FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2067967780> ?p ?o ?g. }

• W2067967780 endingPage "17390" @default.
• W2067967780 startingPage "17381" @default.
• W2067967780 abstract "The region extending from −5.4 to −3.9 kilobase pairs from the transcription start site of the Col6a1 gene has been previously shown to contain sequences activating tissue-specific transcription in articular cartilage, intervertebral disks, subepidermal, and vibrissae mesenchyme and peripheral nervous system (Braghetta, P., Fabbro, C., Piccolo, S., Marvulli, D., Bonaldo, P., Volpin, D., and Bressan, G. M. (1996) J. Cell Biol. 135, 1163–1177). Analysis of expression of deletions of this region in transgenic mice has identified the 383-base pair fragment E–L as the most active sequence of the region. Linker-scanning mutagenesis analysis of segment E–J, which spans the 5′ 245 base pairs of E–L and is sufficient for high frequency expression in articular cartilage, showed that all the mutations reduced transcription considerably, suggesting that the integrity of the entire cluster of elements is necessary for enhancer activity. Electrophoretic mobility shift assays with nuclear extracts derived from various sources showed that fragment E–J binds numerous transcription factors (at least 22). These factors are present in most cells, expressing and nonexpressing α1(VI) collagen mRNA, but in different relative proportions, and none of them appears to be cell type-specific. Several lines of evidence indicate that sequence elements of the enhancer may have different functional roles in various cells. The data configure the −5.4/−3.9 region of theCol6a1 gene as a new type of tissue-specific enhancer, characterized by a variety of tissues supporting its activation and by the dependence of its function only on ubiquitous transcription factors. This type of enhancer is postulated to be particularly important for genes such as those of the extracellular matrix, which are often expressed with broad tissue specificity. The region extending from −5.4 to −3.9 kilobase pairs from the transcription start site of the Col6a1 gene has been previously shown to contain sequences activating tissue-specific transcription in articular cartilage, intervertebral disks, subepidermal, and vibrissae mesenchyme and peripheral nervous system (Braghetta, P., Fabbro, C., Piccolo, S., Marvulli, D., Bonaldo, P., Volpin, D., and Bressan, G. M. (1996) J. Cell Biol. 135, 1163–1177). Analysis of expression of deletions of this region in transgenic mice has identified the 383-base pair fragment E–L as the most active sequence of the region. Linker-scanning mutagenesis analysis of segment E–J, which spans the 5′ 245 base pairs of E–L and is sufficient for high frequency expression in articular cartilage, showed that all the mutations reduced transcription considerably, suggesting that the integrity of the entire cluster of elements is necessary for enhancer activity. Electrophoretic mobility shift assays with nuclear extracts derived from various sources showed that fragment E–J binds numerous transcription factors (at least 22). These factors are present in most cells, expressing and nonexpressing α1(VI) collagen mRNA, but in different relative proportions, and none of them appears to be cell type-specific. Several lines of evidence indicate that sequence elements of the enhancer may have different functional roles in various cells. The data configure the −5.4/−3.9 region of theCol6a1 gene as a new type of tissue-specific enhancer, characterized by a variety of tissues supporting its activation and by the dependence of its function only on ubiquitous transcription factors. This type of enhancer is postulated to be particularly important for genes such as those of the extracellular matrix, which are often expressed with broad tissue specificity. kilobase pair(s) chloramphenicol acetyltransferase base pair(s) 5-bromo-4-chloro-3-indolyl-β-d-galactoside polymerase chain reaction Genes of the extracellular matrix are very often among targets of terminal differentiation programs. In most cases, expression of the genes is the result of transcriptional regulation attained by tissue-specific enhancers. Well characterized examples are genes such as osteocalcin, collagen I, osteopontin, and bone sialoprotein in osteoblasts, and collagen II and XI in chondroblasts. The exclusive transcription of osteocalcin and the high level expression of α1(I) collagen, osteopontin, and bone sialoprotein are controlled by sequences binding Osf2/Cbfa1, a transcription factor necessary for the differentiation of osteoblasts (1.Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3605) Google Scholar), whereas transcription of α1(II) and α2(XI) genes requires sequences recognized by Sox9 and other members of the high mobility group class of transcription factors, which are involved in cartilage differentiation (2.Bell D.M. Leung K.K.H. Wheatley S.C. Ng I.J. Zhou S. Ling K.W. Sham M.H. Koopman P. Tam P.P.L. Cheah K.S.E. Nat. Genet. 1997; 16: 174-178Crossref PubMed Scopus (761) Google Scholar, 3.Lefebvre V. Huang W. Harley V.R. Goodfellow P.N. de Crombrugghe B. Mol. Cell. Biol. 1997; 17: 2336-2346Crossref PubMed Google Scholar, 4.Zhou G. Lefebvre V. Zhang Z. Eberspaecher de Crombrugghe B. J. Biol. Chem. 1998; 273: 14989-14997Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 5.Lefebvre V. Li P. de Crombrugghe B. EMBO J. 1998; 17: 5718-5733Crossref PubMed Scopus (671) Google Scholar, 6.Bridgewater L.C. Lefebvre V. de Crombrugghe B. J. Biol. Chem. 1998; 273: 14998-15006Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Thus, the identification and analysis of enhancers responsible for tissue-specific expression of extracellular matrix components are important not only to understand the regulation of their genes but also to clarify the genetic control of differentiation programs.Our group has undertaken the study of regulation of collagen VI in the mouse and has identified several sequences of the 5′-flanking region of the Col6a1 gene active in transcriptional control (7.Piccolo S. Bonaldo P. Vitale P. Volpin D. Bressan G.M. J. Biol. Chem. 1995; 270: 19583-19590Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 8.Braghetta P. Fabbro C. Piccolo S. Marvulli D. Bonaldo P. Volpin D. Bressan G.M. J. Cell Biol. 1996; 135: 1163-1177Crossref PubMed Scopus (35) Google Scholar, 9.Braghetta P. Vitale P. Piccolo S. Bonaldo P. Fabbro C. Girotto D. Volpin D. Bressan G.M. Eur. J. Biochem. 1997; 247: 200-208Crossref PubMed Scopus (12) Google Scholar, 10.Fabbro C. Braghetta P. Girotto D. Piccolo S. Volpin D. Bressan G.M. J. Biol. Chem. 1999; 274: 1759-1766Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). In particular, analyses in transgenic mice have located three regions responsible for tissue-specific transcription at high frequency (8.Braghetta P. Fabbro C. Piccolo S. Marvulli D. Bonaldo P. Volpin D. Bressan G.M. J. Cell Biol. 1996; 135: 1163-1177Crossref PubMed Scopus (35) Google Scholar). The 0.6 kb1 just upstream of the transcription start site drives expression in the superficial and muscular aponeurotic system and in tendons. A second fragment, from −7.5 to −6.2 kb, induces transcription in joints, intervertebral disks, vibrissae, skeletal muscle, and meninges. Finally, the sequence between about −5.4 to −3.9 kb from the transcription start site contains information for expression in articular cartilage, intervertebral disks, nerves, vibrissae, and subepidermal mesenchyme. The strong activating capacity of the −5.4- to −3.9-kb sequence has been confirmed by experiments with transgenic mice, where promoter-CAT fusions including this region are expressed in several tissues over 100 times more efficiently than constructs lacking it (9.Braghetta P. Vitale P. Piccolo S. Bonaldo P. Fabbro C. Girotto D. Volpin D. Bressan G.M. Eur. J. Biochem. 1997; 247: 200-208Crossref PubMed Scopus (12) Google Scholar). One interesting feature of the −5.4/−3.9 region is that information controlling transcription in cells with different embryological origin and function, including articular chondrocytes, Schwann cells, 2P. Vitale, P, Braghetta, D. Volpin, and G. M. Bressan, manuscript in preparation. 2P. Vitale, P, Braghetta, D. Volpin, and G. M. Bressan, manuscript in preparation. and fibroblasts, is enclosed in a relatively small DNA fragment, 1.5 kb, a size compatible with that of a single enhancer (11.Blackwood E.M. Kadonaga J.T. Science. 1998; 281: 60-63Crossref PubMed Scopus (607) Google Scholar). The question therefore arises whether the −5.4/−3.9 region contains only one enhancer dictating different tissue specificities or multiple enhancers, each one responsible for transcriptional activation in only one tissue. Such enhancers are usually formed by multiple transcription factor binding elements, including positively acting factors that are spatially localized in the organism in addition to ubiquitous ones (12.Arnone M.I. Davidson E.H. Development. 1997; 124: 1851-1864PubMed Google Scholar). Data accumulated so far on genes of the extracellular matrix conform to this condition. Studies on cis-acting sequences of collagen I genes have identified distinct enhancers for activation in cells of calcified tissues, skin, and fascial and interstitial fibroblasts. When characterized, as in bone, the active sequences have been found to bind tissue-specific nuclear factors (1.Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3605) Google Scholar, 13.Liska D.J. Reed M.J. Sage E.H. Bornstein P. J. Cell Biol. 1994; 125: 695-704Crossref PubMed Scopus (59) Google Scholar, 14.Dodig M. Kronenberg M.S. Bedalov A. Kream B.E. Gronowicz G. Clark S.H. Mack K. Liu Y.-H. Maxon R. Pan Z.Z. Upholt W.B. Rowe D.W. Lichtler A.C. J. Biol. Chem. 1996; 271: 16422-16429Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 15.Bou-Gharios G. Garrett L.A. Rossert J. Niederreither K. Eberspaecher H. Smith C.N. Black C. de Crombrugghe B. J. Cell Biol. 1996; 134: 1333-1344Crossref PubMed Scopus (135) Google Scholar, 16.Rossert J. Chen S.S. Eberspaecher H. Smith C.N. De Crombrugghe B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1027-1031Crossref PubMed Scopus (90) Google Scholar). Similarly, discrete elements recognizing tissue-specific transcription factors are necessary for high level transcription of α1(II) and α2(XI) genes in chondrocytes (2.Bell D.M. Leung K.K.H. Wheatley S.C. Ng I.J. Zhou S. Ling K.W. Sham M.H. Koopman P. Tam P.P.L. Cheah K.S.E. Nat. Genet. 1997; 16: 174-178Crossref PubMed Scopus (761) Google Scholar, 3.Lefebvre V. Huang W. Harley V.R. Goodfellow P.N. de Crombrugghe B. Mol. Cell. Biol. 1997; 17: 2336-2346Crossref PubMed Google Scholar, 4.Zhou G. Lefebvre V. Zhang Z. Eberspaecher de Crombrugghe B. J. Biol. Chem. 1998; 273: 14989-14997Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 5.Lefebvre V. Li P. de Crombrugghe B. EMBO J. 1998; 17: 5718-5733Crossref PubMed Scopus (671) Google Scholar, 6.Bridgewater L.C. Lefebvre V. de Crombrugghe B. J. Biol. Chem. 1998; 273: 14998-15006Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). In this study we have analyzed functional and structural features of the −5.4/−3.9 region. Unexpectedly, the region neither contains a unique enhancer sequence for all five tissues nor several distinct enhancers for the different tissues; rather, sequences controlling transcription in different tissues overlap extensively but do not coincide. In addition, no cell type-specific nuclear factors could be identified. These criteria identify a novel class of enhancers responsible for tissue-specific transcription.RESULTSAnalysis of Activating Properties of −5.4/−3.9 Enhancer Region in VivoIn order to define sequences of the −5.4/−3.9 region responsible for transcriptional activation in different tissues, transgenic mice were produced with the constructs outlined in Fig.1 a. In these constructs thelacZ gene containing a nuclear localization signal is fused to the proximal 1.4-kb 5′-flanking sequence and to different deletions of the −5.4/−3.9 region of the Col6a1 gene. The sequence of the −5.4/−3.9 region and the position of the start and end points of the deletions are reported in Fig. 2. The region extends from the BamHI site at −5.4 kb (site A) to the EcoRI site at −3.9 (site P). Embryos were dissected usually at 15.5 days, whole mount-stained with X-gal, and the distribution of transgene-positive cells determined by histological examination of serial sections. As expected by the presence of the proximal 1.4-kb fragment, all expressing embryos exhibited staining in superficial and muscular aponeurotic system and tendons (8.Braghetta P. Fabbro C. Piccolo S. Marvulli D. Bonaldo P. Volpin D. Bressan G.M. J. Cell Biol. 1996; 135: 1163-1177Crossref PubMed Scopus (35) Google Scholar) (data not shown), a condition that allowed easy identification of the number of expressing embryos. The results of the analysis of distribution of staining is summarized in Fig. 1 a for only those tissues that were previously shown to require the −5.4/−3.9 region for high level and high frequency expression of the transgene, i.e.articular cartilage, peripheral nervous system, intervertebral disks, vibrissae, and subepidermal mesenchyme (8.Braghetta P. Fabbro C. Piccolo S. Marvulli D. Bonaldo P. Volpin D. Bressan G.M. J. Cell Biol. 1996; 135: 1163-1177Crossref PubMed Scopus (35) Google Scholar). In addition to constructs with deletions of the −5.4/−3.9 region, the published data obtained with constructs containing the entire region (construct (A–P)lacZ) or lacking it completely (constructs with 1.4–3.9 kb of flanking sequences) are also reported for comparison in Fig. 1 a. Expression patterns from these two types of constructs constitute positive and negative controls, respectively, for the deletion constructs. The results are analyzed here separately for each tissue or group of tissues.Articular CartilageSequences driving expression in articular cartilage were detectable only in fragment E–P. A portion of this fragment, labeled E–L, exhibited the highest frequency (7/10). The 5′-half of the E–L (fragment E–I) was much less active (2/11). Likewise, the 3′-half of E–L (segments I–L and H–M, which is only a few base pairs longer) was expressed at lower frequency (3/10 and 0/5 respectively, overall 3/15). Among subfragments derived from E–L (E–I, I–L, F–H, H–M, and E–J), only E–J was expressed with a frequency comparable to E–L itself (5/6). One conclusion coming from these results is that the main sequence necessary for transcription in articular cartilage is included in segment E–J and that integrity of this fragment is required for high levels of expression. The data indicate that distinct elements are located within E–I and I–L and that neither element reaches high frequency expression in the absence of the other, suggesting synergism. Very weak but detectable inductive activity was also found in segment L–P (1/8) and could be narrowed down to N–O. However, a fragment containing both the E–L and the L–P segments (construct E–P) was not more active than E–L alone (3/5 and 7/10, respectively). As the expression frequency of the entire −5.4/−3.9 region in articular cartilage (construct A–P) was 100%, this suggests that full induction requires not only elements located in E–L but also elements comprised within L–P and A–E, which, when tested per se, are weakly or not inductive.Peripheral Nervous SystemExpression in this tissue at high frequency required the presence of the entire E–L region, as found for constructs (B–P)lacZ (5/5), (E–P)lacZ (4/5), and (E–L)lacZ (5/10). Constructs not containing any portion of the E–L region did not express in this tissue. Finally, constructs enclosing only portions of the E–L region ((A–G)lacZ, (A–K)lacZ, (I–P)lacZ, (E–I)lacZ, (I–L)lacZ, (E–J)lacZ, (F–H)lacZ, and (H–M)lacZ) expressed with very low frequency (only 3 over a total of 60 lines produced). Thus, the main regulatory region for PNS is E–L. However, as B–P is the only deletion with 100% expression frequency, full activation probably requires additional sequences comprised in either L–P and/or B–E.Intervertebral DisksThe E–L region was strongly inducing, whereas its subfragment E–J was less efficient, indicating that, at variance with articular cartilage, elements in the J–L sequence are necessary for high frequency activation in this tissue. Unlike articular cartilage, A–E had autonomous activity, although at low frequency (1/8), in intervertebral disks. Fragment N–O was also weakly inductive in this tissue.Vibrissae and Subepidermal MesenchymeThe distribution of activating regions for these two tissues was similar, although frequencies of expressing over total transgenic lines were not exactly coincident. The E–L region was strongly activating; however, the shorter E–J segment was equally effective, suggesting that sequences of the J–L region are not crucial for high frequency expression in these tissues. As for intervertebral disks, segment A–E induced β-galactosidase expression at low frequency (1/8 in the two tissues). A weak inducing region with slightly different activity in the two locations was contained in fragment N–O (3/15 and 1/15, respectively).The main conclusion that can be drawn from these experiments is that high level expression requires the simultaneous presence of elements contained within segment E–I and I–L and that these distinct elements act in a synergistic manner. This is true for all the five tissues examined, although at different extents. The effect is strong for the peripheral nervous system in which splitting of the E–L fragment abolishes transcription completely, whereas in the other tissues the individual halves of E–L maintain a low degree of activity. A second conclusion is that, in addition to E–L, sequences with weak or no inducing activity when tested in isolation are necessary to reach the maximal enhancer activity measured for the entire −5.4/−3.9 region. It is apparent that strongly and weakly activating sequences controlling transcription in the five tissues overlap, but do not coincide (Fig. 1 b), suggesting that transcriptional activation in the various tissues examined requires the cooperation of distinct but partially common sets of nuclear factor binding elements within the −5.4/−3.9 enhancer region.Linker-scanning Mutagenesis Analysis of the E–J RegionTo map the active sequences of the enhancer region better, a linker-scanning mutagenesis analysis of the E–J segment was carried out. The choice of E–J was dictated by the fact that this was the shortest segment exhibiting considerable inducing activity in vivo in four out of five tissues investigated (Fig. 1) and in transfected cell lines in vitro (Fig. 6). Although E–J was a very weak activator in the peripheral nervous system (only 1 expressing in 26 lines generated with sequences internal to the fragment, which include E–J, E–I, and F–H, Fig. 1), it contains a large portion (about 2/3) of E–L, the smallest fragment highly expressed in this tissue. The results of these experiments are reported in Fig. 3. Each one of the mutations introduced into E–J lowered significantly (at least 50%) transcription compared with either p1.4(E–J)CAT or p1.4(A–P)CAT, indicating that all sequences included elements or part of elements important for full activation. For constructs containing LS11, LS13, LS15, and LS16, CAT expression was similar to that of the enhancerless plasmid p1.4CAT, suggesting that these mutations abolished completely enhancer performance. Comparison of the corresponding sequences with those of transcription factor binding site data bases (28.Quandt K. Frech K. Karas H. Wingender E. Werner T. Nucleic Acids Res. 1995; 23: 4878-4884Crossref PubMed Scopus (2421) Google Scholar) identified a consensus sequence for AP1 in the fragment covered by LS11. No significant homologies with known binding sites for transcription factors were found in the sequence spanning LS13. As for LS15 and LS16, no potential sites were revealed when the individual corresponding sequences were compared; when the data base was probed with the merged sequences a C/EBP-binding site was identified at the boundary of the two segments. Thus, the loss of enhancer activity in both p1.4(LS15)CAT and p1.4(LS16)CAT might be accounted for by the mutation of a transcription factor binding site spanning part of the two mutations.Figure 3Analysis of E–J segment by linker-scanning mutations. Upper panel, sequence of the various mutations. The small size letter sequences marked LS1 to LS16 identify mutations introduced into the E–J segment, whose sequence is given in large size letters. Lower panel, expression of mutated constructs in NIH3T3 cells. Plasmids p1.4(LS1)CAT to p1.4(LS16)CAT contain the corresponding mutations defined in the upper panel. Each of the 16 mutations lowered significantly expression of CAT activity compared with the wild type sequence contained in constructs p1.4(E–J)CAT and p1.4(A–P)CAT.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To investigate further the activating properties of elements in the E–J region, CAT constructs, in which the proximal 1.4 kb were fused with three copies of oligonucleotides I to VIII marked in Fig. 2, were transfected into NIH3T3 fibroblasts and sternal chondroblasts, and CAT activity was determined. Addition of three copies of oligonucleotides to the 1.4-kb proximal sequence was usually activating, but only weakly (≤2-fold), compared with the addition of the E–J segment (≥5-fold) (data not shown). This was also the case of oligonucleotide VIII, which included the potential AP1-binding site mutated in LS11. These results favor the idea that the integrity of the entire E–J segment is necessary for full induction; they also suggest that the enhancer function is not dependent on one in particular but rather requires cooperation of different transcription factors.Complexity of Transcription Factors Binding to the E–J SegmentThe results of the linker-scanning mutagenesis analysis suggest that the E–J segment binds a considerable number of nuclear factors. To test this prediction, protein-DNA interaction assays were performed. DNase I footprinting assays of the E–J region produced weak protections, the major of which encompassed about 90 nucleotides of the E–I fragment (footprint 1 in Fig.4) including completely the LS4 to LS8 segments defined in linker-scanning experiments (see Fig. 3). Other protected regions were shorter and much weaker, and two examples are given in Fig. 4 (footprints 2 and 3). Although the DNase I footprinting experiments showed that a considerable portion of the E–J region binds nuclear factors, the weak intensity of the protections prevented further analysis of the complexity of the binding transcription factors. This aspect was therefore investigated by mobility shift assays using the 12 overlapping double-stranded oligonucleotides defined in Fig. 2, which together span the entire E–J region. A representative gel shift pattern generated with nuclear extracts from chick embryo sternal chondroblasts is given in Fig.5. Similar patterns were obtained with nuclear extracts from other cell types, and the data are summarized in Table I. Main features of the pattern of band distribution were the absence of retarded bands specific for only one of the cell types considered and the variation of the relative intensity of bands with different cell extracts. Moreover, some bands were common to different cell types but absent in one strain of the same cell type, thus increasing the variation among cell strains. One notable example is band Xc, which, in addition to other cell types, is found in RCS, but not in sternal chondroblasts, and in tendon, but not in NIH3T3 fibroblasts. To define better the complexity of nuclear factors binding to the E–J segment, the bands were classified on the basis of their sensitivity to EDTA and heat and of the pattern of oligonucleotides inhibiting their formation in electrophoretic mobility shift assay competition experiments (TableII). The number of bands differing for at least one of these classification criteria is 22, corresponding to an estimate of the minimum number of different types of transcription factors that can bind to the E–J fragment.Figure 5Representative pattern of shifted bands generated with oligonucleotides I–XII spanning the E–J sequence (defined in Fig. 2). The figure reports the results obtained with nuclear extracts from chick embryo sternal chondroblasts. Some of the band were weak with this nuclear extract; however, as summarized in Table I, they were stronger with other nuclear extracts and could therefore be unequivocally defined.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IComparison of retarded bands obtained by electrophoretic mobility shift assay with different oligonucleotides of the E–J fragment and nuclear extracts from various cell typesNuclear extractBandChick embryo sternal chondroblastsRCS (rat chondro- sarcoma)Chick embryo tendon fibroblastsNIH3T3 (fibroblasts)C2C12 (myoblasts)RN22 (rat Schwann cells)EL4 (T-lymphoma)Ia++++++++Ib++++++++++Ic+++++++Id+++++++IIa+++++++++++++++++++IIb±++±++++++IIc−±−−+−−IIIa++++++++++++++++++++++++IIIb+++++++++++++IIIc+++−−+−IIId−++±++±+IVa+−−−−±+IVb++±+±±−±IVc+++++++++++++−IVd++++++++++++++++++++Va−−−−±−+Vb+++++++++++Vc++++++++++++VIa++++++++++++++++++++VIb±±+±±−±VIc++++++++++++++++++++++VId−++±±+±VIIa±±±±++±±VIIb++++++++++VIIc+++++++±±+±VIId++++++±++++++++++++VIIIa++++++++VIIIb±++±±+±±VIIIc++++++++±±VIIId++++++±++VIIIe−−−−+++++++IXa±++±±+±−IXb++±±++++++IXc+−−±−−−IXd−+++++±IXe+++++++Xa+±−+++++Xb+++++++++++++Xc−++±−±+±XIa+++++++++++++XIb±+±+++++XIc−±±−±+−XIIa++++++++++++++++++++XIIb+++±−±++−XIIc+±+++±±XIId±++±+++++XIIe++++++±++++Oligonucleotides I–XII are defined in Fig. 2. Intensity of bands is given on the basis of an arbitrary scale from absence of the band (−) to very strong band (++++). Open table in a new tab Table IIIdentification of the minimal number of transcription factors binding to the E-J segment by electrophoretic mobility shift assayBandEDTA sensitivityaEDTA was added to a concentration of 10 mm or the samples incubated at 95 °C for 5 min just before the assay. The bands were either unaffected (−), decreased of intensity (±), or completely abolished (+) by these treatments.Heat sensitivityaEDTA was added to a concentration of 10 mm or the samples incubated at 95 °C for 5 min just before the assay. The bands were either unaffected (−), decreased of intensity (±), or completely abolished (+) by these treatments.Competitor oligonucleotidebOligonucleotides competing efficiently the indicated bands at a 800-fold molar excess.Transcription factor (TF-#)cBands with similar properties (EDTA, heat sensitivity, and oligonucleotide competition pattern) were assumed to be due to the same transcription factor.Ia+−ITF-1Ib−+ITF-2Ic±+I, XIITF-3Id±+I, XIITF-3IIa+−II, III, XITF-4IIb+−II, III, XITF-4IIc+−II, III, XITF-4IIIa+−II, III, XITF-4IIIb+−II, III, XITF-4IIIc+−II, III, XITF-4IIId±±IIITF-5IVa−+IV, VITF-6IVb±+IVTF-7IVc±+IVTF-7IVd+−IV, VI, VIITF-8Va++VTF-9Vb++VTF-9Vc−+VTF-10VIa−+VITF-11VIb−+IV, VITF-6VIc+−IV, VI, VIITF-8VId+−IV, VI, VIITF-8VIIa±+VIITF-12VIIb±+VIITF-12VIIc+±VIITF-13VIId+−IV, VI, VIITF-8VIIIa++VIIITF-14VIIIb++VIIITF-14VIIIc++VIIITF-14VIIId−+VIIITF-15VIIIe±−VIIITF-16IXa+−IXTF-17IXb+−IXTF-17IXc−+IXTF-18IXd−+IXTF-18IXe+−IXTF-17Xa++XTF-19Xb−+XTF-20Xc+−XTF-21XIa+−II, III, XITF-4XIb+−II, III, XITF-4XIc+−II, III, XITF-4XIIa−−XIITF-22XIIb−−XIITF-22XIIc±+I, XIITF-3XIId±+I, XIITF-3XIIe±+I, XIITF-3All the oligonucleotide probes were tested with nuclear extracts from both chick embryo sternal chondroblasts and C2C12 myoblasts with similar results. For oligonucleotides I and VIII, experiments were also performed with nuclear extracts from EL4 lymphocytes, as some of the bands were particularly intense using extracts from these cells (see Table I).a EDTA was added to a concentration of 10 mm or the samples incubated at 95 °C for 5 min just before the assay. The bands were either unaffected (−), decreased of intensity (±), or completely abolished (+) by these treatments.b Oligonucleotides competing efficiently the indicated bands at a 800-fold molar excess.c Bands with similar properties (EDTA, heat sensitivity, and oligonucleotide competition pattern) were assumed to be due to the same transcription factor. Open table in a new tab Chromatin DNase I Footprinting AssaysExpression of promoter constructs in vivo and the different intensity of gel shift bands in various cell types (Table I) stimulate to hypothesize that elements binding transcription factors in the E–J segment have distinct regulatory relevance in different cells. To confirm this hypothesis chromatin DNase I footprinting and in vitro promoter assays were performed. The first kind of assays were carried out with the rationale that distinct assemblies of protein complexes binding to the enhancer region would imply a different organization of chromatin in the various tissues. Mouse cells employed for this analysis comprised lines derived from tissues where the −5.4/−3.9 enhancer region is stimulatory such as NIH3T3 fibroblasts, MC615 chondroblasts, and the Schwann cell line SCT-1 (8, 9 and see below); all these lines expressed the α1(VI) collagen mRNA, which was particularly abundant in NIH3T3 and MC615 (Fig.6 a). The analysis was also carried" @default.
• W2067967780 created "2016-06-24" @default.
• W2067967780 creator A5025440802 @default.
• W2067967780 creator A5033640808 @default.
• W2067967780 creator A5035804082 @default.
• W2067967780 creator A5037201968 @default.
• W2067967780 creator A5068631630 @default.
• W2067967780 creator A5075234976 @default.
• W2067967780 date "2000-06-01" @default.
• W2067967780 modified "2023-09-30" @default.
• W2067967780 title "Analysis of Transcription of the Col6a1 Gene in a Specific Set of Tissues Suggests a New Variant of Enhancer Region" @default.
• W2067967780 cites W1544935394 @default.
• W2067967780 cites W1964064521 @default.
• W2067967780 cites W1964440437 @default.
• W2067967780 cites W1969770714 @default.
• W2067967780 cites W1971585190 @default.
• W2067967780 cites W1974971415 @default.
• W2067967780 cites W1983177944 @default.
• W2067967780 cites W1985789832 @default.
• W2067967780 cites W1994596418 @default.
• W2067967780 cites W1998162329 @default.
• W2067967780 cites W2006494322 @default.
• W2067967780 cites W2008609802 @default.
• W2067967780 cites W2025404099 @default.
• W2067967780 cites W2025986180 @default.
• W2067967780 cites W2064879068 @default.
• W2067967780 cites W2065030732 @default.
• W2067967780 cites W2069115371 @default.
• W2067967780 cites W2071325750 @default.
• W2067967780 cites W2072457100 @default.
• W2067967780 cites W2082502466 @default.
• W2067967780 cites W2088120545 @default.
• W2067967780 cites W2102047286 @default.
• W2067967780 cites W2105131037 @default.
• W2067967780 cites W2108097859 @default.
• W2067967780 cites W2114513729 @default.
• W2067967780 cites W2124025352 @default.
• W2067967780 cites W2135636594 @default.
• W2067967780 cites W2151625863 @default.
• W2067967780 cites W2151708616 @default.
• W2067967780 cites W2159614480 @default.
• W2067967780 cites W2166180405 @default.
• W2067967780 cites W216922957 @default.
• W2067967780 cites W707389 @default.
• W2067967780 doi "https://doi.org/10.1074/jbc.m000075200" @default.
• W2067967780 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10747869" @default.
• W2067967780 hasPublicationYear "2000" @default.
• W2067967780 type Work @default.
• W2067967780 sameAs 2067967780 @default.
• W2067967780 citedByCount "9" @default.
• W2067967780 countsByYear W20679677802014 @default.
• W2067967780 countsByYear W20679677802018 @default.
• W2067967780 countsByYear W20679677802021 @default.
• W2067967780 crossrefType "journal-article" @default.
• W2067967780 hasAuthorship W2067967780A5025440802 @default.
• W2067967780 hasAuthorship W2067967780A5033640808 @default.
• W2067967780 hasAuthorship W2067967780A5035804082 @default.
• W2067967780 hasAuthorship W2067967780A5037201968 @default.
• W2067967780 hasAuthorship W2067967780A5068631630 @default.
• W2067967780 hasAuthorship W2067967780A5075234976 @default.
• W2067967780 hasBestOaLocation W20679677801 @default.
• W2067967780 hasConcept C104317684 @default.
• W2067967780 hasConcept C111936080 @default.
• W2067967780 hasConcept C138885662 @default.
• W2067967780 hasConcept C179926584 @default.
• W2067967780 hasConcept C41895202 @default.
• W2067967780 hasConcept C54355233 @default.
• W2067967780 hasConcept C70721500 @default.
• W2067967780 hasConcept C86339819 @default.
• W2067967780 hasConcept C86803240 @default.
• W2067967780 hasConceptScore W2067967780C104317684 @default.
• W2067967780 hasConceptScore W2067967780C111936080 @default.
• W2067967780 hasConceptScore W2067967780C138885662 @default.
• W2067967780 hasConceptScore W2067967780C179926584 @default.
• W2067967780 hasConceptScore W2067967780C41895202 @default.
• W2067967780 hasConceptScore W2067967780C54355233 @default.
• W2067967780 hasConceptScore W2067967780C70721500 @default.
• W2067967780 hasConceptScore W2067967780C86339819 @default.
• W2067967780 hasConceptScore W2067967780C86803240 @default.
• W2067967780 hasIssue "23" @default.
• W2067967780 hasLocation W20679677801 @default.
• W2067967780 hasOpenAccess W2067967780 @default.
• W2067967780 hasPrimaryLocation W20679677801 @default.
• W2067967780 hasRelatedWork W1818003313 @default.
• W2067967780 hasRelatedWork W2007849798 @default.
• W2067967780 hasRelatedWork W2017956702 @default.
• W2067967780 hasRelatedWork W2034211922 @default.
• W2067967780 hasRelatedWork W2043249063 @default.
• W2067967780 hasRelatedWork W2255694509 @default.
• W2067967780 hasRelatedWork W2516911135 @default.
• W2067967780 hasRelatedWork W2901726948 @default.
• W2067967780 hasRelatedWork W2903659686 @default.
• W2067967780 hasRelatedWork W3177876754 @default.
• W2067967780 hasVolume "275" @default.
• W2067967780 isParatext "false" @default.
• W2067967780 isRetracted "false" @default.
• W2067967780 magId "2067967780" @default.
• W2067967780 workType "article" @default.