Skip Navigation

Cardiovascular Research 2001 50(1):24-33; doi:10.1016/S0008-6363(01)00204-8
© 2001 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Dellow, K. A.
Right arrow Articles by Barton, P. J.R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dellow, K. A.
Right arrow Articles by Barton, P. J.R.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2001, European Society of Cardiology

Identification of novel, cardiac-restricted transcription factors binding to a CACC-box within the human cardiac troponin I promoter

Kimberley A. Dellow, Pankaj K. Bhavsar, Nigel J. Brand and Paul J.R. Barton*

Cardiothoracic Surgery, National Heart and Lung Institute, Imperial College of Science Technology and Medicine, Dovehouse Street, London SW3 6LY, UK

* Corresponding author. Tel.: +44-0207-351-8184; fax: +44-0207-376-3442 k.dellow{at}ic.ac.uk p.bhavsar{at}ic.ac.uk n.brand{at}ic.ac.uk p.barton{at}ic.ac.uk

Received 22 September 2000; accepted 12 December 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 
Objectives: The expression of the human cardiac troponin I (hTnIc) gene is developmentally regulated and tissue-specific. In analysing the putative binding elements within the proximal promoter, a CACC-box sequence overlapping a consensus Sp1 element has been identified. The aim of this study was to characterise the factors binding to this element and to determine their importance in the transcriptional activity of the promoter. Methods: A combination of supershift and competition electrophoretic mobility shift assays (EMSA) were used to identify the binding of factors to the overlapping CACC-box/Sp1 consensus element. The functional importance of this element was tested by transient transfection into primary neonatal rat cardiac myocytes in culture. Results: At least four factors were able to interact with this region including the zinc finger proteins Sp1, Sp3 and two potentially novel factors. Whereas both Sp1 and Sp3 bound to the consensus Sp1 element, and to a lesser extent the CACC-box, two of the complexes required the intact CACC-box for binding. Site-directed mutagenesis of this region showed that the CACC-box is essential for hTnIc promoter–reporter activity. Further characterisation using EMSA indicated that the factors binding the hTnIc CACC-box are unlikely to be zinc finger proteins as they are insensitive to the addition of divalent cation chelating agents. They were also unable to bind to other known CACC-box elements. These factors are present in both human and rat cardiac muscle but absent from a number of cell lines including several derived from skeletal muscle. Conclusion: The human cardiac troponin I gene promoter requires an upstream CACC-box element for full activity. This element binds at least two complexes which represent novel, tissue-restricted DNA-binding activity present in the heart which we have named HCB1 and HCB2 for heart CACC-box binding factors.

KEYWORDS Cell culture/isolation; Contractile apparatus; Gene expression; Myocytes; Sequence (DNA/RNA/prot.)

This article is referred to in the Editorial by M. Flesch (pages 3–6) in this issue.


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 
The human cardiac troponin I (hTnIc) gene shows cardiac-specific and developmentally regulated expression. Accumulation of mRNA and protein follow the same pattern suggesting that overall expression is mainly regulated at the level of transcription in the human heart [1]. Work on the human promoter region from this laboratory has shown that the first 100-bp region is sufficient to drive reporter activity following transfections into primary neonatal rat cardiac myocytes [2]. A similar situation was observed with the mouse gene promoter where a 230-bp proximal promoter region was shown to be sufficient to drive reporter activity and recapitulate the tissue-specificity of the endogenous gene [3]. A combination of mutation and transient transfection studies using the hTnIc proximal promoter region identified a number of functionally important cis-acting elements including two GATA elements and an A/T-rich element overlaying a canonical TATA box, all of which are highly conserved in rodents [2]. The hTnIc proximal promoter region also contains a putative CACC-box (–97 CCCACCCC –90) overlapping a Sp1 consensus element (–93 CCCCGCCCC–85) not present in the equivalent region of the rat or mouse proximal promoters [3,4].

CACC-box elements have been identified in the regulatory regions of many muscle-specific genes including those encoding the slow and fast skeletal muscle troponin I isoforms [5], slow skeletal muscle myosin light chain 2 [6], slow skeletal/cardiac troponin C [7], cardiac {alpha}-actin [8] and myoglobin [9]. Functional activity through a number of these CACC-boxes has been attributed to the binding of the ubiquitous zinc finger factor Sp1 and the identification of other CACC-box binding factors, particularly muscle-specific factors, has been limited to date. The myocyte nuclear factor (MNF) and the uncharacterised CBF40 were originally identified in nuclear extracts from skeletal muscle cell lines by their interactions with the myoglobin CACC-box [9,10]. MNF belongs to the winged-helix family of factors and although it was originally identified binding a CACC-box, it was later found to have a high affinity for an A/T-rich element [11]. All factors identified to date which bind to CACC-box and CACC-box like elements (CACACC) have been characterised as zinc finger factors, for example, the Krüppel-like factors (KLF). KLF proteins are a family of tissue-restricted and ubiquitously expressed transcription factors that belong to the Sp1/KLF superfamily of C2H2 zinc finger factors and recognise a variety of CACC-boxes (CCCACCC) and CACC-box like (CACACC) elements. A number of Krüppel-like factors have been identified including the erythroid EKLF [12], gut-enriched GKLF [13,14], the lung LKLF [15] and FKLF-2 which is highly expressed in bone marrow and striated muscle [16]. The KLF proteins have been shown to be involved in a number of tissue-specific functions including regulating cell growth and differentiation. EKLF is the best characterised of these factors, it binds the CACACC-box found in the β-globin gene regulatory region, and has been shown to be intrinsically involved in erythropoiesis [17,18].

In this study two potentially novel, tissue-restricted factors have been identified. These bind an overlapping CACC-box (CCCACCCC) and consensus Sp1 element (CCCCGCCCC) present in the hTnIc gene promoter. Electrophoretic mobility shift assay (EMSA) shows that these factors require an intact CACC-box as well as flanking sequence for binding. Transient transfections of promoter–reporter constructs into neonatal cardiac myocytes show that the CACC-box is essential for promoter activity. Further characterisation using competition EMSA has demonstrated that these factors are unable to recognise other known CACC-boxes or GC-rich regions and that binding is not disrupted by the addition of chelating agents to the EMSA binding reaction. This suggests that these factors, which we have named HCB1 and HCB2 (heart CACC-box binding factors), are unlikely to be zinc fingers and may represent a new class of CACC-box binding proteins.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 Acknowledgments
 References
 
2.1 Maintenance of cell lines
Several cell lines were used for the preparation of nuclear extracts. The cells were maintained in a 5% (v/v) CO2 humidity incubator at 37°C in growth medium: DMEM supplemented with 2 mM L-glutamine, 0.1 U/ml penicillin and 0.1 mg/ml streptomycin as well as the appropriate serum: 10% fetal calf serum (FCS) for the skeletal muscle cell lines C2C12 derived from mouse C3H leg muscle myoblasts [19] and Sol 8 derived from rat soleus muscle [20], 5% FCS for African green monkey kidney cells COS-1 cells [21] and 10% neonatal calf serum for mouse Swiss NIH embryo NIH 3T3 [22]. Skeletal muscle cells lines were induced to differentiate by decreasing the serum content to 2% (v/v) horse serum and cells were fully differentiated after 72 h. The cells were grown to 60–70% confluency before passaging, differentiation or harvesting. Cells were harvested by using trypsin–EDTA solution (0.05 mg/ml Sigma) and incubated for up to 5 min at 37°C 5% (v/v) CO2. Once the cells were in suspension they were gently triturated to break-up clumps and then centrifuged at 800 g for 5 min. The cell pellet was then washed in PBS and was ready for making nuclear extracts (see Section 2.3).

2.2 Preparation of cardiac myocytes, plasmids and transfection protocol
The investigation conforms with the Guide For The Care And Use Of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996). Cardiac myocytes were isolated from hearts taken from 1-day-old Sprague–Dawley rats and cultured as previously described [23]. The plasmids for transfection were purified by alkaline-SDS lysis and polyethylene glycol precipitation [24]. The promoter–reporter constructs were made as previously reported [2] using the –531 to +67 region of the hTnIc promoter cloned into the pBLCAT3+vector. Nucleotide substitutions were created within the CACC/Sp1 element using the QuikChange site-directed mutagenesis kit (Stratagene). The plasmid were transfected in duplicate for a total of six complete experiments. Variability in transfection efficiency was corrected for by co-transfection with the β-galactosidase expression plasmid pON 249 [25]. Transfections were performed using the calcium phosphate following the protocol described by Decock et al. [26]. Briefly, cardiac myocytes (<95% pure) were seeded at 750 000 cells per well in Falcon 3046 multiwell dishes (Becton Dickinson), left overnight and refed. The cells were transfected with 8 µg of test plasmid and 2 µg of pON and left for 16 h. Precipitates were removed by repeated washing with warmed media and cells harvested 48 h later into 300 µl of 1x reporter lysis buffer (Promega). All assays were performed as previously reported [2].

2.3 Preparation of nuclear extracts and EMSA
Nuclear protein was extracted from primary cells or cell line cultures using a method adapted from Schreiber et al. [27]. Briefly, 1x106 cells were washed in PBS, pelleted at 4°C for 1 min at 11 000 g and carefully resuspended in 400 µl of ice-cold cell lysis buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 1 mM aprotinin, 1 mM leupeptin). The suspension was incubated on ice for 15 min with occasional mixing by inversion. Then 24 µl 10% NP-40 was added, the cells mixed by inversion and nuclei immediately pelleted at 4°C for 1 min at 11 000 g. The supernatant was discarded and the pellet containing nuclei was resuspended by dropwise addition of 50 µl ice-cold nuclear extraction buffer (20 mM HEPES, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 1 mM aprotinin, 1 mM leupeptin). This was then incubated on ice for 30 min and nuclear debris pelleted at 11 000 g as above for 15 min.

Fetal heart samples were obtained from elective terminations and age determined by anatomical measurement with reference to standard growth curves. Crude nuclear extracts were made from human tissue by a method similar to that used for cell lines. Briefly, small amounts of myocardial explant tissue (~0.5–1.0 g) were homogenised in 400 µl of cell lysis buffer and incubated for 30 min on ice. Cells were lysed by adding 33 µl of 10% NP-40 and centrifuging at 4°C 11 000 g for 1 min. The pellet was resuspended in 100 µl of nuclear extraction buffer and incubated on ice for 30–45 min then centrifuged at 4°C 11 000 g for 15 min. The protein concentration of nuclear extracts was determined with the BCA protein assay kit (Pierce) using the manufacturer's instructions. The nuclear extracts were stored at –80°C until use.

EMSA binding reactions were incubated in 20 µl final volume for 20 min at room temperature containing 4 µg nuclear extract and 10 fmol end-labelled oligonucleotide double-stranded cassette in 10% (v/v) glycerol and 1 µg poly[dI–dC]·[dI–dC]. The binding buffer used for the CACC/Sp element contained 25 mM HEPES, pH 7.6, 100 mM KCl, 10 mM MgCl2 and 25 mM DTT. Supershift assays were performed by adding 1 or 2 µl of polyclonal antibodies for the Sp family members Sp1, Sp2, Sp3 and Sp4 (Santa Cruz). The reactions were incubated at room temperature for 15 min prior to the addition of the radiolabelled probe. Competition was performed in the presence of a 100-fold excess of unlabelled oligonucleotide cassette (see Tables 1 and 2Go) and incubating for 20 min at room temperature prior to addition of the labelled probe. Complexes were resolved using an 8% non-denaturing polyacrylamide gel in 1xTBE and electrophoresis at 10 V/cm for 3 h. Gels were dried and exposed overnight at –80°C to autoradiography film.


View this table:
[in this window]
[in a new window]

  		Table 1 DNA sequences used as probes to investigate the binding of factors to the hTnIc overlapping CACC-box and consensus Sp1 element in EMSAa

 

View this table:
[in this window]
[in a new window]

  		Table 2 DNA sequences used in competition against the hTnIc overlapping CACC-box and consensus Sp1 element (CS WT) in EMSAa

 

   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 Acknowledgments
 References
 
3.1 Four specific complexes bind the hTnIc CACC-box–Sp1 element
An overlapping CACC-box and consensus Sp1 binding element was initially identified in the hTnIc proximal promoter and the factors able to interact with this region were identified by using EMSA. A 34-bp double-stranded oligonucleotide cassette encompassing the C-rich region was radiolabelled and incubated with nuclear extracts from neonatal rat cardiac myocytes (CS WT; see Table 1). A number of oligonucleotide cassettes were designed to contain nucleotide changes within the putative binding element and these cassettes were used at 100-fold excess to the radiolabelled probe in competition analysis (Fig. 1a and b).


Figure 1
View larger version (62K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 EMSA analysis of the hTnIc CACC-box/Sp1 element. The radiolabelled CS WT cassette was incubated with either rat or human cardiac extracts in the absence or presence of EDTA. (a) Competition analysis of complexes identified from rat neonatal cardiac myocytes using 100-fold excess of cold mutated cassettes in the absence of EDTA. The EMSA pattern obtained using human fetal cardiac nuclear extracts is also shown. (b) Competition analysis performed in the presence of EDTA. (c) EMSA analysis performed in the presence of 75–750 µM 1,10-phenanthroline. FP, free probe.

 
Preliminary EMSA studies of this region determined the binding of at least three complexes. Upon optimisation of the electrophoresis conditions, including increasing the concentration of both the buffer salts and polyacrylamide gel and extending the run time of the electrophoresis, the resolution of the complexes formed was greatly improved. Using these optimised conditions at least four complexes were identified which show specific binding to the CACC-box/Sp1 element (complexes a–d; Fig. 1a). Competition with the wild-type element CS WT (self) removed all of the complexes, whereas an unrelated Oct-1 consensus binding element did not compete for any of the factors. In contrast, a cassette containing two nucleotide changes within the 5' end of the CACC-box (CS M1) was unable to compete for complexes b and c, whilst completely disrupting the formation of complexes a and d. This demonstrates that factors involved in the formation of complexes b and c required an intact CACC-box element.

As the C-rich region contains a consensus Sp1 binding element, nucleotide changes were made which had been previously shown to disrupt the binding of Sp family members (CS M2; [28]). The addition of CS M2 competitor disrupted the formation of complexes b and c and decreased the intensity of complexes a and d. From this it can be concluded that the CS M2 cassette was able to bind the factors involved in complexes b and c and retained the ability to bind factors involved in complexes a and d, albeit with reduced affinity.

Following studies on the myoglobin CACC-box, where a four nucleotide base change was used to ablate binding to the CACC-box [9], a similar mutation was introduced within the hTnIc CACC-box/Sp1 element to encompass the overlapping region (CS M3). The addition of this mutant cassette in competition did not disrupt the formation of the four specific complexes. Similarly, when the CS M3 cassette was radiolabelled and used directly in binding experiments none of the complexes were observed (data not shown). Therefore the nucleotide changes within the overlapping region removed the ability of this cassette to bind the factors involved in the formation of all of the complexes a to d.

As the gene being studied is human it was important to determine if similar binding patterns could be observed with nuclear extracts prepared from human tissue. To this end a number of myocardial tissue samples were obtained from fetal human hearts. Crude nuclear extracts were made from these samples and an EMSA analysis, similar to that used with the rat samples, was performed using the CS WT probe. An identical pattern of complexes was obtained which demonstrated the same binding requirements as shown with the rat nuclear extracts, an example of which is shown in Fig. 1a. This demonstrates that the same specific DNA–protein complexes appear to be detected using human cardiac extracts as have been observed with rat.

To date all of the factors identified binding to CACC-boxes and GC-rich regions contain zinc finger DNA-binding domains, which require zinc ions for DNA-binding. Chelating agents added to the binding reactions in EMSA will disrupt the formation of such complexes by depleting available divalent cations including zinc. The binding of factors to the hTnIc CACC-box/Sp1 element was therefore tested in the presence of the chelating agents EDTA and 1,10-phenanthroline. Complexes b and c were clearly observed in EMSA performed in the presence of 5 mM EDTA using CS WT as radiolabelled probe incubated with neonatal rat cardiac nuclear extracts (Fig. 1b). The formation of complexes a and d, however, were disrupted under these conditions. The efficacy of competitor cassettes to compete (CS M2, self) or not (CS M1, CS M3, Oct1) for the binding of b and c in the presence of EDTA was similar to that observed under standard conditions without EDTA (Fig. 1a), supporting the sequence requirements determined for the formation of complexes b and c. Removal of complexes a and d also allowed complexes b and c to be observed more clearly. The addition of the chelating agent 1,10-phenanthroline (75–750 µM; Fig. 1c) also destabilised the formation of complexes a and d but not b and c. The use of chelating agents therefore allowed the four DNA-binding complexes to be characterised as those that were sensitive to chelators, and which might involve a zinc-finger tertiary structure in their DNA-binding domains (complexes a and d), and those that were insensitive (complexes b and c).

3.2 Sp family is involved in the formation of complexes a and d
The competition EMSA demonstrated that the factors involved in the formation of complexes a and d preferentially recognise the 3' consensus Sp1 element. Their mobility pattern is similar to that observed for Sp1 and Sp3 [29,30] and they appear to require divalent cations for DNA-binding activity (Fig. 1). To test if Sp factors were involved in the formation of any of the complexes, supershift analysis was carried out using a variety of specific antibodies (Fig. 2). The addition of anti-Sp1 antibody to the EMSA binding reaction resulted in a slight decrease in the intensity of complex a with no apparent change in any of the other complexes. Addition of an anti-Sp3 antibody completely removed complex d. Addition of the anti-Sp1 and anti-Sp3 antibodies together resulted in the loss of most of complex a and all of complex d without affecting complex b or c. Addition of the anti-Sp2 and Sp4 antibodies had no apparent effect on complex intensities, neither did addition of the preimmune serum. These data indicate that factors immunologically indistinguishable from Sp1 and Sp3 are involved in the formation of complexes a and d, but not in the formation of complexes b and c.


Figure 2
View larger version (57K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Supershift EMSA analysis of the factors binding to the hTnIc CACC-box/Sp1 element. Antibodies that recognise Sp family of factors were incubated in the EMSA binding reaction with nuclear extracts from rat neonatal cardiac myocytes. FP, free probe.

 
3.3 Intact CACC-box is essential for the full activity of the hTnIc promoter
To test the functional importance of the CACC-box–Sp1 element, wild-type and mutant promoter–reporter constructs were transfected into primary cultures of neonatal rat cardiac myocytes. The mutations used were the same as those used in the competition analysis and were generated by site-directed mutagenesis within a –531/+67 hTnIc promoter–reporter construct. Transfection data were normalised to β-galactosidase activity and are shown relative to the wild-type construct (CS WT). A construct containing the two nucleotide change within the 5' end of the CACC-box (CS M1), which abrogated binding of factors involved in the formation of complexes b and c in EMSA, showed 45% activity as compared to the wild type construct (Fig. 3) indicating that these factors contribute at least 50% of the functional activity of this element. The construct containing the mutation within the overlapping region (CS M3) which abrogate binding of all four factors in EMSA, demonstrated the lowest activity at 15% relative to wild-type suggesting that Sp1/Sp3 also contribute to functional activity. In contrast the two nucleotide mutation within the 3' end of the element (CS M2) which reduced Sp1/Sp3 binding in EMSA, did not significantly alter activity as compared to the wild-type construct suggesting that the lower affinity binding of Sp1/Sp3 seen with this construct is sufficient to maintain full promoter activity (see Discussion). In conclusion, the functional analysis demonstrates that the CACC-box element is important for promoter activity and that complexes b and c contribute at least 50% of the activity observed.


Figure 3
View larger version (25K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Promoter/reporter analysis using transient transfections into primary neonatal rat cardiac myocytes. Wild-type and mutant constructs used are depicted schematically on the left. The experiments were normalised to β-galactosidase activity and shown relative to the wild-type construct (CS WT). Each experiment was performed in duplicate six times and error bars demonstrate the standard error of mean.

 
3.4 Complexes b and c do not compete with other CACC-boxes in EMSA and may require sequences outside the CACC-box
A number of CACC-box elements have been described within the regulatory elements of different genes. It was therefore necessary to determine the ability of various CACC-boxes to compete for complex b and c. To this end a number of CACC-box and GC-rich elements (see Table 2) were used as competitors at 100-fold excess of the radiolabelled wild-type CACC-box/Sp1 element CS WT (Fig. 4). In contrast to CS WT, which when used as self-competitor was able to remove binding of complexes b and c, as well as the complexes involving Sp1 and Sp3 (complex a and d), none of the other competitors were able to compete for complexes b and c. These included the proximal and distal CACC-box elements from the rat TnIc gene (rCACprox and rCACdist), the slow skeletal/cardiac troponin C (cTnC), the myoglobin, {varepsilon}-globin and β-globin genes and the consensus binding elements for the zinc finger proteins Egr-1 and Sp1. Taken together, the data indicate that the proteins involved in complexes b and c are novel, potentially cardiac-restricted CACC-box binding proteins unrelated to previously described CACC-box binding factors.


Figure 4
View larger version (103K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Competition analysis of the hTnIc CACC-box/Sp1 element. A number of known CACC-box and GC-rich elements were used at 100-fold excess against the radiolabelled CS WT cassette. Oct-1 was used as an unrelated control. FP, free probe.

 
It is interesting to note that the distal CACC-box from the rat TnIc promoter region, the {varepsilon}-globin, myoglobin and cTnC CACC-boxes as well as the Sp1 consensus GC-rich box were all able to compete for the binding of complexes a and d (Sp1 and Sp3) to various degrees. The consensus Egr-1 element, the MNF A/T rich element and the unrelated Oct-1 consensus element were all unable to compete for any of the complexes.

Three of the sequences used as competitors, rCACdist, cTnC and myoglobin, contain CACC-boxes almost identical to that found in the hTnIc CACC-box/Sp1 element. Their failure to compete for complexes b and c suggests that the core CCCACCCC sequence is insufficient by itself for the formation of complexes b and c. In order to test this, an extra nucleotide was added 5' to the {varepsilon}-globin CACC-box sequence thereby changing it to the sequence of the hTnIc CACC-box element ({varepsilon}-Glob/hTnIc). This cassette was also unable to compete for complexes b and c (Fig. 4), indicating that sequences outside the CACC-box itself are also required for the binding of proteins involved in the formation of complexes b and c.

3.5 Complexes b and c are not detected in nuclear extracts from various cell lines
To examine tissue specificity of the proteins forming complexes b and c EMSA was performed using nuclear extracts from a variety of cell lines (Fig. 5). Complexes with similar mobility patterns to those involving Sp1/Sp3 and Sp3 (complexes a and d, respectively) from neonatal rat cardiac myocytes were observed from all the extracts tested which included the skeletal-muscle derived Sol 8 and C2C12, the NIH 3T3 fibroblast line and the kidney cell line COS-1. In contrast, complexes b and c were not observed using any of the nuclear extracts including the skeletal-muscle cell lines either as extracts from undifferentiated myoblasts (Mb) or differentiated myotubes (Mt). Complexes a and d were confirmed as containing Sp1/Sp3 and Sp3, respectively, in the Sol 8 extracts by supershift analysis (data not shown). All extracts were tested using an Oct-1 consensus element cassette (Fig. 5). These results, taken together with data presented in Fig. 1, suggest that complexes b and c are enriched, if not specific to cardiac muscle.


Figure 5
View larger version (63K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 EMSA analysis of a range of nuclear extracts made from different cell lines. The cell line extracts were tested with the wild type hTnIc CACC-box/Sp1 probe (CS WT) and the viability of each of the extracts was tested with a consensus Oct-1 probe. FP, free probe.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
Acknowledgments
 References
 
Even though a number of muscle-specific gene regulatory regions contain functionally important CACC-box elements, such as the 5'-CCCACCCC-3' element found within the hTnIc proximal promoter, the factors which bind to these elements have not been completely determined. In this study several specific complexes were identified binding to an overlapping CACC-box/Sp1 consensus element in the human TnIc gene using both rat and human cardiac tissue extracts in EMSA. Competition analysis determined that two of the complexes (b and c) required an intact CACC-box element for binding and were not affected by changes within the consensus Sp1 binding element. These complexes, which we have named HCB1 and HCB2, appear to represent novel, cardiac-restricted factors binding the CACC-box in the human TnIc gene.

Two other specific complexes (a and d) were identified binding to the overlapping CACC-box/Sp1 element which were distinct from HCB1 and HCB2 and bound preferentially to the consensus Sp1 element. Evidence obtained from a combination of supershift analysis, competition analysis and the use of chelating agents, identified the involvement of Sp1 and Sp3 in the formation of these complexes. Both Sp1 and Sp3 are present in cardiac tissue and at least three isoforms of Sp3 have been identified, a full-length version and two truncated isoforms [29,31]. Only the addition of anti-Sp1 and Sp3 antibodies together was able to interfere with the formation of complex a. This would appear to indicate that complex a was formed by the comigration of Sp1 with the longer isoform of Sp3 and this complex can be resolved into two close running complexes after 5 h of electrophoresis (data not shown). Sp1 and Sp3 are also able to recognise CACC-box elements to varying degrees and this was demonstrated in the competition analysis using various CACC-box elements (Fig. 4). This may also explain why the cassette designed to ablate binding of factors to the hTnIc consensus Sp1 element (CS M2) only reduced the affinity of Sp1/Sp3 binding, as the CACC-box element is unaffected in this mutant cassette. The apparent lack of effect of the CS M2 mutation in promoter–reporter activity (Fig. 3) may therefore be due to binding of Sp1 and Sp3 to the C-rich region. Whatever the true functional importance of Sp1 and Sp3, the nucleotide changes in the CACC-box element (CS M1) which ablated the binding of HCB1 and HCB2, without affecting the binding of Sp1 and Sp3, had a significant effect on the activity of promoter–reporter construct in transient transfections into primary cultures of neonatal rat cardiac myocytes. This demonstrates the importance both of the CACC-box element and of HCB1 and HCB2 in the activity of the hTnIc gene promoter.

HCB1 and HCB2 do not correspond to any of the known CACC-box binding factors on two counts. First, all of the factors identified to date that are able to bind CACC-box elements have been shown to contain zinc finger DNA-binding domains. This includes the members of the C2H2 zinc finger Sp1/KLF superfamily, such as Sp1, Sp3, GKLF and related factors [30,32], the human T-cell receptor factor htβ and the related rat ZF89 and human BERF-1 [33]. The immediate early gene zinc finger factor Egr-1 recognises a GC-rich sequence but, as with Sp1 and Sp3, also has a low affinity for CACC-box sequences [34]. The addition of chelating agents such as 1,10-phenanthroline and EDTA has been regularly used to demonstrate the requirement of zinc in the DNA-binding activity of a number of factors [35–37]. As reported here, the addition of 1,10-phenanthroline or EDTA to the EMSA binding reaction disrupted the formation of the complexes involving Sp1 and Sp3. In contrast, binding of HCB1 and HCB2 was not disrupted by the addition of these chelating agents. This would appear to indicate that divalent cations, such as zinc, are not required for the DNA-binding activity of these potentially novel factors and that they are unlikely to be zinc finger factors, although this will be fully confirmed once these factors have been cloned. One fully characterised factor which binds to a CACC-box element and which does not belong to a zinc finger family is the myocyte nuclear factor (MNF). MNF belongs to the winged-helix family of transcription factors and although originally identified as binding to the myoglobin gene promoter CACC-box, preferentially recognises an A/T-rich element [10,11]. The HCB1 and HCB2 complexes were not competed by the addition of an excess of the MNF consensus A/T-rich element therefore are unlikely to represent MNF proteins.

Second, the cell line distribution of many of the known CACC-box binding factors is different from HCB1 and HCB2 in particular, MNF, CBF40 and Egr-1 which have all been detected by EMSA in both Sol 8 and C2C12 cell lines [9,10,38]. However, neither HCB1 nor HCB2 were detected by EMSA from these cell lines.

The precise function of HCB1 and HCB2 has yet to be determined. However, a number of synergistic interactions involving factors binding CACC-box elements and adjacent cis-acting elements have recently been reported. These include the interactions of factors binding the SERCA1 CACC-box and E-box which together have been indicated as a weight-bearing response element in vivo [39] and the relative proximity of CACC-box elements with A/T-rich elements recognised by the myocyte enhancer factor 2 (MEF2). The myoglobin CACC-box and an A/T-rich element, able to interact with MEF2, have been demonstrated to synergistically co-ordinate muscle-specific regulation [40]. Synergistic activation has also been observed for the nerve-specific regulation in skeletal muscle of the myosin light chain 2 slow gene through a CACC-box and the binding of MEF2 to an adjacent A/T-rich element [6]. The human cardiac troponin I proximal promoter contains an A/T-rich element, which is a weak binding site for MEF2, located 50 bp downstream of the CACC-box [2]. It is therefore of interest to speculate as to whether there is synergistic co-operation between these cis-acting elements and the role that the HCB factors may play in the cardiac-specific and/or developmental regulation of the hTnIc gene.

In summary, the work presented here demonstrates the identification of two novel factors, HCB1 and HCB2, that bind to a functionally important CACC-box present in the human cardiac troponin I gene proximal promoter. These factors do not appear to require cations for binding DNA and are therefore unlikely to be zinc finger factors. This is unusual as all factors identified to date binding to CACC-box elements are zinc finger factors. HCB1 and HCB2 appear to be cardiac-restricted and have not been detected from skeletal muscle cell lines or fibroblast cell lines. Work is continuing to clone and characterise these factors further.

Time for primary review 25 days.


   Acknowledgments
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
References
 
We are grateful to Dr. Leslie Wong (MRC Fetal Tissue Bank) for providing fetal heart samples. We would also like to thank Una Sahye for her technical assistance. This work was supported by the British Heart Foundation (PG99007, FS297, FS96010, PG98194).


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 

  1. Sasse S., Brand N.J., Kyprianou P., et al. Troponin I gene expression during human cardiac development and in end-stage heart failure. Circ. Res. (1993) 72:932–938.[Abstract/Free Full Text]
  2. Bhavsar P.K., Dellow K.A., Yacoub M.H., et al. Identification of cis-acting DNA elements required for expression of the human cardiac troponin I gene promoter. J. Mol. Cell Cardiol. (2000) 32:95–108.[CrossRef][ISI][Medline]
  3. Di Lisi R., Millino C., Calabria E., et al. Combinatorial cis-acting elements control tissue-specific activation of the cardiac troponin I gene in vitro and in vivo. J. Biol. Chem. (1998) 273:25371–25380.[Abstract/Free Full Text]
  4. Murphy A.M., Thompson W.R., Peng L.F., et al. Regulation of the rat cardiac troponin I gene by the transcription factor GATA-4. Biochem. J. (1997) 322:393–401.[ISI][Medline]
  5. Nakayama M., Stauffer J., Cheng J., et al. Common core sequences are found in skeletal muscle slow- and fast-fiber-type-specific regulatory elements. Mol. Cell Biol. (1996) 16:2408–2417.[Abstract]
  6. Esser K., Nelson T., Lupa-Kimball V., et al. The CACC box and myocyte enhancer factor-2 sites within the myosin light chain 2 slow promoter cooperate in regulating nerve-specific transciption in skeletal muscle. J. Biol. Chem. (1999) 274:12095–12102.[Abstract/Free Full Text]
  7. Parmacek M.S., Ip H.S., Jung F., et al. A novel myogenic regulatory circuit controls slow/cardiac troponin C gene transcription in skeletal muscle. Mol. Cell Biol. (1994) 14:1870–1885.[Abstract/Free Full Text]
  8. Biesiada E., Hamamori Y., Kedes L., et al. Myogenic basic helix-loop-helix proteins and Sp1 interact as components of a multiprotein transcriptional complex required for activity of the human cardiac {alpha}-actin promoter. Mol. Cell Biol. (1999) 19:2577–2584.[Abstract/Free Full Text]
  9. Bassel-Duby R., Hernandez M.D., Gonzalez M.A., et al. A 40-kilodalton protein binds specifically to an upstream sequence element essential for muscle-specific transcription of the human myoglobin promoter. Mol. Cell Biol. (1992) 12:5024–5032.[Abstract/Free Full Text]
  10. Bassel-Duby R., Hernandez M.D., Yang Q., et al. Myocyte nuclear factor, a novel winged-helix transcription factor under both developmental and neural regulation in striated myocytes. Mol. Cell Biol. (1994) 14:4596–4605.[Abstract/Free Full Text]
  11. Yang Q., Bassel-Duby R., Williams R.S. Transient expression of a winged-helix protein, MNF-β, during myogenesis. Mol. Cell Biol. (1997) 17:5236–5243.[Abstract]
  12. Miller I.J., Bieker J.J. A novel, erythroid cell-specific murine transcription factor that binds to the CACCC element and is related to the Kruppel family of nuclear proteins. Mol. Cell Biol. (1993) 13:2776–2786.[Abstract/Free Full Text]
  13. Garrett-Sinha L.A., Eberspaecher H., Seldin M.F., et al. A gene for a novel zinc-finger protein expressed in differentiated epithelial cells and transiently in certain mesenchymal cells. J. Biol. Chem. (1996) 271:31384–31390.[Abstract/Free Full Text]
  14. Shields J.M., Christy R.J., Yang V.W. Identification and characterisation of a gene encoding a gut-enriched Kruppel-like factor expressed during growth arrest. J. Biol. Chem. (1996) 271:20009–20017.[Abstract/Free Full Text]
  15. Anderson K.P., Kern C.B., Crable S.C., et al. Isolation of a gene encoding a functional zinc finger protein homologous to erythroid Kruppel-like factor: identification of a new multigene family. Mol. Cell Biol. (1995) 15:5858–5965.[Abstract]
  16. Asano H., Li X.S., Stamatoyannopoulos G. FKLF-2: a novel Kruppel-like transcription factor that activates globin and other erythroid lineage genes. Blood (2000) 95:3578–3584.[Abstract/Free Full Text]
  17. Nuez B., Michalovich D., Bygrave A., et al. Defective haematopoiesis in fetal liver resulting from inactivation of the EKLF gene. Nature (1995) 375:316–318.[CrossRef][Medline]
  18. Perkins A.C., Sharpe A.H., Orkin S.H. Lethal β-thalassaemia in mice lacking the erythroid CACCC-transcription factor EKLF. Nature (1995) 375:318–322.[CrossRef][Medline]
  19. Yaffe D., Saxel O. Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature (1977) 270:22–29.[CrossRef]
  20. Mulle C., Benoit P., Pinset C., et al. Calcitonin gene-related peptide enhances the rate of desensitization of the nicotinic acetylcholine receptor in cultured mouse muscle cells. Proc. Natl. Acad. Sci. USA (1988) 85:5728–5732.[Abstract/Free Full Text]
  21. Gluzman Y. SV40-transformed simian cells support the replication of early SV40 mutants. Cell (1981) 23:175–182.[CrossRef][ISI][Medline]
  22. Jainchill J.L., Aaronson S.A., Todaro G.J. Murine sarcoma and leukemia viruses: assay using clonal lines of contact-inhibited mouse cells. J. Virol. (1969) 4:549–553.[Abstract/Free Full Text]
  23. Bhavsar P.K., Brand N.J., Yacoub M.H., et al. Isolation and characterisation of the human cardiac troponin I gene (TNNI3). Genomics (1996) 35:11–23.[CrossRef][ISI][Medline]
  24. Sambrook J., Fritsch E.F., Maniatis T. Molecular cloning: a laboratory manual. (1989) vol. 2. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
  25. Cherrington J.M., Mocarski E.S. Human cytomegalovirus iel transactivates the {alpha} promoter–enhancer via an 18-base-pair repeat element. J. Virol. (1989) 63:1435–1440.[Abstract/Free Full Text]
  26. Decock J.B., Gillespie Brown J., Parker P.J., et al. Classical, novel and atypical isoforms of PKC stimulate ANF- and TRE/AP-1-regulated-promoter activity in ventricular cardiomyocytes. FEBS Lett. (1994) 356:275–278.[CrossRef][ISI][Medline]
  27. Schreiber E., Matthias P., Muller M.M., Schaffner W. Rapid detection of octamer binding proteins with ‘mini-extracts’ prepared from a small number of cells. Nucleic Acids Res. (1989) 17:6419–6419.[Free Full Text]
  28. Vinals F., Fandos C., Santalucia T., et al. Myogenesis and MyoD down-regulate Sp-1: a mechanism for the repression of Glut 1 during muscle cell differentiation. J. Biol. Chem. (1997) 272:12913–12921.[Abstract/Free Full Text]
  29. Kennett S.B., Udvadia A.J., Horowitz J.M. Sp3 encodes multiple proteins that differ in their capacity to stimulate or repress transcription. Nucleic Acids Res. (1997) 25:3110–3117.[Abstract/Free Full Text]
  30. Suske G. The Sp-family of transcription factors. Gene (1999) 238:291–300.[CrossRef][ISI][Medline]
  31. Saffer J.D., Jackson S.P., Annarella M.B. Developmental expression of Sp1 in the mouse. Mol. Cell Biol. (1991) 11:2189–2199.[Abstract/Free Full Text]
  32. Philipsen S., Suske G. A tale of three fingers: the family of mammalian Sp/XKLF transcription factors. Nucleic Acids Res. (1999) 27:2991–3000.[Abstract/Free Full Text]
  33. Passantino R., Antona V., Barbieri G., et al. Negative regulation of β enolase gene transcription in embryonic muscle is dependent upon a zinc finger factor that binds to the G-rich box within the muscle-specific enhancer. J. Biol. Chem. (1998) 273:484–494.[Abstract/Free Full Text]
  34. Rafty L.A., Khachigian L.M. Zinc finger transcription factors mediated high constitutive platelet-derived growth factor-B expression in smooth muscle cells derived from aortae of newborn rats. J. Biol. Chem. (1998) 273:5758–5764.[Abstract/Free Full Text]
  35. Law L.G., Itoh H., Law D.J., et al. Transcription factor ZBP-89 regulates the activity of the ornithine decarboxylase promoter. J. Biol. Chem. (1998) 273:19955–19964.[Abstract/Free Full Text]
  36. Shi Y., Berg J.M. A direct comparison of the properties of natural and designed zinc-finger proteins. Chem. Biol. (1995) 2:83–89.[CrossRef][ISI][Medline]
  37. Wieczorek E., Lin Z., Perkins E.B., et al. The zinc finger repressor, ZBP-89, binds to the silencer element of the human vimentin gene and complexes with the transcriptional activator, Sp1. J. Biol. Chem. (2000) 275:12879–12888.[Abstract/Free Full Text]
  38. Tounay O., Benezra R. Transcription of the dominant-negative helix-loop-helix protein Id1 is regulated by a protein complex containing the immediate-early response gene Egr-1. Mol. Cell Biol. (1996) 16:2418–2430.[Abstract]
  39. Mitchell-Felton H., Hunter R.B., Stevenson E.J., et al. Identification of weight-bearing-responsive elements in the skeletal muscle sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA1) gene. J. Biol. Chem. (2000) 275:23005–23011.[Abstract/Free Full Text]
  40. Grayson J., Bassel-Duby R., Williams R.S. Collaborative interactions between MEF-2 and Sp1 in muscle-specific gene regulation. J. Cell Biochem. (1998) 70:366–375.[CrossRef][ISI][Medline]
  41. Yu C.-Y., Motamed K., Chen J., et al. The CACC box upstream of human embryonic {varepsilon} globin gene binds Sp1 and is a functional promoter element in vitro and in vivo. J. Biol. Chem. (1991) 266:8907–8915.[Abstract/Free Full Text]
  42. Van Vliet J., Turner J., Crossley M. Human Kruppel-like factor 8: a CACCC-box binding protein that associates with CtBP and represses transcription. Nucleic Acids Res. (2000) 28:1955–1962.[Abstract/Free Full Text]
  43. Rolland V., Liepvre X.L., Jump D.B., et al. A GC-rich region containing Sp1 and Sp1-like binding Sites is a crucial regulatory motif for fatty acid synthase gene promoter activity in adipocytes. J. Biol. Chem. (1996) 271:21297–21302.[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Dellow, K. A.
Right arrow Articles by Barton, P. J.R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dellow, K. A.
Right arrow Articles by Barton, P. J.R.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?