PART II. STRUCTURE AND REGULATION OF COLLAGEN GENES

Fibrillar Collagen Genes
Structure and Expression in Normal andDiseased States“
FRANCESCO RAMIREZ,~SHARON BOAST,
MARINA D’ALESSIO, BRENDAN LEE,JAMES PRINCE, MING-WAN SU,HENRIK VISSING, ANDHIDEKATSU YOSHIOKA
Department of Microbiology and Immunology andDepartment of Orthopedic Surgely
Morse Institute of Molecular Genetics
State Universiv of New York
Health Science Center at Brooklyn
Brooklyn, New York 11203

INTRODUCTION
The collagens represent a multigene family whose members are segregated into
evolutionarily distinct groups. The largest of these groups (group 1) includes thegenes that specify the procollagen precursors of the fibril-forming molecules (types I,
11, 111, V, and XI). To date seven of the group 1 genes have been cloned, and theircorresponding loci have been mapped. Despite some important differences, the overall
organization of the fibrillar collagen genes is highly similar and remarkably conservedin Deuterostomia. This substantiates the hypothesis that the group 1 collagens arosefrom a common multi-exon progenitor prior to vertebrates’ radiation. The characterization of several collagenopathies has elucidated some of the selective pressuresthat have rigidly maintained such a complex organization during evolution. Thesestudies have also defined the metabolic consequences of collagen mutations and theunique contribution of each collagen type to the integrity of the body architecture.
In contrast to their structural similarities, the differential expression of the group 1
genes correlates with the presence of distinct networks of interactions between cisacting sequences, as well as between these elements and specific trans-acting nuclear
factors. Relevant to this, the finding that fibrillar collagen genes are activated during‘This work was supported by grants from the National Institutes of Health (AR 38648, H D
22657, and HL 41104) and the March of Dimes Birth Defects Foundation (1-1042).
bPresent address: Brookdale Center for Molecular Biology, Mt. Sinai Medical Center, 1
Gustave Levy Place, New York, New York 10029.
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early animal embryogenesis supports the notion that this ubiquitous and ancient groupof proteins plays an active role in critical morphogenetic programs of all metazoa, as
well.
Our work on these three topics (gene structure and evolution; pathogenesis andphysiology; regulation and developmental expression) will be briefly reviewed here,with particular emphasis on the human system.

GENE STRUCTURE AND EVOLUTION
A fibrillar procollagen molecule can be generalized as consisting of three major
domains, a long triple-helical region made of several Gly-X-Y repeats and two shortterminal projections.’
The structure of the C-terminal propeptide is fairly similar in all procollagetl
chains, and it is characterized by the presence of highly conserved elements, notably
the C-protease cleavage site, the cysteinyl residues, and the N-linked carbohydrate
attachment site. C-propeptides play a key metabolic function since intracellular foldingof procollagens into the triple helix begins with the formation of inter- and intrachain
disulfide bonds.’ The spatial organization of the cysteinyl residues thought to be
involved in these interactions is seemingly invariant among fibril-forming procollagens;
to be specific, the first four residues (C1 to C4) are believed to participate in interchain
linkages, the remaining (C5 to C8) in intrachain bonding.‘ The pattern of the first
four cysteines, however, displays some exceptions in that pro-a2( I) and pro-a 1(XI)lack C2, and pro-a2( V) lacks C3.”-’ Interestingly, the C-propeptides of two echinoid
fibrillar collagens exhibit the same structural features as their vertebrate counterparts,
thus suggesting that intra- and intermolecular junctions, as well as N-linked glycosylation, are fundamental structural/functional elements of the molecular architecture
in vertebrate and invertebrate species.&’ At the gene level, the C-propeptide coding
sequences are similarly divided into four exons (numbers 49 to 5 2 ) , the first of whichcontains both collagenous and C-propeptide sequences?
With the exception of two fused exons in COLlAl, the triple-helical coding exonsof the group 1 genes conform to an organizational pattern in which both their number
The exons are mostly 54 bp in length or multiples
and size are rigidly maintained.2~4*s*8~9of it (45 bp, 99 bp, 108 bp, 162 bp). This strongly suggests that the triple-helicalcoding domain evolved by processes of duplication, fusion, and deletion of an ancestral
54-bp unit that codes for six Gly-X-Y triplets, or 1% turns of the helix.” Relevant tothis, it has recently been found that a bacterial hydrolase is stabilized into a homotrimeric conformation by six Gly-X-Y repeats, as well.” Irrespective of individual
sizes, each of the collagenous coding exons begins with a Gly codon and ends witha Y codon, thus resulting in an in-frame “cassette” type of organization. Such a motif
has recently been identified in sea urchin collagen genes but not in those of Drosophilamelanogaster and Caenorhabditis elegan~.~-’~’~-‘~
Hence, the different organization of
the invertebrate collagen genes seems to reflect more fundamental differences in theevolution of the Protostomia and Deuterostomia genomes.
In contrast to the other two domains, the N-propeptides differ greatly in size andcomposition among group 1 chains and appear to adhere to one of two basic architectures.I5 The first, characteristic of pro-al( I), pro-al( 111), and pro-a2(V), displays
the longitudinal arrangement of a cysteine-rich globular domain, a collagenous sequence, and a short, nonhelical ~egment.9.””~The second, characteristic of pro-a2( I)16

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and pro-al( 11), lacks the entire cysteine-rich globular domain, and, as a consequence,the signal peptide is directly connected to the collagenous ~equence?"~
More recently,
a third architecture has been recognized in pro-al(XI), whose N-propeptide is significantly longer than the others; it contains short, dispersed clusters of collagenous
sequences and lacks the cysteine-rich globular domain (H. Yoshioka et al., manuscript
in preparation). It should be noted that interrupted collagenous sequences are also
observed in pro-a2( V) and pro-a 1(II).17s19
Interestingly, a phylogenetic comparisonof the latter chain in man, chicken, and frog has revealed that the loss of the cysteinerich globular region is a relatively recent event, for this domain is present in theamphibian, but not in the avian and human chains (M. W. Su et al., manuscript in
preparation). At the genomic level the N-propeptide coding sequences of COLl Al,
COLlA2, COL2A1, and COL3A1 are organized in distinct manners with consequent
divergency in both the number and the size of the corresponding ex0ns.2'~*'~.'~
The
numerical heterogeneity of the N-terminal coding units seems, at the present time, tocorrelate more with the heterotrimeric assembly of their products than with theparticular architecture of individual N-pro pep tide^.'^From the data hitherto gathered, it is therefore clear that the evolutionary historyof the group 1 collagen genes is ancient, in that it predates vertebrates' radiation. In
contrast to the triple-helical domain, whose 54-bp exon seems to have been selected
as an optimal unit for chain interaction, the N- and C-propeptides seem to havefollowed distinct and more relaxed evolutionary pathways. The detail of such a scenario
is likely to be elucidated when the organization of additional members of the group
1 family is characterized in different metazoa.

PATHOGENESIS AND PHYSIOLOGY
Because of the metabolic, structural, and genomic similarities of the group 1
collagens, the well-characterized type I collagenopathies serve as paradigms for the
analysis of other connective tissue disorders. To date two clinically distinct conditions
are associated with mutations in the subunits of the type I collagen, namely osteogenesis
imperfecta (01) and Ehlers-Danlos syndrome Type VII (EDS VII)?' In broad terms
the former, characterized by bone fragility, results from mutations that affect the
intracellular assembly of the trimers; whereas the latter, characterized by loose joints,
is caused by defects that interfere with extracellular conversion of procollagen tocollagen.
01 mutations, which include deletions, mis-splicing, and single amino acid substitutions, are all believed to decrease the rate of helical assembly and thus to expose
greater regions of unassembled chains to overmodification?' Hence, the relative location of the defect within the triple-helical domain is thought to determine the overall
rate of modification and consequently the degree of phenotypical severity?' 01 mutations have also provided some relevant information on the evolution of the genes.
First, the essential role of the highly conserved cysteinyl residues of the C-propeptide
has been demonstrated by the finding that loss of C8 in pro-a2(I) collagen leads tothe exclusion of this chain from trimer assembly?* Second, the absolute preservationof the organization of the triple-helical coding domain has been inferred from deletionmutants in which shortened but in-frame procollagen chains lead to the formation ofout-of-phase parental trimers that are incompatible with s~rvival.2~
Some exceptions to the aforementioned rules are now being discovered in type I1
and type I11 collagenopathies. In two EDS VII patients, large heterozygous deletions

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77

in the triple-helical domain of COL3A1 call into question the validity of the so-called
location rule, since they exhibit an inverse relationship between clinical severity andC-terminal proximity of the mutati0ns.2'~' Similarly, the finding that a single-exon
deletion and a Gly-to-Ser substitution at nearly the same location in al(I1) lead tosevere (spondyloepiphyseal displasia) and lethal phenotypes (type I1 achondrogenesishypochondrogenesis), respectively, implies that the nature of the mutation may also
play an important r01e.**-~~EDS VII has provided novel information on the mechanisms of splice-site selections
in multi-exon genes. The five heterozygous cases hitherto studied display in fact thesame splicing abnormality, exon-skipping, which is, however, caused by three distinct
mutations. The first, which involves the obligatory GT dinucleotide of the 5' splicesite of the sixth intron of COLlA2, leads to precise outsplicing of exon-6 sequenceswith the consequent elimination of the N-proteinase cleavage site and the N-telopeptide
cross-linking site." A similar event is seen in the COLlA2 allele of another EDS VII
patient,32in which all of the sequences believed to regulate splicing are surprisingly
normal. Conceivably, the mutation resides within the large intervening sequence thatflanks exon 6, thus suggesting that splicing of such a complex transcript might bedependent on as-yet-uncharacterized elements. The remaining three EDS VII variants
bear the same mutation in either COLl A1 or COLlA2, notably a G-to-A substitution
in the last codon of exon 6.33-34Such a finding reiterates the importance of the wholeexon/intron junction sequence in conferring specificity to the splicing reaction. The
exon change decreases but does not abolish the relative strength of the splicing signal,in that corrected spliced transcripts are demonstrable in both the COLlA2 (50%)
and COLlAl (20%) mutated allele^.'^-^^ In line with this and in contrast to the intron
mutation, the mis-splicing caused by the exon mutation can be effectively suppressedin cellula by lowering the temperature at which the fibroblasts are grown?' It canalso be argued, by analogy to in vitro splicing experiment^:^ that the differential
effectiveness of the exon 5/intron 5 junction to compete in cis for splice-site recognition
in COLlA2 vs. COLlAl may reflect the difference in relative distance of the splicing
signals in the two gene^.^,^ Accordingly, we suggest that splicing mutations in collagengenes can have variable expression depending on which of the sequences are affected,
on the relative strength and distance of the cis-competing signal, and plausibly on
exogenous factors such as temperature, which may modulate mis-splicing in different
tissues and/or at different developmental stages.

REGULATION AND DEVELOPMENTAL EXPRESSION
The sequences around the transcription start site of human type I collagen genes
have been functionally analyzed in search of cis-acting elements that regulate optimaland tissue-specific expression.
We have found that the COLlA2 promoter (-3800 +54), linked to a reporter
gene- the bacterial chloroamphenicol acetyltransferase (CAT)-and transfected into
human fibroblasts and lymphoblasts, displays tissue-specific expression. A series of
CAT constructs containing 5' COLIA2 deletions identified the region necessary forproper transcription from position -772 to +54 (S. Boast et al., manuscript in
preparation). In the same experiments we also demonstrated that the type I and typeI1 collagen genes possess distinct regulatory elements, in that CAT activity of theminimal COLlA2 promoter is effectively abolished by competition with the promoter
sequences of COLlAl but not COL2A1. When COLlA2-driven CAT activity wascompeted by internal fragments of the COLlA2 promoter, inhibition was observed

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with all competing molecules in fibroblasts. In parallel experiments utilizing immortalized chondrocytes as recipient cells, all but one of the COLlA2 fragments (- 104
54) displayed efficient competition of the basal CAT activity. This finding suggeststhat chondrocytes lack fibroblasts’ specific factor( s) binding to the sequences between-104 +54 of COLlA2. In a SV40-transformed human fibroblast cell line, which
does not produce pro-a2(I) c0llagen,3~the COLlAZ/CAT construct was found to
mimic the downregulation of the endogenous gene when transfected at low concentrati~n.~’At higher concentrations of the chimeric construct CAT activity was fully
restored, indicating the presence of (a) titratable transacting factor( s) present onlyin the nuclear lysate of the transformed cells.)7 Competition experiments and bandshift assays revealed that binding of the factor(s) occurs in the region of COLlA2
comprised between -360 and +54.37 Hence, most of the sequences regulating its
expression appear to reside within this short segment of the COLlA2 promoter.
Similar experiments have demonstrated the presence of a downstream cis-acting
enhancer element in COLl Al. Briefly, transcriptional activity of the COLl A1 promoter was originally assessed on an a globin gene after injection of the construct into
Xenopus Zaevis oocytes or transfection into NIH 3T3 cells. When the activity of thisreference construct was compared to that of a similar one that contains in addition
782 bp of the first intron of COLlAl, a significant enhancement in globin productionwasSubsequently, however, others have shown that in chicken embryo
fibroblasts the COLl A 1 intervening sequence fails to enhance transcription?+” To
reconcile this discrepancy, the activity of the 782-bp element positioned in bothorientations upstream from a COLl A1 promoter/CAT construct has recently beentested in a variety of cells, including human fibroblasts, human lymphoblasts, andchicken embryo fibroblasts ( S . Boast et aZ., manuscript in preparation). The results
showed that the 782-bp element enhances-in a preferential but not exclusive orientation-its own promoter without tissue specificitybut rather with species specificity,
for it does not display activity in avian cells.
To complement and extend our study on collagen regulation the pattern of collagen
onset during the development of sea urchin and Xenopus faevis embryos has recently
been ascertained. In the former organism two fibrillar collagen genes were found to
be expressed in the mesenchyme cell lineage during early stages of ernbryogenesi~5~
On the basis of these data, we concluded that the two genes specify molecules likely
to be involved in both gastrulation and skeletogenesis.4*Likewise, in the frog’s embryo
type I1 collagen transcripts were detected at the time of mesodermal induction:’ thus
suggesting that this fibrillar collagen is probably involved in the spatial organizationof the developing embryo prior to organogenesis (M. W. Su et aZ., manuscript in
preparation). It will be of interest to further elucidate the function and regulation ofcollagen in these two organisms in which activation of cell lineage-specific genes
results from either determinative or inductive responses.
In conclusion, the analysis of the biology of the fibrillar collagen genes in vertebrate
and invertebrate species, as well as in pathological and physiological conditions, hasdemonstrated the unique versatility of this system in addressing a variety of questions
that greatly impact on our understanding of eukaryotic gene expression and evolution.

+

ACKNOWLEDGMENTS
The authors thank Ms. R. Lingeza for preparing the manuscript.

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