From the Renal Division, Department of Medicine, Brigham and
Women's Hospital and Harvard Medical School, Boston, Massachusetts
02115 and INSERM U489, Hôpital Tenon, Paris,
France
Received for publication, August 16, 2000, and in revised form, November 21, 2000
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ABSTRACT |
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Tissue-specific expression patterns of the
paired type IV collagen genes COL4A5 and COL4A6
form the basis for organ involvement in X-linked Alport syndrome, a
disorder in which these genes are mutated. We investigated the proximal
promoter region of COL4A5 and COL4A6 using
glomerular visceral epithelial cells, in which COL4A5 alone
is transcribed; keratinocytes, in which the genes are co-transcribed;
and additional model cell lines. By RNase protection assays, the
intergenic region is 292 base pairs. Transcription start sites for two
5' splice variants of COL4A6 are 1 kilobase apart.
Transient transfections with reporter gene constructs revealed that the
minimal promoters for COL4A5 and COL4A6 are
within 100 base pairs of their respective transcription start sites and
are functionally distinct. In further transfection, gel shift and footprinting assays, we defined a bidirectional positive regulatory element, which functions in several cell types, but not in glomerular visceral epithelial cells selectively transcribing COL4A5.
The existence of separate promoters for COL4A5 and
COL4A6 permits fine control over their expression.
Activation through the bidirectional element can bring about
co-expression of the genes, exploiting their paired arrangement.
Features of the proximal promoter region frame its roles in a
hierarchy regulating type IV collagen gene expression.
Type IV collagen is a major constituent of basement
membranes (1). Its lattice network is assembled from six The "major" chains The mechanisms accounting for the specialized distribution of the minor
It is likely that the pairing of the type IV collagen genes facilitates
their coordinated regulation. The regulation of COL4A1 and
COL4A2, separated by 130 bp, is served by a shared
bidirectional promoter, which is subject to the effects of remote
cis-acting elements (25-34). Transcriptional mechanisms
regulating the minor chains have not been elucidated. In contrast to
the COL4A1-COL4A2 promoter, which applies to obligately
co-expressed isoforms, a framework for the minor chains must account
for coordinate expression in several tissues, as well as the discordant
expression pattern of the Using established models for keratinocytes, in which Cell Culture--
Human GVE cells from the 56/10A1 line
(35) were maintained in a 1:1 mixture of Dulbecco's modified Eagle's
medium and Ham's F12 supplemented with 5 µg/ml insulin, 5 µg/ml
transferrin, 5 ng/ml selenium, 10 ng/ml dexamethasone, and 1% fetal
bovine serum. SCC-25 keratinocytes (ATCC CRL-1628) were maintained in a
1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12
supplemented with 0.4 µg/ml hydrocortisone and 10% fetal bovine
serum. SK-N-SH brain neuroblastoma cells (ATCC HTB-11) were maintained
in minimal essential Eagle's medium with Earle's balanced salts, 2 mM L-glutamine and 1 mM sodium
pyruvate, supplemented with non-essential amino acids and 10% fetal
bovine serum.
RNase Protection Assays--
Total cellular RNA was
isolated from nearly confluent monolayers, using Ultraspec RNA
(Biotecx). Antisense riboprobes were transcribed using T3 or T7 RNA
polymerase (Ambion) in the presence of [ Amplification of Nascent Transcripts--
Transcription of
COL4A5 and COL4A6 in model cell lines was
assessed by reverse transcription-polymerase chain reaction (RT-PCR) amplification of heterogeneous nuclear RNA, using intron-directed primers (37). RT was carried out on 1-µg samples of DNase I-digested total RNA, using the Superscript Preamplification System (Life Technologies). PCR amplifications were carried out on 5% of the RT
products, in the presence of [ Reporter Gene Constructs and Transient Transfections--
Parent
constructs p
Scanning substitution mutations MT1 through MT5, containing
SacII restriction sites in place of native 6-bp sequences,
were introduced into p
For transient transfections, cell monolayers grown to 60-90%
confluence on 60-mm dishes were incubated for 6 h with 2.5 µg of
the luciferase test construct and 1.5 µg of the normalizing vector
pSV- Nuclear Extracts--
Nuclear extracts were prepared by standard
protocols, with minor modifications (39). Ten 175-cm2
flasks with nearly confluent monolayers were used for each preparation, and all steps were carried out at 4 °C. Cells were washed twice and
scraped into phosphate-buffered saline, collected, centrifuged at
1000 × g for 15 min, consolidated, and centrifuged
again, yielding packed cell volumes of ~0.5 ml. Following
resuspension in 2.5 ml of Buffer A (10 mM KCl, 10 mM HEPES, pH 7.9, 1.5 mM MgCl2, 0.5 mM dithiothreitol, 1 mM phenylmethylsulfonyl
fluoride), cells were placed on ice for 10 min, then pelleted. They
were resuspended in 1 ml of Buffer A, Dounce homogenized (A type
pestle), then centrifuged at 1000 × g for 15 min. The
supernatant was removed, and the pellet was centrifuged at 30,000 × g for an additional 10 min, yielding a crude nuclear
fraction. This was resuspended in 0.5 ml of Buffer C (420 mM NaCl, 20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 20% glycerol), Dounce homogenized (B type pestle), then extracted in Buffer C for 30 min. Following centrifugation at 30,000 × g for 30 min, the supernatant was collected,
and dialyzed overnight against a buffer containing 100 mM
KCl, 10 mM HEPES, pH 7.9, 1.5 mM
MgCl2, 10 mM Gel Shift and Chemical Footprinting Assays--
Probes for gel
shift and chemical footprinting assays were prepared from 73-bp
MslI-FokI fragments (nt 728-800,
GenBankTM/EBI accession D28116), which were isolated from
p
For gel shift assays, 2,000 cpm of labeled probe was added to 8 µg of
nuclear extract, 5 µg of poly(dI·dC), and 3 µg of bovine serum
albumin in 20 µl of gel shift buffer containing 50 mM
KCl, 20 mM HEPES, pH 7.9, 5 mM
MgCl2, 1 mM dithiothreitol, and 20% glycerol.
Mixtures were incubated at 4 °C for 30 min, then electrophoresed on
8% non-denaturing polyacrylamide gels in 1× TGE buffer (25 mM Tris, 190 mM glycine, 1 mM EDTA).
We found that coupled gel shift/footprinting assays were required to
delineate DNA elements interacting with proteins in nuclear extracts,
because of the low occupancy of these sites. We used a previously
described copper phenanthroline footprinting technique, with minor
modifications (40). Specialized reagents were purchased from Aldrich,
and all steps were carried out at 4 °C. Scaled-up gel shift assays
were carried out using 100,000 cpm of labeled probe, 40 µg of nuclear
extract from SCC-25 cells, 15 µg of poly(dI·dC), and 15 µg of
bovine serum albumin in 100 µl of gel shift buffer. After
electrophoresis, gels were immersed in 400 ml of 10 mM
Tris-HCl, pH 8.0, followed by addition of 40 ml of DNA cleavage reagent (36 ml of distilled water, added to 2 ml of 9 mM
CuSO4, and 2 ml of 40 mM 1,10-phenanthroline
monohydrate in ethanol). The cleavage reaction was initiated by
addition of 40 ml of 58 mM 3-mercaptopropionic acid, and
terminated at 3-90 min by addition of 40 ml of 30 mM 2,9-dimethyl-1,10-phenanthroline. Bands corresponding to unbound DNA
and the major bandshift S1 (see "Results") were
visualized by autoradiography, then excised. DNA was eluted overnight
at 37 °C in 0.5 M ammonium acetate, 10 mM Mg
acetate, 1 mM EDTA, 0.1% SDS and isolated by sequential
phenol/chloroform extraction, chloroform extraction, and ethanol
precipitation, with 10 µg of glycogen as a carrier. Pellets were
rinsed twice in 85% ethanol and electrophoresed on 10% sequencing
gels. Digestion patterns were visualized by autoradiography and
analyzed by scanning densitometry.
Characterization of Model Systems--
COL4A5 and
COL4A6 expression was analyzed in several cell lines by
RNase protection assay. We report results for GVE cells, in which
COL4A5 alone is expressed; SCC-25 keratinocytes, in which both genes are expressed; and SK-N-SH neuroblastoma cells, in which
neither gene is expressed (Fig. 1). To
ascertain whether transcription accounts for differences among these
mRNA expression patterns, we amplified nascent COL4A5
and COL4A6 transcripts, using intron-directed primers. In
this surrogate for the nuclear runoff assay (37, 41), detection of
nascent transcripts corresponded to steady-state mRNA levels (Fig.
2), indicating that transcription, rather
than mRNA processing or degradation, is the major determinant of
steady-state mRNA levels. The mRNA expression profiles in GVE and SCC-25 cells provide evidence that the type IV collagen composition of corresponding renal glomerular and epidermal basement membranes arises at the transcriptional level.
Structure of the Proximal Promoter Region--
Transcription start
sites were mapped by RNase protection assays, using RNA from SCC-25 and
GVE cells (Fig. 3). Start sites for
transcripts of COL4A5 and 5' splice variants A and B of
COL4A6 are upstream relative to earlier determinations by
cDNA sequencing (4, 42). The intergenic region is 292 bp. By
inspection, the 5'-flanking regions of exons E1 of COL4A5
and E1a of COL4A6 lack classical TATA boxes but lie
downstream from AT-rich regions, which could confer specificity to
sites of transcriptional initiation (Fig.
4). Additional features of the intergenic
region include a tandem array of CCAAT boxes and a central CTC box, a
functional motif, which is conserved in the intergenic region
separating the COL4A1 and COL4A2 genes (28, 31).
Transcript B of COL4A6 is associated with at least two
closely situated start sites. The 5'-flanking region of exon E1b also
lacks a classical TATA box, but is associated at the upstream start
site with a sequence that is identical to a transcriptional initiator
(Inr) at 7 of 8 nucleotides (43).
We undertook a detailed analysis of the proximal promoter region, as
this is where cell-specific regulatory elements are expected to exert
their effects. In transient transfections with luciferase reporter gene
constructs, we sought to characterize the minimal COL4A5 and
COL4A6 promoters and to identify important proximal regulatory elements.
A parent construct p
Construct p
We refined localization of the minimal promoters for COL4A5
and COL4A6 by deletion analysis. In SCC-25 and SK-N-SH
cells, deletion of nt 755-876 (GenBankTM/EBI accession
D28116), in construct p
By comparison to serial deletions of p
Taken together, our findings support a configuration of non-overlapping
minimal promoters for COL4A5 and COL4A6. In
SCC-25 and SK-N-SH cells, the positive regulatory effects of an
intergenic, potentially bidirectional, element are evident. This
configuration has valuable attributes for regulation of the paired
genes, although it is by itself insufficient to explain cell-specific
expression patterns (see "Discussion").
A Positive Regulatory Element in the Intergenic Region--
To
investigate further the positive regulatory element between nt 755 and
876, we utilized constructs with directed substitutions in this region,
in SCC-25 cells (Fig. 6). Four sites MT1
through MT4 were chosen to span the region, and a fifth, containing an AP-1 consensus site (MT5), was targeted directly. The MT2 mutation had
a clear bidirectional effect, reducing luciferase activity 82% in the
COL4A5 orientation, and 68% in the COL4A6
orientation, relative to the parent constructs. Among the remaining
constructs, we observed appreciable changes relative to the parent
constructs with MT4 only, which caused a 2-fold increase in luciferase
activity, in the COL4A6 orientation. This mutation was
associated with the recruitment of additional shifted bands in gel
shift assays (data not shown), suggesting the introduction of binding
sites for spurious transcriptional activation. Substitution of the AP-1
site had no significant effects, suggesting that this element is not
required for basal transcription.
To investigate the interactions of nuclear factors with the
bidirectional element, gel shift assays were carried out, using labeled
probes containing the region of the MT2 mutation (nt 728-800). Several
band shifts were observed following incubation of wild-type probes with
nuclear extracts from SCC-25 or GVE cells but not following incubation
of MT2 mutant probes (Fig. 7). The
banding patterns were complex and differed between the two cell types, suggesting multiple interactions between the DNA fragments and cell-specific nuclear factors. Most of the band shifts were abolished by competition with excess unlabeled wild-type fragment, but not with
MT2 mutant fragments, supporting the specificity of the interactions, and the importance of the region affected by the MT2 mutation.
To delineate the boundaries of the bidirectional enhancer, a coupled
gel shift/chemical footprinting assay was carried out, using the
predominant shifted band S1 (Fig. 7). The
shifted probe was protected from cleavage at the site 5'-TAGC-3'
(Fig. 8), a site of preferential
cleavage for copper phenanthroline (44). Analysis by scanning
densitometry indicated strong protection of the region
5'-TAGCGGATGGATCTCA-3', with weaker protection of the adjacent region
5'-GAGGGG-3'. This site lacks known regulatory elements, by database
search (TRANSFAC, release 3.5; Ref. 45). The MT2 mutation likely
disrupts binding at the site secondarily.
Several divergently transcribed gene pairs have been described in
higher eukaryotes. Within the type IV collagen gene family itself,
pairing has been conserved through an evolutionary pattern of
duplications, across mammalian species (46). This implies biological
advantage, deriving perhaps from stoichiometric requirements for
related gene products. Pairing has been conserved within other gene
families as well, including the histone genes, which are arranged
within duplicated clusters as oppositely transcribed pairs, which share
common promoter elements (47, 48). Additional examples such as that
afforded by the functionally related genes GPAT-AIRC (49), whose products catalyze steps in
the purine nucleotide biosynthetic pathway, and TAP1-LMP2
(50), whose products are central in antigen presentation, reinforce the
notion that paired gene arrangements have been conserved in response to
evolutionary selection pressures. For the type IV collagen genes
specifically, regulatory schemes operating within this framework
account for a highly specific subunit distribution, among tissues that
vary considerably in their basement membrane composition and turnover requirements.
We have demarcated the promoter region of the genes COL4A5
and COL4A6 and defined a novel positive regulatory element
situated in the intergenic region. Our results are consistent with a
scheme in which COL4A5 and COL4A6 are associated
with functionally distinct promoters, which can be regulated separately
or coordinately. The existence of separate promoters for Transcripts A
and B of COL4A6 in particular affords a basis for their
differential expression in several tissues, including kidney, lung,
skin, and placenta (42). If the chief benefit of type IV collagen gene
pairing is to facilitate coordinated transcription, then the
overlapping COL4A1-COL4A2 promoter, and the
distinct COL4A5 and COL4A6 promoters, represent
minor but important variations permitting finely regulated synthesis of
their respective gene products. The positive element defined here may
confer the capacity for co-expression of COL4A5 and
COL4A6 genes in keratinocytes and other cell types (12, 13).
The complexity of DNA binding in the region of the bidirectional
element is evidenced by the appearance of multiple band shifts in our
gel shift assays, each reflecting one of multiple DNA-protein interactions. Abolition of these band shifts by the MT2 mutation, with
corresponding functional effects in SCC-25 cells, suggests that binding
of this cis-acting element is a condition for bidirectional transcription. If the site is viewed as a nidus for the binding of
transcription factor complexes, then differential effects in SCC-25 and
GVE cells can reflect the activity of a transcriptional activator in
SCC-25 cells, and/or the activity of a transcriptional repressor in GVE
cells. Inasmuch as the footprints in our assays do not correspond to
consensus regulatory elements, we cannot posit roles for specific
transcription factors in binding the bidirectional element. Moreover,
the activity of this element in nonexpressing SK-N-SH cells implicates
higher-order regulation by sequence-specific transcriptional repressors
operating through distal elements, or by general, i.e.
chromatin-mediated, mechanisms of transcriptional repression (51).
We have also demonstrated selective transcription of COL4A5
in GVE cells, corresponding to the discordant expression pattern of the
A working model for the proximal promoter region is presented in Fig.
9. Its major features are separability of
the minimal promoters for the two genes, and provision for the
bidirectional element, which can act as a shared near-upstream positive
regulator. In this model, as applied to GVE cells, inactivity of the
bidirectional element is an important feature of selective
COL4A5 transcription, which is mediated by distal enhancers
(panel A). In other cell types, activity of the
bidirectional element is determined by the activation state of the
locus as a whole, and the presence of specific MT2-binding
transcription factors (panels B, C). Distinctions between SCC-25 and SK-N-SH cells reside in the mechanisms by which the
genes are "pre-activated," i.e. rendered
activator-responsive, by favorable local chromatin structures and DNA
methylation patterns (51, 52). These mechanisms could involve
keratinocyte-specific enhancers, and/or somatic silencing mechanisms in
cells like those from the SK-N-SH line, derived from the central
nervous system. Predictions from the working model can now be addressed
in further studies, including comparisons among DNase I-hypersensitive
sites in chromatin from these model cell lines, and analysis of
promoter constructs in vivo, in transgenic mouse lines.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(IV)
chains,
1(IV)-
6(IV), encoded by the genes COL4A1
through COL4A6 (2). In humans, these genes are arranged as
head-to-head pairs COL4A1-COL4A2, COL4A3-COL4A4, and
COL4A5-COL4A6, transcribed from opposite strands of chromosomes 13, 2, and X respectively. The functional
differentiation of the individual
(IV) chains is evidenced in
genetic diseases. Mutations in COL4A5 give rise to X-linked
Alport syndrome, a disorder of renal glomerular basement membrane
(GBM),1 variably associated with hearing loss and ocular
abnormalities (3). Deletions involving COL4A5 and
COL4A6 occur in Alport syndrome associated with diffuse
leiomyomatosis (4). Mutations in COL4A3 or COL4A4
have been implicated in autosomal recessive Alport syndrome (5) and
benign familial hematuria (6).
1(IV) and
2(IV) are present ubiquitously
in basement membranes, as [
1(IV)]2
2(IV)
heterotrimers (1). In contrast the "minor" chains
3(IV)
through
6(IV) are expressed in a tissue-restricted fashion. The
3(IV) and
4(IV) chains have been co-localized in basement
membranes of the kidney, lung, choroid plexus, and neuromuscular
junction (7-10). The
5(IV) chain is present in renal glomerular and
distal tubular basement membranes, Bowman's capsule, and in basement
membranes of the skin, trachea, eye, and neuromuscular junction
(10-13). The
6(IV) chain has been localized in each of these
structures except the GBM (12, 13). The importance of the discordant
expression pattern in the GBM is illustrated by a recently described
case of Alport syndrome associated with ectopic
6(IV) expression,
and a putative regulatory mutation in the COL4A6 promoter
region (14).
(IV) collagen chains are poorly understood. Expression of the
3(IV),
4(IV), and
5(IV) chains is activated in the developing renal glomerulus, as basement membranes of distinct ultrastructure and
type IV collagen subunit composition are elaborated by endothelial and
visceral epithelial cells (10, 12, 15-19). Developmental regulation of
minor chain expression has been characterized in other tissues as well
(20, 21). Special considerations relating to expression of the minor
chains arise in Alport syndrome. In this condition, pathogenic
mutations in one of the minor chains generally lead to loss of the
remaining chains (11, 12, 22). This effect can be achieved by global
downregulation at the mRNA level, as demonstrated in a recent study
of a canine model with a COL4A5 mutation (23), or by
post-transcriptional mechanisms (19, 24).
5(IV) and
6(IV) chains in GBM.
5(IV) and
6(IV) are co-expressed, and renal glomerular visceral epithelial (GVE) cells, in which
5(IV) is expressed selectively, we provide evidence that tissue-specific expression patterns of type IV collagen arise at the transcriptional level. By detailed investigation of the
proximal promoter region, we arrive at an initial understanding of the
mechanisms regulating COL4A5 and COL4A6
transcription. We demonstrate the existence of two promoters for
COL4A6, which are functionally separable from the
COL4A5 promoter, and report the identification of a positive
regulatory element, which can effect coordinated regulation. Finally,
on the basis of findings in these and other cell lines, we frame
potential roles for the proximal promoter region, in a hierarchy
required to explain cell-specific transcription of this gene pair.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]UTP and
freed from unincorporated nucleotides using Sephadex G-50 columns
(Roche Molecular Biochemicals). Assays were carried out using Ambion
RPAII kits. To assess expression in model cell lines, 40-µg samples
of RNA were incubated with 50,000 cpm of
5(IV) (nt 252-455,
GenBankTM/EBI accession U04520) and
6(IV) (nt
5353-5511, GenBankTM/EBI accession U04845) riboprobes, and
500 cpm of low specific activity
-actin (nt 1464-1601,
GenBankTM/EBI accession NM_001101) riboprobe.
Hybridizations were carried out at 60 °C overnight and digested with
RNase T1 for 90 min at 37 °C. Products were analyzed on 6-8%
denaturing polyacrylamide gels. Transcription start sites were
determined by similar methods, using genomic subclones, shown in Fig.
3, as templates for riboprobe synthesis (36).
-32P]dCTP. Primers were
designed on the basis of previously described genomic sequence (38). By
3'-terminal nucleotide position (GenBankTM/EBI accession
AL034369), these were for intron 1 of COL4A5: RT,
5'-CAAGAATGTTTCTGACTTGGC-3' (72935); PCR forward, 5'-TAGTTATT TAGTTGCAGAGGCAGAG-3' (72749); and PCR reverse,
5'-GCAATTGGATTGAAAATGGTAGGC 3' (72913). For intron 2 of
COL4A6 these were: RT, 5'-GAGGGGGCAAAGTGTGAAG-3' (54281);
PCR forward, 5'-CTAACTATCTAAGGGGCTGTGC-3' (54521); and PCR reverse,
5'-ATTGGCTGATGGTGAGATTTGTATC-3' (54355). Control amplifications of
-actin mRNA were carried out using oligo(dT)-primed RT products.
Primers were, by 3'-terminal nucleotide position (GenBankTM/EBI accession NM_001101): PCR forward,
5'-CAGCAGTCGGTTGGAGCGAGCATC-3' (1389); and PCR reverse,
5'-ACACGAAAGCAATGCTATCACCTC-3' (1557). In preliminary experiments using
RNA from HepG2 cells, which express both genes, we ascertained
exponential phases of the PCR reactions. These ranges of cycle number
were used in assaying the model cell lines. RT-PCR products of nascent
transcripts were analyzed on 6% polyacrylamide gels and detected by autoradiography.
5E1, p
6E1a, and p
6E1b contain genomic restriction
fragments upstream from a luciferase reporter gene, in the vector
pGL3-Basic (Promega), as shown in Fig. 5. Deletion constructs were
produced by excision of appropriate restriction fragments, with
subsequent blunt-ending if necessary, and religation (Fig. 5).
5E1 and p
6E1a (see Fig. 6). To generate
each mutant, two amplifications A and B were carried out in parallel, with p
5E1 or p
6E1a as template, so as to yield products
overlapping at primer-encoded SacII restriction sites.
Reaction A primers (designated by 3'terminal nucleotide position
(GenBankTM/EBI accession D28116) were the conserved
5'-ACTTCCAGACTAGTTGACTGA-3' (637), and for the respective mutants: MT1,
5'-TCCCCGCGGTAGATCTATTTGTAATTGGCTTTG-3' (738); MT2,
5'-TCCCCGCGGTAGTCCCAGTTTAGATCTATTTG-3' (750); MT3, 5'-TCCCCGCGGTCTGAGATCCATCCGCTAAA-3' (777); MT4,
5'-TCCCCGCGGAAGCACGGCCCCTCTGAG-3' (791); and MT5,
5'-TCCCCGCGGACAATAAAAAGCACGGCCCC-3' (797). Reaction B primers from the
multiple cloning site of pGL3-Basic were the conserved
5'-ATCGATAGGTACCGAGCTCT-3' for p
5E1 templates and
5'-TACCGGAATGCCAAGCTTAC-3' for p
6E1a templates, and for the
respective mutants: MT1, 5'-TCCCCGCGGGACTATTTTTTTAGCGGATGGA-3' (789);
MT2, 5'-TCCCCGCGGTAGCGGATGGATCTCAGAGG-3' (798); MT3,
5'-TCCCCGCGGGTGCTTTTTATTGTTACTCATTA-3' (825); MT4,
5'-TCCCCGCGGGTTACTCATTAGAAACAAATTTTG-3' (838); and MT5,
5'-TCCCCGCGGTTAGAAACAAATTTTGGTCGGT-3' (844). Amplification products
were digested with appropriate restriction enzymes and reinserted into
the parent constructs in compound ligations. Mutations were verified by
direct sequencing. All plasmids were prepared for transient
transfections using Qiagen plasmid kits.
galactosidase (Promega), in 1.5 ml of Opti-MemI medium (Life
Technologies) containing 10 µg/ml Polybrene (American Bioanalytical). Cells were exposed to 30% dimethyl sulfoxide for 3 min, restored to
complete medium and harvested 32 to 34 h thereafter in Reporter Lysis Buffer (Promega). Luciferase activities were determined using a
Monolight 2001 luminometer (Analytical Luminescence Laboratory Inc.)
with the Luciferase Assay System (Promega). They were normalized for
transfection efficiency using
-galactosidase activities, determined
by the
-Galactosidase Enzyme Assay System (Promega). Luciferase
activities are expressed relative to paired promoterless controls, and
represent mean ± S.E. for at least three transfections.
-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, and 20% glycerol.
Extracts were stored in aliquots at
80 °C, and protein
concentrations were estimated using the Bio-Rad protein assay kit.
6E1a or mutant p
6E1a(MT2) by multiple-step restriction digest
and gel purification. Fragments were labeled at one end by fill-in of a
CCCC overhang, using Klenow fragment (New England BioLabs) in the
presence of [
-32P]dGTP, followed by chase with excess
cold dGTP. Labeled fragments were isolated from 9% non-denaturing
polyacrylamide gels. Unlabeled fragments were used in competition assays.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Expression profiles of model cell lines.
RNase protection assays for simultaneous detection of 5(IV),
6(IV), and
-actin mRNA expression in the indicated cell lines
were carried out as described under "Experimental Procedures."
Numbers refer to molecular size in bases.
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Fig. 2.
Identification of nascent transcripts.
Transcriptional activity of COL4A5 and COL4A6 was
assessed by RT-PCR of nascent transcripts, as described under
"Experimental Procedures." Products are shown for the indicated PCR
cycles, with negative RT controls obtained at the highest cycle. RT-PCR
amplifications of -actin mRNA served as controls. Results are
representative of three experiments. Molecular sizes of the products
are indicated.
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Fig. 3.
Determination of transcription start
sites. A, map of the promoter region of
COL4A5 and COL4A6. Numbering is according to
GenBankTM/EBI accession D28116. Restriction fragments used
as templates are indicated, with arrows denoting
transcription in the antisense orientation. TA
and TB refer to Transcripts A and B of
COL4A6. B, analysis of protected bands. The major
protected species are indicated by arrows. Sizing was by
comparison against an end-labeled HinfI-digested ØX174 DNA
ladder (Promega), giving 200 bp for COL4A5, 105 bp for
Transcript A of COL4A6, and 209 and 234 bp for predominant
bands associated with Transcript B of COL4A6. The data are
from two different gels, which were run under similar conditions and
gave overlapping molecular size markers.
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Fig. 4.
Structure of the COL4A5 and
COL4A6 promoter region. Transcription start
sites, sequence bearing identity to a transcriptional initiator
(Inr) at 7 of 8 nucleotides, and CCAAT, AP-1, Sp-1, and CTC
consensus regulatory elements are indicated (see text).
5E1 reporting in the COL4A5
orientation gave luciferase activity 17- and 47-fold higher than the
promoterless vector pGL3-Basic, in SCC-25 and SK-N-SH cells,
respectively (Fig. 5). This finding
localizes the minimal COL4A5 promoter to the intergenic
region. Inasmuch as the two cell types differ in COL4A5 expression (Figs. 1 and 2), overlying mechanisms are implicated in
regulation of the endogenous promoter. Transfections with construct p
6E1a gave luciferase activity 17- and 40-fold higher than the promoterless control, in SCC-25 and SK-N-SH cells (Fig. 5). These activities place the minimal COL4A6 promoter in the
intergenic region as well and implicate additional mechanisms in
repressing COL4A6, in SK-N-SH cells. In GVE cells,
luciferase activity was only 5- to 6-fold over promoterless controls,
in both the COL4A5 and COL4A6 orientations (Fig.
5). The lack of orientation specificity for these constructs suggests
that distal elements are involved in the selective activation of
COL4A5 (see "Discussion").
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Fig. 5.
Promoter activity in model cell lines.
Parent constructs containing the 5'-flanking regions of the
COL4A5 and COL4A6 genes were transiently
transfected into the indicated cell lines. A map of the region and
restriction sites used in generating the constructs are shown.
Numbering is according to GenBankTM/EBI accession D28116.
Hatched areas denote 5'-untranslated regions. A,
COL4A5 constructs; B, COL4A6 E1a
constructs; C, COL4A6 E1b constructs. Luciferase
activities represent mean ± S.E. (n = 3).
6E1b containing the 5'-flanking region of E1b was used to
assess whether transcription of COL4A6 is directed by two
promoters. Reporter gene activity was 7- to 9-fold higher than the
promotorless control in the three cell lines (Fig. 5), indicating the
presence of a functional promoter, which is distinct from that upstream
of E1a and thus a site of differential regulation for Transcript B. Given comparable promoter activities among the cell lines, additional
regulatory mechanisms must apply, to explain differences in
steady-state levels of Transcript B.
5E1, caused 82 and 87% reductions in
activity, respectively (Fig. 5), indicating the presence of a positive
regulatory element in this region. Further deletion to nt 625 did not
alter reporter gene activity appreciably. Construct p
5E1(625), which
contains 99 base pairs upstream from the transcription start site, gave
luciferase activity significantly higher than promoterless controls,
suggesting that the minimal promoter resides within this fragment. In
GVE cells, serial deletions of p
5E1 to nt 625 had no significant
effects, with activity for p
5E1(625) remaining significantly higher
than control. These results consign the minimal promoter to the
immediate 5'-flanking region, as in the other cell lines, but provide
evidence against the function of a positive regulatory element.
Deletion of the tandemly arranged CCAAT boxes or of the CTC box (see
Fig. 4) had no significant effects on reporter gene activity.
5E1, those of p
6E1a were
associated with only modest (<55%) reductions in luciferase activity
(Fig. 5). Plasmid p
6E1a (755), containing 67 bp upstream of the
transcription start site, gave luciferase activity significantly above
promoterless controls. These data indicate the presence of a minimal
promoter immediately upstream of exon E1a, which is thus functionally
separable from the COL4A5 promoter, as it is from a second
downstream COL4A6 promoter.
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Fig. 6.
Mutational analysis of a putative
bidirectional element. A, substitution mutations. The
region containing a positive regulatory element is indicated by an
oval. MT1 through MT5 were introduced into p 5E1 and
p
6E1a, as described under "Experimental Procedures." Numbering
is according to GenBankTM/EBI accession D28116.
B, luciferase activity for wild-type and mutant constructs.
Transient transfections were carried out in SCC-25 cells, and
activities are shown for constructs reporting in the COL4A5
or COL4A6 orientation.
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Fig. 7.
Binding of the bidirectional element.
Nuclear extracts from SCC-25 or GVE cells were incubated in gel shift
assays with wild-type or MT2-mutant probes, in the absence or in the
presence of excess ~150-fold unlabeled competitor. Prominent band
shifts are designated S1 and
S2 for SCC-25 cells, the latter representing a
triplet, and G1 for GVE cells, representing a
doublet. Nonspecific band shifts (NS) remained in the
presence of excess competitor. U indicates unbound
probe.
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Fig. 8.
Footprinting of the bidirectional
element. S1-bound and unbound probes were resolved by
gel shift assays, subjected to chemical cleavage in situ,
for the indicated periods, and isolated as described under
"Experimental Procedures." Approximately equal amounts of the
recovered products were analyzed on 10% sequencing gels. A strongly
protected region is indicated by the filled box, with a
weakly protected region indicated by the open box. The
shaded sequence 5'-TAGC-3' is cleaved preferentially by
copper phenanthroline (44). The site of the MT2 mutation is
indicated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5(IV) and
6(IV) chains in the renal GBM. On the basis of our
promoter studies, distal elements are required to account for selective
activation of the COL4A5 promoter. Nonetheless, features of
the proximal promoter region may afford a partial explanation. In GVE
cells, inactivity of the bidirectional element is evidenced
functionally in transient transfections, and is corroborated by
differential patterning in gel shift assays. This inactivity could be
permissive for selective COL4A5 activation, ensuring absence
of the
6(IV) chain in GBM, by uncoupling transcription of the two
genes. In principle, selective transcription could also be accomplished
through asymmetric effects of cis-acting elements on a
shared promoter; however, this scheme, described for other co-expressed
gene pairs including COL4A1-COL4A2 (31, 32), may
not afford sufficient selectivity for COL4A5 and
COL4A6.
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Fig. 9.
Model of the
COL4A5-COL4A6 proximal promoter
region. The model contains non-overlapping sites for recruitment
of transcriptional initiation complexes (large boxes), and a
positive regulatory element near the MT2 site (small box).
A, uncoupled activation of COL4A5 transcription,
as in GVE cells, depends on inactivity of the bidirectional element. A
proposed glomerular activator of COL4A5 (GA) acts through a
distal enhancer. B, coupled transcriptional activation, as
in SCC-25 cells, occurs through the MT2 site, bound by a bidirectional
activator (BA). C, in SK-N-SH cells the genes are repressed
(darkened symbols) and activator-unresponsive.
An interesting issue raised by this and earlier work on other paired genes is the mechanism by which a proximate bidirectional element coordinates transcription. One possibility is that elements bound at the MT2 site alternate among minimal promoters, contributing to assembly and/or activation of basal transcription factors at each of these sites discretely. Another possibility is that promoter modulation occurs simultaneously, even to the point of interlocking divergent transcriptional initiation events. Simultaneous interactions may provide a more compelling case for conservation of the paired gene arrangement, as has been suggested for other gene pairs (53).
The bidirectional element is the most potent
cis-acting element in the intergenic region, in SCC-25 and
SK-N-SH cells. By deletion analysis, we could not demonstrate roles for
a tandem array of CCAAT boxes, or for a central CTC box. These elements may play important roles in directing COL4A5 and
COL4A6 transcription under different experimental
conditions, or in cellular response to external mediators, such as have
been shown to affect the COL4A1-COL4A2 promoter
(54). A putative regulatory mutation causing ectopic 6(IV)
expression in the GBM is in close proximity to tandem CCAAT boxes (14),
raising the possibility that the mutation facilitates transcriptional
activation through these sites. It remains to be seen whether any of
these promoter elements are conserved in the intergenic region
separating the COL4A3 and COL4A4 genes, therein
providing a possible means of coordinating the expression of the four
minor chains. Such elements could be targets of altered regulation in
cases of Alport syndrome in which decreased mRNA levels for all of
the minor chains accompany mutation in a single type IV collagen gene
(23). Whereas it is possible that this effect arises at the level of
mRNA stability, as has been proposed (23), it is also possible that
the presence of a mutation generates a signal feeding back on the
transcription of related genes.
In summary, we have shown that the transcription of the
COL4A5 and COL4A6 genes is directed by distinct
promoters, which permit highly selective expression of their gene
products. In keratinocytes, co-expression of 5(IV) and
6(IV)
correlates with the activity of a bidirectionally active proximal
promoter element, which is bound at a previously unrecognized site.
This element is likely to be important in establishing tissue-specific
patterns of
5(IV) and
6(IV) co-expression. Nonetheless, it is
clear from the complexity of binding, as well as from the largely
unexplained phenomenon of discordant expression in the GBM, that the
proximal promoter region functions within a transcriptional hierarchy,
which is largely responsible for the specialized distribution of type
IV collagen.
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ACKNOWLEDGEMENTS |
---|
We thank Christine Herzog, Ursula Kaiser, and Stephen Reeders for critical review of the manuscript.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants DK02419 and DK48317.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.
§ To whom correspondence should be addressed: Renal Division, Brigham and Women's Hospital, Harvard Medical School, HIM 522, 77 Ave. Louis Pasteur, Boston, MA 02115. Tel.: 617-525-5860; Fax: 617-525-5861; E-mail: zhou@rics.bwh.harvard.edu.
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M007477200
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ABBREVIATIONS |
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The abbreviations used are: GBM, glomerular basement membrane(s); bp, base pair(s); GVE, glomerular visceral epithelial; RT, reverse transcription; PCR, polymerase chain reaction; nt, nucleotide(s).
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