core homology region, which are based on the relative
number of integrated transgenes expressing
-galactosidase, as well
as on the intensity and tissue distribution of X-gal staining. It
should be noted that the last two features were fairly reproducible
among different transgenic embryos that harbor the same construct;
moreover, the number of integrated copies averaged between 2 and 5 without much variation in transgene expression. These observations
enabled us to segregate with some confidence
-galactosidase-positive
embryos into high and low expressors (Fig. 5). Visual documentation of
our estimates is provided in the whole-mount X-gal staining of
illustrative transgenic embryos belonging to the groups of low
(
378LAC and 20.2/18.8pLAC) and high (21.1/18.8pLAC) expressors (Fig.
6). These data were subsequently
confirmed at the tissue level by histological examination of
-galactosidase-positive transgenic embryos (see below). Similarly,
the percentage of
-galactosidase-positive embryos separated the
constructs into low (33-40%) and high (71-88%) transgenic
expressors (Fig. 5).
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Fig. 6.
Expression of human COL1A2 transgenes.
A, photomicrograph of -galactosidase staining of
the whole E15.5 embryo harboring the
378LAC construct. B,
photomicrograph of
-galactosidase staining of the whole E15.5 embryo
harboring the 21.8/18.8pLAC construct. C, photomicrograph of
-galactosidase staining of the whole E15.5 embryo harboring the
20.2/18.8pLAC construct. Transgenic embryos were processed under the
same experimental conditions, and stained for the same length of
time.
378LAC construct directed low transcription of the reporter gene
(Fig. 6A); expression was limited to a subset of type I
collagen-producing cells, often with a mosaic pattern (Fig. 7). Positive sites include the
interstitial tissue of the submandibular gland, skin fascia, and,
occasionally, tendons and periosteum of developing ribs (Fig. 7).
Noticeably, none of the four
378LAC transgenic embryos displayed
ectopic
-galactosidase activity in type I collagen-negative tissues.
Furthermore, the percentage of integrated
378LAC construct expressing
-galactosidase was low, 4 embryos out of 10 (Fig. 5). The staining
intensity and tissue distribution, as well as the percentage of
378LAC transgenic expressors are almost identical to the values
previously obtained with eight transgenic embryos carrying the mouse
350 Col1a2 promoter (11).
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Fig. 7.
Tissue distribution of human 378LAC
transgene expression. X-gal staining can be seen in a few cells of
type I collagen-producing tissues. In A, the characteristic
blue staining can be seen in the interstitial cells of the
submandibular gland (arrow) but is not apparent in skeletal
muscle (m). Staining can also be seen in the outer lining of
the stomach, which is made up of fibroblasts and smooth muscle cells
(B, g). No staining in liver lto cells,
nor in the capsule (B, l). Little staining can be
seen in the fascia of the skin (C, f) along the
back of the embryo, whereas keratinocytes of the epidermis
(arrow) are negative. In D, mesenchymal cells
show blue staining in the ear region (ch). Some,
but not all tendons, express the transgene as shown in the footpad of
the hind limb (E). In F, osteoblasts of the
clavicle bone are intensely blue. The periosteum of ribs
also show blue staining prior to ossification, as
illustrated in G. Osteoblasts surrounded by the osteum of
the mandible (H) and frontal bone (I) appear
blue (arrows). The retina of the eye
(e) is not stained. The strong staining of the nose
(J) is localized in the cells surrounding the nasal passage
(arrows), as well as the fascia of the skin.
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Fig. 8.
Tissue distribution of human 21.8/18.8pLAC.
X-gal staining can be seen in almost all type I collagen-producing
cells. In A, section of the neck region shows staining
in the clavicle (arrow), arterial wall of the carotid artery
(arrowhead), mesenchymal cells of the submandibular gland
and skeletal muscle, as well as the osteoblasts of the mandible
(star). In B, staining is present in osteoblasts
of the frontal bone, but neither in brain (b) nor eye
(e). In C, a cross section of the heart and
surrounding region shows staining in the aorta (asterisk).
Staining can also be seen in the heart valves (arrow) and
pericardium (arrowhead); C1 is a higher
magnification of this area. In D, osteoblasts in the
mandibular bone are intensely blue, as well as the ribs
(F, arrow). E1 shows a higher
magnification of the diaphragm (d) and liver capsule being
stained. E2 is also a higher magnification of the two
adjacent ribs, nearest to the arrow in E. The
section of the head shows staining of the meninges (F,
m) but not the brain tissue (F, b). In
G, the section of the abdomen shows staining in several
organs, such as the layers of the stomach wall (g), splenic
premordium (s), and pancreas (p). Interstitial
cells of the lung were stained, as well as the pulmonary artery
(H, arrow). In I, the osteoblasts of
the femoral head show blue staining (arrow).
Section through layers of the skin along the back of the embryo shows
blue staining in the fascia and various skeletal muscle
layers (J) but not in chondrocytes of the ribs
(c) or in the epidermis. In K, kidney capsule
(arrowhead) and interstitial cells of the mesengium and
tubules are also stained.
-galactosidase-positive (Fig. 5). This
finding raised the possibility that insulator-like element(s) may be
present in the divergent sequences that flank the core homology region. Conveniently located restriction sites were therefore used to generate
constructs containing either the 5'-flanking sequence and first four
footprints of the core homology region (22.8/20.8pLAC) or the remaining
of the core homology region and 3'-flanking sequence (20.8/17.5pLAC)
(Fig. 5). Only the latter construct yielded high
-galactosidase
levels and in a large percent (88%) of transgenic embryos (Fig. 5).
Inspection of the divergent 3'-flanking sequence apparently responsible
for enhanced position-independent expression of the transgene revealed
that it consists of two AluI repeats organized in a
head-to-head orientation (26). Interestingly, AluI repeats
are also interspersed throughout the
-globin locus (13-15). The
lack of expressors with the 22.8/20.8pLAC may be due to strong
silencer(s) within the 5'-flanking sequence or to the relatively low
number of transgenics examined.
-galactosidase is similar to the values reported for
mouse constructs with different lengths of the far-upstream enhancer
(11). Importantly, however, the mouse analysis was carried out with
sequences that extend well beyond the 3'-boundary of the human core
homology region (11). Accordingly, it remains to be determined whether
or not DNA element(s) contributing to enhanced position independence
are also present immediately 3' of the Col1a2 core homology
region. Indeed, the shortest far-upstream construct of
Col1a2 used previously includes only the region encompassing HS3-4 and ~2 kb of the divergent 3'-flanking sequence (11). Similarly to transgenes with the whole core homology region, this shorter mouse construct was reported to direct high levels of
-galactosidase and in 80% of transgenic embryos (11). Lacking information about the distribution of nuclear protein binding sites in
the core homology region of Col1a2, we tested the activity of a comparable human construct (20.2/18/8pLAC) without the
HS5-containing region and 3'-flanking sequence (Fig. 5). Consistent
with the results of 21.1/18/8pLAC, deletion of the 3'-flanking sequence correlated with decreased percent of transgenics expressing
-galactosidase (Fig. 5). At variance with the mouse data, however,
loss of the HS5-containing region -and consequently, loss of FU1-7
-resulted in significantly reduced X-gal staining (Fig.
6C).
378LAC, but
less intense and uniform than constructs with the entire core homology
region (Fig. 9). Positive sites include
muscles, bones, and kidney; however, expression in skin fascia was
abrogated in all five transgenic embryos (Fig. 9). This last result
contrasts with the consistent, albeit low and mosaic, expression in
skin of
378LAC (Fig. 7). Sequence inspection revealed the presence of
a nuclear matrix attachment region (MAR) consensus sequence in the
5'-third of the core homology region (Fig. 3) (27). It remains to be
determined whether or not the MAR sequence participates together with
the AluI repeats in conferring position independence to the
transgene. Awaiting additional data, we interpreted the transgenic
results to suggest that full enhancer activity depends on the integrity
of the whole core homology region and on the presence of the
immediately 3'-flanking sequence.
View larger version (101K):
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Fig. 9.
Tissue distribution of human
20.2/18.8pLAC. X-gal staining can be seen in a large subset of
type I collagen-producing cells. In A, osteoblasts of the
calvarium (arrows) are stained blue but not the brain
(b). Other osteoblasts are also stained, as seen in the
clavicle bone (C). The mandibular bone (D,
arrow) and the cortical bone of a lumbar vertebra
(F). In B, cells in the tongue are stained. In
D, the muscular wall of the abdominal aorta (a)
is stained. The fascia of the skin (G, f) along
the back of the embryo and keratinocytes of the epidermis
(arrow) are both negative. The intercostal muscles
(m) between the ribs appear blue (G)
as do the shoulder girdle muscles (J, m). In the
abdomen, the only staining is seen in the mesonephric tissue
(H, ms) but not the interstitial cells of the
testis (t). The other organ that shows strong staining is
the kidney (I), mainly in the interstitium.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2(I)
collagen genes. They have also suggested differences in functional
organization of the cis-acting elements that control
tissue-specificity in the two genes.
-galactosidase-positive transgenic embryos. We also
noted the presence of an MAR sequence within IS2. These kinds of
sequences are commonly found at the boundaries of transcription units
and/or near enhancers (13, 27); however, they do not confer by
themselves position independence (13, 31). Accordingly, we propose that
the AluI repeats may protect the transgene from position
effect by organizing chromatin in concert with core homology region
element(s), such as the MAR sequence. Similar data in transgenic models
have suggested the existence of comparable elements in the
1(I)
collagen gene (12).
378LAC
transgenic embryos. We believe that these observations are indirect
evidence that the far-upstream enhancer works in concert with the
proximal promoter. We rest our conclusion on the widely held view that
high and tissue-specific gene expression depends on local
concentrations of protein complexes that are bound to enhancer and
promoter sequences and that, in turn, favor interaction between these
physically distinct DNA entities (32). A case in point is the mutually
exclusive interactions of the upstream LCR with the proximal promoters
of the
-globin gene cluster during development (13-16). It follows
that eliminating a substantial number of cis-acting elements
(i.e. FU1-7) could dramatically subvert the natural
architecture of the enhancer/promoter interaction and, for example,
lead to loss of expression in one or more tissues. Such a strict
enhancer/promoter interdependence would explain the more severe effect
of deleting a portion as opposed to the entirety of the core homology
region. Tissue specificity is therefore more likely to depend on
synergism among multiple cis-acting elements that reside in
both enhancer and promoter, rather than only on individual sites in
each of them.
-responsive element was located to nearly identical promoter sequences of Col1a2 and COL1A2 but was associated with
CTF/NF-I binding in the former and with the Sp1·Smad2-Smad4
complex in the latter (16, 19-21). Although the location of the
nuclear protein binding sites in the mouse far-upstream enhancer is
currently unknown, the differential expression of construct with
similar deletions of the far-upstream enhancer may indicate subtle
differences in the regulation of COL1A2 and Col1a2.
378 COL1A2 promoter contains elements that
control tissue specificity in subsets of mesenchymal cells, and this
activity is significantly augmented when the promoter is linked to the
far-upstream enhancer. We therefore propose that the predominant
function of the far-upstream element is to broaden and intensify
tissue-specific transcription from the proximal promoter. Indeed,
promiscuous expression of the
378LAC transgene was never observed in
tissues that do not express the endogenous collagen gene. Our study
provides the first indication for the evolutionary conservation of the
functional domain of the mammalian
2(I) collagen genes and the first
characterization of critical DNA elements in the far-upstream enhancer.
Work in progress is focusing on the characterization of
trans-acting factors binding to the core homology region of
COL1A2, as well as on deciphering the functional relationship between
the far-upstream enhancer and proximal promoter.
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ACKNOWLEDGEMENTS |
---|
We thank Cindy Else for excellent technical assistance and Karen Johnson for typing the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants AR386481, AR44888, and HL41262 and by the Medical Research Council and Arthritis Research Campaign (UK).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF345994.
§ Both authors contributed equally to the work.
Published, JBC Papers in Press, March 28, 2001, DOI 10.1074/jbc.M101397200
To whom correspondence should be addressed: Dept.
of Biochemistry and Molecular Biology, Mount Sinai School of
Medicine-New York University, One Gustave L. Levy Place, Box 1020, New
York, NY 10029. Tel.: 212-241-1757; Fax: 212-722-5999; E-mail:
ramirf01@doc.mssm. edu.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
kb, kilobase(s);
COL1A2, human 2(I) collagen gene;
FU, footprint;
HS, DNase
I-hypersensitive sites;
IS, identity island;
LCR, locus control region;
TGF
, transforming growth factor-
;
TNF
, tumor necrosis
factor-
;
HUVEC, human umbilical vascular endothelial cells;
X-gal, 5-bromo-4-chloro-3-indolyo-
-D-galactoside solution;
bp, base pair(s);
MAR, matrix attachment region.
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REFERENCES |
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