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J. Biol. Chem., Vol. 280, Issue 16, 15749-15760, April 22, 2005
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From the
Department of Histology, Microbiology, and Medical Biotechnologies, University of Padova, 35131 Padova and the Division of Experimental Oncology, CRO-IRCCS, 33081 Aviano, and the ||Department of Biomedical Sciences and Technologies, University of Udine, 33100 Udine, Italy
Received for publication, November 5, 2004 , and in revised form, January 28, 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For several years the authors have been studying Emilin1, a protein of elastic fibers that is preferentially detected by electron microscopy between the amorphous core and the coat of microfibrils (the name is an acronym from Elastin microfibrils interface located protein) (9). The primary sequence shows that Emilin1 comprises five domains (10). A C1q-like domain similar to those of type VIII and type X collagens is located at the carboxyl-terminal end. A short collagenous domain separates the C1q domain from a long region with a high probability of coiled-coil conformation. Finally, two domains are found at the amino terminus: a signal peptide and a new type of domain called the EMI domain (11). The latter, a cysteine-rich sequence of about 75 amino acids, is shared by a number of genes (seven in mammals) that have been grouped into the Emilin gene family (www.gene.ucl.ac.uk/nomenclature/genefamily/emilin.html#HGNC_table2). Gene targeting experiments suggest that the protein has a role in the assembly of elastic fibers, particularly in blood vessels; in its absence, elastic lamellae of aorta are interrupted and their outline is irregular (12). A function of Emilin1 in elastogenesis is also indicated by the finding that the protein binds to other components of elastic fibers such as Elastin and Fibulin-5 (12). However, analysis of the distribution of Emilin1 mRNA during mouse development induces us to suggest additional functions of Emilin1 not related to elastic fibers; in fact, early after implantation, Emilin1 mRNA is detected not only in the cardiovascular system but also in extra-embryonic tissues (extra-embryonic visceral endoderm, ectoplacental cone, and spongiotrophoblast), epithelial tissues that do not produce elastic fibers (13). During organogenesis high level expression is found in interstitial connective tissue, perichondrium, and mesenchymal condensations, sites containing elastic fibers. This brief description of Emilin1 expression during development testifies to a complex gene regulatory setup. Nevertheless, the Emilin1 gene and flanking sequences are a compact unit; the gene, consisting of 8 exons, is about 8 kb long and is separated by the nearby 2310015E02Rik and Khk genes by about 6.3 kb at the 5'-end and 0.7 kb at the 3'-end, respectively (14) (see Fig. 3C). This feature makes the Emilin1 gene a good candidate for analysis of the mechanisms of tissue-specific transcriptional regulation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A fragment spanning positions –402 (corresponding to a BamHI restriction site) to –1 from the first codon was generated from the 16.8-kb fragment by PCR with the reverse primer comprising mutagenized bases that change the sequence corresponding to the translation start site ATGGCC to a PstI restriction site. After digestion with BamHI and PstI, the amplified 407-bp fragment and an 8249-bp HindIII/BamHI fragment extending from –403 to –8651 bp were ligated into the promoterless vector pBLCAT6 (15) to give plasmid p8.7-CAT. 5'-End deletions extending to about –6.0, –5.1, –4.0, –3.0, and –1.1 kb and to –630 bp were generated by exonuclease III treatment ("Erase a Base" kit, Promega) of the 8.7-kb fragment using the procedures recommended by the manufacturer.
The 630-bp deletion was inserted into pBLCAT6 to give p630-CAT. Deletions of p630-CAT from 5'- and 3'-ends were developed by Bal31 digestion following established protocols (16). p(630–470)-CAT and p(630–441)-CAT were generated from the 16.8-kb fragment by PCR with appropriate primer oligonucleotides.
For site-directed mutagenesis of Initiator1 and Initiator2 sequences (Fig. 1), the mutated p630-CAT constructs were obtained by ligation of a HindIII/NcoI fragment carrying sequences from –630 to –459 bp, an NcoI/XbaI fragment extending from –458 to –1, and the promoterless vector pBLCAT6. For p630Inr2-CAT, the mutant fragment was generated by PCR with a forward primer comprising mutagenized bases that change the native sequence of Initiator2 (–453/–447) TCACTCT to Tgtggtt (where lowercase letters indicate mutated bases) and a reverse primer comprising mutagenized bases that change the sequence corresponding to the translation start site ATGGCC to an XbaI restriction site TCTAGA. To derive the mutant fragment of p630Inr1-CAT, the Initiator1 sequence CCAGACA (–517/–511) was replaced with Cgtggtt (17) following a two-step PCR-based site-directed mutagenesis procedure (16) using the following primers: F1 (–630/–612), 5'-GAAGCTTCACATTTCTCTGTTTTTCC-3', forward (HindIII site underlined); M1 (–522/–502), 5'-CCTTCCCCCaaccacGCTGGC-3', reverse; M2 (–524/–504), CCGCCAGCgtggttGGGGGAA-3', forward; R2 (–458/–441), 5'-GGGATCCGGCACAAGAGTGACCATG-3', reverse (BamHI site underlined).
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-lacZ, the 407-bp BamHI/PstI fragment was replaced with a 238-bp fragment extending from –402 to –170 generated by PCR with appropriate primer oligonucleotides. To derive the transgene construct p8.7-lacZ-intron, the third intron of Emilin1 gene was synthesized by PCR from the 16.8-kb fragment as DNA template and inserted downstream from the lacZ gene into the p8.7-lacZ plasmid.
To obtain the transgene construct p0.6-lacZ gene, the 8.0-kb fragment that contains the coding region of Emilin1 from +78 in the first exon to the intergenic region between Emilin1 and Khk genes (Fig. 3C) was generated by digestion of the 16.8-kb fragment with XhoI and HindIII and cloned into the SmaI site of pBluescript vector. The transgene 0.6-lacZ was excised with HindIII and NotI and inserted upstream of the XhoI/HindIII fragment of the above plasmid.
To derive the construct p8.7-lacZ-gene, the BamHI fragment of the p0.6-lacZ plasmid, including 402 bp of proximal promoter region and the lacZ gene, was cloned into the same site of pBluescript after removal of the HindIII site. The XhoI/HindIII fragment spanning positions +78 to +8111 was then inserted downstream the lacZ sequence. Finally, the BamHI fragment from –402 to +1299 of the 16.8-kb fragment was replaced with the BamHI fragment of the above plasmid containing 402 bp of the proximal promoter region, the lacZ gene, and the coding region from +78 to +1299. All constructs were sequenced in both directions to verify correct cloning.
Cells—The following cell lines were used: NIH3T3 fibroblasts, C2C12 myoblasts, EL4 lymphocytes (8), MC615 chondrocytes (19), BC3H1 smooth muscle cells (ATCC, Manassas, VA), and H.end (20) and cEnd (21) murine endothelial cell lines. The cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, with the exception of EL4 cells, which were maintained in RPMI 1640, 10% fetal calf serum, 4 mM glutamine, and 20 mM 2-mercaptoethanol.
5'-RACE Analysis—To determine the transcription start sites of the Emilin1 gene, 5'-RACE was performed using the "5'-RACE system for rapid amplification of cDNA ends" (Invitrogen) and total RNA from E14.5 mouse embryos. First strand cDNA was synthesized from 1 µgof total RNA using SuperscriptTM II reverse transcriptase (Invitrogen) and an antisense primer (RACE1, 5'-GCCGTGGTGAGTTCTGGG-3', which corresponds to nucleotides 137–154) at 37 °C for 1 h. After (dC)-tailing with terminal dideoxynucleotide transferase at 37 °C for 10 min, the cDNA was PCR-amplified with internal antisense primers (RACE2, 5'-GCTTCCGTGGCTATGGTCAG-3', which corresponds to nucleotides 43–62; RACE3, 5'-GCAGACAGCAGAGATAGCA-3', which corresponds to nucleotides 25–43) and a 5'-anchor oligo(dG) primer provided with the kit. PCR products were resolved in a 2% agarose gel, isolated, and subcloned into pGEMT-Easy vector (Promega), and 40 clones were sequenced.
RNase Protection Assay—Four different partially overlapping DNA sequences were used to generate the riboprobes. Probe 1 was derived by subcloning a blunted 891-bp StuI/XhoI fragment extending from –814 to +77 into the SmaI site of pGEM3 vector (Promega). Probe 2 was similarly obtained from a 681-bp StuI/PstI fragment extending from –814 to –133. To generate probe 3 and probe 4, a 561-bp fragment from –815 to –254 and a 470-bp fragment from –815 to –345, respectively, were PCR-amplified and subcloned into pGEMT-Easy vector (Promega). Linear DNA templates were prepared by XbaI (probe 1), HindIII (probe 2), and SalI (probe 3 and probe 4) digestion. 32P-Labeled RNA probes were transcribed using 40 units of T7 polymerase (Ambion, Inc.) in the presence of 1 µg of linearized plasmid, 10 µl of [
-32P]UTP (400 Ci/mmol; Amersham Biosciences), ATP, GTP, and CTP (400 µM each), 10 mM dithiothreitol, and 40 units of RNasin (Promega) in a total volume of 20 µl. The hybridization of the probe with total RNA (10 µg from E14.5 mouse embryos) was carried out at 42 °C overnight according to the standard procedure of RPAII RNase protection assay kit (Ambion Inc.). RNase digestion was performed using an RNase A/RNase T1 mixture in RNase digestion buffer (Ambion Inc.), and protected fragments were separated on a 6% nondenaturing polyacrylamide gel.
Transfections and Promoter Assays—NIH3T3 and C2C12 cells were grown as described above; 3 x 105 cells were plated into 10-cm Petri dishes and transfected the following day with the CAT constructs and the control plasmid pRSV-luciferase (15 and 1.2 µg, respectively) using the calcium phosphate method (22). All subsequent manipulations and assays were performed as described (23).
Generation and Analysis of Transgenic Mice—Fertilized B6D2F1 x B6D2F1 mouse oocytes were microinjected with lacZ constructs and implanted in the uterus of CD1 pseudopregnant mothers using standard procedures (24). Transgenic mice were identified by PCR analysis on genomic DNA from yolk sac or tail biopsies using primers derived from the lacZ sequence (forward, 5'-CGGTGATGGTGCTGCGTTGGA-3'; reverse, 5'-ACCACCGCACGATAGAGAGATTC-3') and reaction conditions as described (25). Whole mount and histological examinations of 
-galactosidase expression were carried out exactly as described (18).
Other Assays—Northern blotting and analysis of chromatin DNase I-hypersensitive sites were performed as described previously (8, 23).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In order to analyze the elements that control the transcriptional regulation of Emilin1, a 16,762-bp HindIII fragment (for brevity identified in this study as the 16.8-kb fragment) encompassing the entire gene was subcloned from a BAC construct (12). In addition to the Emilin1 gene, which spans 7479 bp and includes eight exons, the fragment contains 8651 bp of 5'-flanking sequence and 632 bp of the intergenic region between Emilin1 and Khk (Fig. 3C). The characterization of the regulatory sequences contained in the 16.8-kb fragment started with the identification of transcription start sites and promoter assays in cell cultures using appropriate deletions.
Determination of the Transcription Start Sites—To map the transcription start site(s) of the Emilin1 gene, 5'-RACE and RNase protection assays were employed. 40 clones obtained with 5'-RACE were sequenced. Although variable, the 5'-end of the clones was frequently found within two regions as follows: one is a broad region between –90 and –160 bp from the start codon, and the other corresponds to the short sequence of putative Initiator2 (Fig. 1). RNase protection analysis showed more than 60 different protection products falling between position –515 and the first codon. Three major transcription start sites were identified at –40, –101, and –123 bp; they were embedded in a region between –38 and –152 bp containing more than half of the transcription start sites determined by RACE (Fig. 1). A group of protected bands coincided with a major clustering of RACE products at putative Initiator2 (Fig. 1). A single protected band was located at position –515, corresponding to putative Initiator1. These results strongly suggest that multiple transcription start sites are utilized for mouse Emilin1 transcription. They also indicate that transcription begins preferentially at putative Initiator2 but also at the other upstream putative Initiator1.
Functional Analysis of the 5'-Flanking Region—To characterize the functional role of Emilin1 regulatory regions, different cell lines were transfected with CAT constructs, including various portions of the 5'-flanking sequence. Our initial analysis was focused on the proximal 630-bp fragment, from which several CAT chimeric constructs were synthesized carrying 5' and 3' deletions (Fig. 2A). The constructs were used for transient promoter assays in NIH3T3 cells, and the results are reported in Fig. 2B. Expression of the reporter gene was halved when the sequence upstream of –537 was cut out (compare constructs p630-CAT and p537-CAT). Further deletion of the region from –537 to –503 determined a considerable decrease of CAT activity (compare constructs p537-CAT and p503-CAT). Additional removal of 5'-sequences from –503 to –480 lowered gene reporter expression to background levels. This analysis indicates the presence of a core promoter region between –480 and –630. This sequence includes putative Initiator1 and several potential binding sites for transcription factor Sp1 (Fig. 1). The importance of the Initiator1 sequence was confirmed by the observation that mutation of the site significantly decreased CAT activity (Fig. 2, construct p630Inr1-CAT). On the contrary, the Initiator2 sequence did not contribute appreciably to core promoter function, as shown by the analysis of 5' deletions (compare constructs p480-CAT and p438-CAT in Fig. 2) and the lack of reduction of promoter activity when the site was mutagenized (Fig. 2, p630Inr2-CAT).
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We then investigated CAT constructs containing larger portions of 5'-flanking sequences (1.1, 3.0, 4.0, 6.0, and 8.7 kb). The plasmid carrying the 1.1-kb fragment exhibited 2-fold CAT expression compared with p630-CAT, indicating the presence of activating elements between –0.63 and –1.1 kb. On the other hand, no difference was detected between 1.1 kb and the longer constructs (data not shown). Because of this limitation, alternative methods were used to highlight regions potentially relevant for transcription, including the determination of chromatin DNase I-hypersensitive sites, sequence analysis using computer programs, and expression of promoter constructs in transgenic mice. These data are presented below.
Analysis of Chromatin DNase I-hypersensitive Sites—DNase I-hypersensitive sites in chromatin are structural landmarks indicative of control regions involved in constitutive and tissue- and/or stage-specific transcription (26, 27). For several ECM genes, DNase I-hypersensitive sites have been found associated with regions that control transcriptional regulation, particularly in a tissue-specific manner (7, 8, 28, 29).
DNase I-hypersensitive sites were analyzed within the 16.8-kb fragment using cell lines of different origin, including NIH3T3 fibroblasts, C2C12 myoblasts, MC615 chondrocytes, BC3H1 smooth muscle cells, EL4 lymphocytes, and endothelial cell lines cEnd and H.end. Northern blotting analysis revealed that all cell lines expressed Emilin1 mRNA with the exception of H.end (Fig. 3A). In addition to the uncleaved 16.8-kb fragment, the probe hybridized to six species (11.6, 7.7, 5.5, 3.9, 3.5, and 2.5 kb) in DNA samples of NIH3T3 nuclei treated with DNase I (Fig. 3B). These species corresponded to hypersensitive sites at approximately +3000 (HSi), –950 (HS1), –3100 (HS2), –4750 (HS3), –5150 (HS4), and –6150 (HS5) from the first codon (Fig. 3C). Sites HSi and HS5 were present in all the cell lines analyzed with similar intensity, including the nonexpressing cell H.end, and are to be considered constitutive (i.e. present in chromatin independently from Emilin1 gene expression) hypersensitive sites. Remarkable differences depending on the cell type could be noted for the other four sites. HS1 was present in all cell lines expressing Emilin1 mRNA and appeared very strong in NIH3T3 and MC615 cells, less evident in C2C12 myoblasts, and very faint in BC3H1, cEnd, and EL4 cells. It was lacking in nonexpressing H.end cells. The band corresponding to hypersensitive site HS2 was very weak in NIH3T3, C2C12, and BC3H1, stronger in MC615, and absent in the other lines. HS3 was faint in all cell lines. HS4 was present only in NIH3T3, C2C12, and MC615 cells, being strongest in the last one. The presence of hypersensitive sites exclusively in Emilin1-expressing cells and the variable intensity of each band depending on the cell type suggest that the sites may correspond to regulatory regions involved in tissue-specific transcriptional regulation of the Emilin1 gene.
Sequence Comparisons—The 5'-flanking region was compared with the corresponding rat and human sequences using the MultiPipMaker (bio.cse.psu.edu/) (30) (Fig. 1B) and Blast softwares (data not shown). Three regions of low similarity (<50%) divided the sequence into four regions of high similarity (Fig. 1B). Moreover, two of the high similarity regions could be further subdivided into subregions by the presence of peaks of very high similarity; as a consequence, the sequence comparison distinguished 14 sequences in the 5'-flanking region as summarized in Fig. 1B and in Table I. One important result was apparent from this analysis: three out of four chromatin DNase I-hypersensitive sites mapping in the 5'-flanking region (HS1, HS3, and HS4) coincided with peaks of higher similarity (Fig. 1B and Table I).
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Analysis of Regulatory Regions Using Transgenic Mice—To locate transcriptionally significant regions, we also exploited expression of promoter-reporter gene constructs in vivo. The series of constructs tested is depicted in Fig. 4. One group of constructs included 5'-flanking sequences extending for different lengths upstream of the first codon (constructs 0.6-lacZ, 1.1-lacZ, 3.0-lacZ, and 8.7-lacZ). Deletion of the proximal 169 bp from 8.7-lacZ produced construct 8.7-lacZ that was derived in order to investigate the role of the small sequence that showed a dramatic influence on promoter-CAT construct activities in vitro (see Fig. 2). Finally, a group of constructs included sequences downstream of the first codon. The third intron was included in one construct (8.7-lacZ-intron) because of a relatively higher density of putative transcription factor binding sites detected in the mouse in this intron compared with the other introns (data not shown). Two additional constructs (0.6-lacZ-gene and 8.7-lacZ-gene) contained the entire region of the Emilin1 gene (excepting for the first 77 coding bases included in the first exon) and most of the intergenic sequence between Emilin1 and Khk genes. Expression of promoter-lacZ constructs was studied mainly on founder embryos at E14.5. Mouse lines were derived with some of the constructs for analysis of transgene expression at different developmental stages, considering that expression of Emilin1 mRNA begins soon after implantation (13).
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-lacZ) gives rise to ectopic expression of the transgene at high frequency. However, this is not the only sequence involved, because the shorter constructs (0.6-lacZ, 1.1-lacZ, and 3.0-lacZ) also exhibit frequent ectopic expression. Therefore, it can be hypothesized that sequences limiting ectopic expression are located in the proximal 169 bp and between –3.0 and –8.7 kb. Paradoxically, ectopic expression was present in 50% of lines generated with the largest construct, 8.7-lacZ-gene (see "Discussion"). -lacZ in Table II). Of interest, the sharp margins of staining obtained with the nuclear lacZ marker gene revealed a previously unrecognized feature of Emilin1 expression in the heart, whereas endocardial cells of both left and right ventricle are equally active in transcription, only the myocardium of the right ventricle expresses the gene at high levels (Fig. 5, B and C).
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-lacZ), removal of this region decreased intensity (less cells and fainter staining) but did not abolish expression of the transgene in endocardium and cardiac cushions at E12.5 (2/3 embryos scored positive); however, no staining was present in the myocardium (data not shown). These results suggest that timely transcriptional activation of Emilin1 in the heart during development does not require the proximal 169 bp of the 5'-flanking sequence and the gene region. Nevertheless, both regions are necessary for sustained transcription at later stages.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our report describes the first characterization of the regulatory setup of mouse Emilin1. The DNA fragment investigated comprised the gene and the 5'- and 3'-flanking sequences separating it from adjacent 2310016E02Rik and Khk genes. The major transcription start sites were located in association with two Initiator consensus sequences identified as Initiator1 (–517 to –511) and Initiator2 (–453 to –447). Besides Initiator1, promoter assays in cell cultures have assigned important functional roles to sequences just upstream of it and between Initiator1 and Initiator2. These sequences contain putative binding sites for transcription factor Sp1 (nucleotides –551 to –540 and –489 to –479). Sp1-binding sites are often located close to the transcription initiation site in TATA-less promoters and contribute to transcriptional initiation (34). On the basis of these results, a reasonable suggestion is that the core promoter of the Emilin1 gene is located between nucleotides 551 and 447 upstream of the first codon.
In addition to providing information on the core promoter region, assays in cultured cells using CAT deletion constructs have identified the proximal 169 bp as an important regulatory fragment. In vitro the activity of the fragment is repressive; in vivo its function is more complex, as it stimulates transgene expression in some tissues (mainly the heart) and, at the same time, it lowers the frequency of ectopic expression. It is likely that the fragment contains both stimulatory and inhibitory elements and that its overall function depends on the cellular context. This conclusion is also suggested by the presence of matches for transcription factors Ets-1 and Runx1, the latter of which is often involved in transcriptional repression (35). These sites are not conserved in rat and human, and the entire 167-bp sequence also exhibits species variations. Hence, the fragment may have a relevant function only in the mouse.
Promoter assays in vitro have also located an activating region between about –0.6 and –1.1 kb from the first codon. This region contains a conserved putative binding site for transcription factor AP1. The actual function of this element has not been investigated. However, the close matching of its sequence with the position of DNase I-hypersensitive site HS1 suggests that the AP1 recognition motif may be functional in the regulation of Emilin1. The –0.6 to –1.1 region is also important for expression in mesenchymal and endothelial cells at several locations in vivo (Table II).
Unfortunately, in vitro promoter assays were not informative for the sequence upstream of –1.1 kb. Nevertheless, a combination of different methods, namely mapping of chromatin DNase I-hypersensitive sites, sequence comparisons, and expression of lacZ promoter constructs in transgenic mice, has succeeded in outlining the functional and structural features of this region. Overall, the three methods have given concurrent results that are also in agreement with the data obtained for the proximal 1.1-kb fragment using promoter assays. Sequences hypersensitive to DNase I are believed to reflect a local rearrangement of nucleosomes and possibly a local distortion in DNA topology due to binding of a large number of transcription factors (36). This suggestion was matched for hypersensitive sites of the Emilin1 5'-flanking region, which mostly map in small fragments with very high sequence conservation and are enriched for transcription factor DNA-binding motifs.
Sequence comparisons between species (mouse, rat, and human) have revealed four regions with higher similarity in the 5'-flanking sequence (Fig. 1B and Table I). These regions could be further subdivided into subregions because of the presence of short peaks of very high similarity (Table I). The region proximal to the first codon is 1055 nucleotides long and corresponds largely to the sequence that we have characterized more thoroughly using promoter assays in vitro (see above). The transcription start sites and two conserved Initiator elements, as well as putative binding sites for other transcription factors, are located within –156 to –679, a subregion of high interspecies similarity. The segment further upstream, which stimulates CAT activity by 2-fold in promoter assays, harbors the conserved AP1 recognition site mapping very close to DNase I-hypersensitive site HS1. The function of the second and third high similarity regions (nucleotides –1340 to –1847 and –2704 to –3619, respectively) were not well established in our analysis because of the fact that constructs were not derived in which the two regions were separated. In fact, the information from these regions comes from the comparison of the expression pattern of 3.0-lacZ (that contains the entire region two and part of regions three) with 1.1-lacZ and 8.7-lacZ (that lack and include both regions respectively. Nevertheless, the observation that construct 3.0-lacZ was expressed at slightly increased frequency in endocardial cushions compared with 1.1-lacZ suggests that either region may contribute to transcription levels in this tissue. The fourth region (from –3894 to –6379) is of key importance for expression in several tissues, particularly the circulatory system, and for achievement of high transcription levels in transgenic mice. Two hypersensitive sites (HS3 and HS4) are enclosed in this region. Most strikingly, both map within short sequences (132 and 241 bp, respectively), exhibiting very high similarity between species (about 80%) and containing conserved consensus sequences for a few transcription factors binding sites. The function of each individual hypersensitive site has not been investigated here. As a consequence, it is not known whether they contribute to regulation in specific tissues or act just to boost transcription levels.
The examination in transgenic mice has also shown that relevant activating regions lie within the gene sequence. However, no DNase I-hypersensitive sites related with expression of the gene have been identified within the gene sequence. The explanation may be due to either the complete absence of such sites within the gene or to the fact that hypersensitive sites are detectable only in tissues that are strongly dependent on the gene region for expression, and these tissues have not been examined here.
It is clear from our data that none of the different regulatory regions have the peculiar property necessary and sufficient for transcription at high frequency and intensity in a given tissue. The exception of the proximal 0.6-kb 5'-flanking region, which drives production of the transgene in tendons and ligaments with maximal frequency (see Table II), is only apparent, as staining in 0.6-lacZ lines was usually limited to a portion of potentially expressing cells and was of variable intensity. Instead, more than one region was required for high level expression in different tissues, and the same region could cooperate with other region(s) to activate transcription in a set of distinct tissues.
If regulatory regions act in such a combinatorial way, how is tissue specificity achieved? The best explanation comes from a model derived from studies on the hemoglobin genes (27). This hypothesis proposes that the key feature to establish an independent expression profile is the ability of the core promoter for a specific gene to communicate with strongly activating cis-regulatory elements in a particular tissue. In the model, intervening chromatin stretches are supposed to loop out even when they contain important regulatory sites for other nearby genes, the reason being that these regions cannot positively interact with the promoter of the specific gene. The spatial unit of activating regulatory DNA regions for a specific gene is referred to as an active chromatin hub (ACH). Productive ACH formation underlies the correct gene expression, requiring the presence of protein factors with the appropriate affinities for each other bound to their cognate DNA sequences. The data reported here can be explained by suggesting that different regulatory sequences participate in the formation of an ACH, depending on the set of protein factors produced in each tissue where Emilin1 is expressed.
An interesting aspect of our study is that the DNA segment analyzed contains information for transcription in every tissue where Emilin1 products have been detected. This may suggest that the fragment harbors the complete set of regulatory regions necessary for appropriate Emilin1 transcription. To establish whether this is the case, additional studies must be carried out for investigating the quantitative aspects of transcription stimulated by the 16.8-kb fragment as part of a suitable transgene. Such transgenes should exhibit position-independent and copy number-dependent expression, as expected for a locus control region. In the ACH model the locus control region is viewed as a part of ACH itself, contributing combinations of cis-regulatory elements that can establish position-independent gene expression. These studies are underway in our laboratory.
One feature of the ACH model is that stable formation of ACH is the critical event buffering against position effects in transgenic experiments and that stable enhancer-promoter interactions, rather than the presence of insulating borders, determine appropriate gene expression. This proposition explains an unexpected finding in our study concerning ectopic expression. Constructs comprising short stretches (less than 3 kb) of the 5'-flanking region were incorrectly transcribed at high frequency (more than 70% of transgenic mouse lines). This can be attributed to interaction of activating sequences unable to form an ACH with nearby promoters. The frequency dropped to zero with longer constructs, including 8.7 kb of 5'-flanking sequence, likely due to the fact that regulatory regions of the transgene were involved in an ACH and were therefore not available for interactions with promoters at the insertion site. Deleting the 5' proximal 169 bp causes destabilization of the ACH and reappearance of ectopic expression. Most unexpectedly, the addition of the gene region (construct 8.7-lacZ-gene) reactivated ectopic expression in some lines (50% frequency). This paradox can be accounted for by observing that the extremities of the investigated 16.8-kb fragment contain possible regulatory regions for the two genes contiguous to Emilin1, 2310015E02Rik, at the 5'-end and Khk (a housekeeping gene) at the 3'-end. In the above model, these sequences are assumed not to interact with the ACH, as they belong to a different functional transcription unit and can consequently synergize with local promoters. Consistent with this suggestion, unpublished work3 has shown that the short intergenic region between Emilin1 and Khk contains an activating sequence for Khk that is located within the 16.8-kb fragment studied here.
During development, the set of regulatory regions that induces maximal levels of Emilin1 expression in some tissues may change. This conclusion is suggested by the analysis of transgenic embryos at different stages. One example is heart, where transgene production at early organ formation is driven mainly by the 5'-flanking region, whereas it becomes dependent on the gene region at mid-advanced organogenesis. A second example is mesenchyme, where initial activation relies mainly on the 5'-flanking region, whereas different arrays of regulatory regions direct expression in the various organs at later stages. These observations are also consistent with the ACH model, where the key event is a productive assembly of regulatory regions rather than the presence of a cis-acting module responsible for tissue specificity.
The analysis of transgenic mice has also revealed details of Emilin1 expression that were not fully appreciated by using in situ hybridization (13). A significant one concerned myocardium, where expression was particularly high in the bulbous cordis and in the right ventricle, which largely derives from it (37). The functional meaning of this finding remains unknown, considering that the heart is normal in Emilin1-deficient mice (12). Nevertheless, it is tempting to speculate that Emilin1 may be involved in the acquisition of local differences of the right ventricle.
In addition to suggesting that transcription of Emilin1 in different tissues is achieved through combinatorial cooperation between various regions, rather than being dependent on a single cis-activating region specific for each (or a small group of) tissue(s), the results reported here are relevant as they define a strong and compact regulatory region that can be exploited for expression of genes in tissues overlapping with Emilin1 expression domains. Examples of such tissues are the ectoplacental cone, extra-embryonic visceral endoderm, megakaryocytes, and endothelial and mesenchymal cells during development. Further characterization of the various regulatory regions identified in this study will allow the design of more detailed sets of regulatory sequences for better tissue- and stage-specific gene expression.
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FOOTNOTES |
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¶ To whom correspondence should be addressed: Dipartimento di Istologia Microbiologia e Biotecnologie Mediche, Università di Padova, Viale G. Colombo 3, 35131 Padova, Italy. Tel.: 39-49-8276086; Fax: 39-49-8276079; E-mail: bressan{at}civ.bio.unipd.it.
1 The abbreviations used are: ECM, extracellular matrix; ACH, active chromatin hub; CAT, chloramphenicol acetyltransferase; E, embryonic age as days post-coitum; Khk, Ketohexokinase gene; X-gal, 5-bromo-4-chloro-3-indolyl--D-galactoside; RACE, rapid amplification of cDNA ends.
2 A. D'Urso, P. Braghetta, and G. M. Bressan, unpublished data.
3 P. de Gemmis, C. Fabbro, D. Volpin, P. Bonaldo, and G. M. Bressan, manuscript in preparation.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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