Skip Navigation

Cardiovascular Research 1998 38(3):736-743; doi:10.1016/S0008-6363(98)00058-3
© 1998 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 Clifford, C.P.
Right arrow Articles by Nunez, D.J.R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Clifford, C.P.
Right arrow Articles by Nunez, D.J.R.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 1998, European Society of Cardiology

Human β-myosin heavy chain mRNA prevalence is inversely related to the degree of methylation of regulatory elements

C.P. Clifford and D.J.R. Nunez*

Section on Clinical Pharmacology, Division of Medicine, Imperial College of Science, Technology and Medicine, Hammersmith Campus, Du Cane Road, London W12 0NN, UK

* Corresponding author. Tel.: +44 (181) 383 3219; Fax: +44 (181) 383 2066; E-mail: d.nunez@rpms.ac.uk

Received 2 October 1997; accepted 23 January 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Methylation of cytosine in CG dinucleotides within regulatory elements is believed to silence gene expression. These dinucleotides occur in certain important regulatory elements in the promoter region of the human β-myosin heavy chain (β-MHC) gene. We therefore investigated whether methylation of these elements correlates with β-MHC gene transcription in human ‘expressing’ (right atrial) and ‘non-expressing' (peripheral blood leucocytes) cells. Methods: We employed 2 techniques to assess promoter methylation: (i) analysis of the susceptibility to digestion of a particular CCGG restriction site in the promoter region when genomic DNA is cleaved with the restriction endonucleases MspI (methylation-insensitive) and HpaII (methylation-sensitive), and (ii) the bisulphite-PCR method to examine in detail the methylation patterns of 3 important regulatory elements that contain CG dinucleotides. β-MHC mRNA expression in right atrium and leucocytes was assessed using reverse-transcription-PCR with specific primers that do not detect {alpha}-MHC cDNA. Results: The digestion pattern observed with MspI or HpaII indicated that the CCGG site was almost completely methylated in leucocytes, but relatively unmethylated in atrial myocardium from the same patients. When methylation was examined with the bisulphite-PCR method we found a reciprocal relationship between the level of β-MHC mRNA expression in leucocytes and atrial myocardium and the degree of methylation of CG dinucleotides in the 5' regulatory elements of the gene. Conclusions: Tissue-specific methylation of the human β-MHC gene promoter may play a role in determining the pattern of expression of this gene. Furthermore, alteration of the level of methylation may underlie the changes in transcription of this gene that occur, for example, when atrial or ventricular myocardium hypertrophies.

KEYWORDS Human; Myosin; DNA methylation; Hypertrophic cardiomyopathy; Bisulphite; Gene expression; Heart; RT-PCR


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Myosin molecules in the thick filament of the sarcomere are encoded by 2 closely related genes, the {alpha}- and β-myosin heavy chain genes ({alpha}-MHC and β-MHC) which are expressed in a tissue specific manner. In man, slow twitch skeletal muscle is the only tissue, apart from the heart, that expresses β-MHC. In the normal adult human ventricle β-MHC predominates, whilst in atrial tissue {alpha}-MHC is more prevalent, although there is still substantial expression of β-MHC [1]. In rodents, normal left ventricular myocardium expresses mainly {alpha}-MHC. In both ventricles and atria, the MHC genes are differentially expressed in response to stimuli such as pressure overload and thyroid hormone.

The transcriptional control mechanisms responsible for these differences in β-MHC expression are not well understood [2–4], although several regulatory sequences at the 5'-end of the gene have been identified, including a thyroid response element which represses the β-MHC gene, but activates the {alpha}-MHC gene, and strong positive and negative elements between positions –340 and –200 upstream from the transcription start site [3]. Some of these regulatory elements contain CG dinucleotides (Table 1) that are susceptible to methylation at the 5' position of the cytosine base, a process which can silence the expression of a gene [5, 6]. We therefore reasoned that differential methylation of one or more CG-containing regulatory elements in the β-MHC gene may affect its transcription and could underlie the differences in expression of this gene between tissues.


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

  		Table 1
 

   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Preparation of DNA from human peripheral blood leucocytes and cardiac muscle
Right atrial tissue and a 10 ml peripheral venous blood sample were obtained at the time of cardiac surgery from 3 patients undergoing coronary artery bypass surgery. Genomic DNA was then extracted using the Nucleon II genomic DNA extraction kit (Scotlab) according to the manufacturer's protocol. At the end of the protocol the DNA was precipitated with ethanol and pelleted by centrifugation, before being resuspended in 1 ml nuclease-free sterile water.

Genomic DNA was extracted from 1 g blocks of human right atrial tissue using a similar protocol designed for solid tissues as recommended by the supplier (Nucleon II kit, Scotlab).

2.2 Preparation of RNA from human peripheral blood leucocytes and cardiac muscle
Human peripheral blood leucocytes were concentrated using a dextran density gradient [7]. The white blood cells were pelleted by centrifugation at 1200 rpm for 6 min.

The white cell pellet and right atrial tissue were stored at –80°C until RNA extraction was performed using the Hybaid Ribolyser kit according to the manufacturer's protocol (Life Sciences). At the end of the procedure, the RNA was precipitated with isopropanol. The RNA pellet was then washed with salt/ethanol solution, air-dried, and resuspended in 100 µl nuclease-free water.

2.3 Reverse transcription-polymerase chain reaction (RT-PCR)
The reverse transcription reaction was performed as previously described [8], using 150 pmol of 12 mer randomer, 4 µl RNA and 9 U MMLV reverse transcriptase (Pharmacia). The reaction was incubated at 37°C for 2 h, and then made up to 100 µl with nuclease-free distilled water.

For amplification of β-MHC cDNA, 2 primers were designed from the human β-MHC gene sequence targeted to the adjacent ends of exons 5–6 and 11–12 [9](forward primer: 5'-CAGTACATGCTGACAGACAGA; reverse primer: 5'-GCACATCAAAAGCGTTATCAG). The PCR reaction mixture contained 1.5 mmol/l MgCl2, 200 µmol/l deoxynucleotides, 1 unit of Prozyme DNA polymerase (Bioline), 2.5 µl of cDNA and 1 µm each primer. PCR amplification was performed for 35 cycles (denaturation: 93°C 30 s; annealing 58°C 30 s; extension 72°C 1 min; final extension 72°C 10 min). The PCR products were resolved by electrophoresis through a 1.8% agarose gel and visualised using ethidium bromide staining. In addition to flanking intronic sequences, there were 3 mismatches of the primers at the 3' end when compared with the {alpha}-MHC cDNA sequence, which, as a consequence should not be amplified. After electrophoretic separation, representative samples of the amplified atrial DNA bands were purified from the agarose gel using GeneClean glass milk (Bio 101). The source of the PCR products was determined by dideoxy sequencing with the Amplitaq FS Cycle Sequencing kit as recommended by the supplier (Perkin Elmer).

2.4 Analysis of methylation patterns using isoschizomeric restriction enzymes and PCR
Primers (forward: 5'-TTCTAGTGACAACAGCCC; reverse: 5'-CATACCCTTTCTCACATTC) were designed to flank a CCGG MspI/HpaII restriction site within the promoter region of the human β-MHC gene (bases 2462 to 2465 of the gene sequence [9]). Cleavage with HpaII will only occur if the cytosine within the CG dinucleotide is unmethylated, whilst MspI cleavage is not dependent on the methylation status. Therefore, using this pair of restriction enzymes it is possible to determine whether the cytosine base is methylated or not.

Genomic DNA (2 µg) was pre-incubated with 1 µl of MspI (20 U) or HpaII (10 U) and 1 µl of 10x restriction enzyme buffer (New England Biolabs) at 37°C for 2 h. The partially-digested DNA was denatured at 95°C for 5 min before adding a further 1 µl of restriction enzyme and leaving the reagents to incubate overnight at 37°C. The restriction enzyme was then inactivated by incubating the reaction mix at 95°C for 30 min. In the positive ‘no-enzyme’ control the restriction enzymes were omitted, and 2 µl of the buffer were used instead. 2.5 µl of the digested or ‘buffer-only' DNA were then added to a standard PCR mixture containing 0.75 mmol/l MgCl2 and the specific primers flanking the CCGG site in the promoter region of the gene. PCR amplification was performed for 35 cycles (denaturation: 93°C 30 s; annealing 58°C 30 s; extension 72°C 1 min; final extension 72°C 10 min). The PCR products were resolved by electrophoresis through a 1.8% agarose gel and visualised using ethidium bromide staining.

Under the conditions described above, no amplification band should be seen after digesting the genomic DNA with MspI; this acts as a negative control. However, the extreme sensitivity of the PCR means that on occasions low residual levels of undigested DNA are detectable. If the site is completely unmethylated, the DNA should be cleaved by HpaII, and no band will be seen after PCR amplification. The target sequence will be amplified normally (i) if the site is completely methylated or hemi-methylated and no digestion occurs with HpaII, and (ii) when the genomic DNA is incubated with restriction enzyme buffer only.

2.5 Analysis of methylation patterns using the bisulphite-PCR method
This method relies on the ability of bisulphite to convert efficiently a cytosine residue to uracil in single-stranded DNA, whilst leaving 5-methylcytosine residues unaltered [10, 11]. The conversion of cytosine to uracil produces 2 non-complementary modified DNA strands (uracils cannot pair with guanines on the opposite strand) which can then be amplified separately by PCR with primer pairs specific for each converted strand (during PCR a uracil base is read as thymine by the DNA polymerase, while 5-methylcytosine, which remains unchanged, is read as cytosine).

Twenty-four µl of genomic DNA (250 mg/ml) was digested for 1 h at 37°C with 3 µl of the restriction enzyme BamHI and 3 µl of 10x restriction enzyme buffer in a final volume of 30 µl. 6 µl of 1.8 M NaOH was added (final concentration 0.3 M in a final volume of 36 µl) and incubated at 37°C for 15 min to denature the DNA. 4 µl 10 mmol/l hydroquinone (final concentration 0.5 mmol/l) and 40 µl 4 M sodium bisulphite pH 5 (final concentration 2 M) were added (final volume of 80 µl). All solutions were freshly prepared (with minimal shaking of the sodium bisulphite solution). The sample was then covered with oil and incubated for 6 cycles of temperature cycling at 55°C for 3 h and 95°C for 3 min. Our unpublished preliminary results showed that this protocol ensured complete conversion of cytosine bases to uracil by bisulphite, whilst methylcytosines remained unchanged. The DNA was purified using the Wizard DNA Clean-Up system according to manufacturer's instructions (Promega), and eluted in 50 µl of Tris HCl (50 mmol/l, pH 8.0) /EDTA (1 mmol/l) buffer. 10 µl of 1.8 M NaOH were added, and the reagents were incubated at 37°C for 15 min to ensure complete desulphonation. The solution was neutralised by adding 40 µl 7.5 M ammonium acetate pH 7, and the DNA was then precipitated with 100 µl of isopropanol. After the DNA was pelleted by centrifuging at 14 000 g for 15 min, the supernatant was removed and the pellet was resuspended in 10 µl of distilled water. This bisulphite-converted DNA was amplified for 40 cycles by PCR (denaturation: 93°C 30 s; annealing 55°C 30 s; extension 72°C 1 min; final extension 72°C 10 min) using a standard reaction mix containing 1 U Prozyme and 2 mmol/l MgCl2. Primers were designed to recognise only the bisulphite-converted DNA according to the protocol of Frommer et al. [10](forward: 5'-AGTAGTGTTTAGGGTTAGAAGTGTTGTG; reverse: 5'-ATCTCAAAAACTATATATATAAAACAAC). Finally, the amplified DNA was sequenced using the AmpliTaq FS automated cycle-sequencing kit (Perkin-Elmer) as recommended by the supplier. The fluorescent dideoxy terminated products were resolved on an ABI 373 automated sequencer.

2.5.1 Declaration
The investigation conforms with the principles outlined in the Declaration of Helsinki (Cardiovascular Research 1997;35:2–3).


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Methylation patterns in lymphocytes and myocytes
Amplification of genomic DNA after digestion with the restriction enzymes MspI and HpaII permits assessment of the degree of methylation of specific CCGG restriction sites. Fig. 1 shows the results of amplifying a ~450 bp segment spanning a CCGG site (bases 2462 to 2465) in the promoter region of the β-MHC gene using myocardial and leucocyte DNA. In DNA samples pre-digested with MspI, an enzyme which cleaves the CCGG site irrespective of the level of methylation of the CG dinucleotide, very little, or no, DNA amplification is observed. Where weak bands are observed (e.g. patients 2 and 3), this is likely to represent amplification of very small amounts of undigested DNA. On the other hand, a bright amplification band is observed in the HpaII-digested samples from leucocytes, but not atrial myocardium, indicating that the CG dinucleotide is methylated in leucocyte, but not myocardial genomic DNA. An amplification band is observed in all the samples pre-treated with restriction enzyme buffer alone; this acts as a positive control.


Figure 1
View larger version (57K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Analysis of the extent of methylation at a CCGG site using the restriction enzymes MspI and HpaII. After digestion with the restriction enzymes MspI and HpaII, a ~450 bp segment spanning a CCGG site (bases 2462 to 2465) in the promoter region of the β-MHC gene was amplified using myocardial and leucocyte DNA from 3 individuals. No amplification is observed with genomic DNA samples after digestion with MspI (M), a methylation-insensitive restriction. A PCR band was observed in the HpaII (H)-digested samples from leucocytes, but not atrial myocardium. An amplification band is observed in all the samples pre-treated with restriction enzyme buffer alone (B); this acts as a positive control. The very faint PCR products observed in the leucocyte (M) lanes of patients 2 and 3 are due to small amounts of residual undigested genomic DNA which have been amplified by the very sensitive PCR technique. L=molecular weight ladder.

 
Bisulphite converts unmethylated cytosine to uracil (detected as thymine after PCR), whereas methylated cytosine remains unchanged. At the CCGG site in the β-MHC promoter, the results were consistent with those obtained by restriction analysis with HpaII and MspI and indicated that there was almost complete demethylation of the CG dinucleotide in atrial tissue, the reverse being the case in leucocytes (data not shown). Fig. 2 shows the electropherograms of samples from the same individual in the region of the CG dinucleotides in the 3 regulatory elements of the β-MHC gene that contain these dinucleotides (Table 1). In the leucocyte samples, the CG dinucleotides remain largely unchanged after bisulphite treatment (i.e. C peak>>T peak). On the other hand, in the atrial samples we have found that there is variable C -> T conversion in the regulatory elements, but with a significant T peak present in all cases, indicating that many of the CG dinucleotides are unmethylated. When the degree of methylation was compared between individuals, we observed consistent methylation in leucocyte DNA in the 3 elements similar to that shown in Fig. 2. However, in atrial DNA samples the level of methylation in each of the elements varied between individuals (Fig. 3 shows the results for one of the elements in 3 different individuals).


Figure 2
View larger version (22K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Bisulphite-PCR analysis of methylation of the promoter region of the β-MHC gene. Unmethylated cytosines occurring outside CG dinucleotides are converted by bisulphite to uracil (which is detected as thymine after PCR), whereas methylcytosines remain unchanged. Typical electropherograms from the ABI 373 automated sequencer are shown in the region of the CG dinucleotides in the 3 regulatory elements of the β-MHC gene that contain these dinucleotides. In the leucocyte samples, the CG dinucleotides remain largely unchanged after bisulphite treatment. However, in the atrial samples from the same patient there is greater C->T conversion in CG dinucleotides, indicating that the degree of methylation of CG dinucleotides is relatively low.

 

Figure 3
View larger version (22K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Inter-individual variation of methylation of the β-MHC promoter in atrial DNA. Typical sequencing electropherograms obtained from bisulphite-PCR products in the region of the CG dinucleotide in one of the regulatory elements of the human β-MHC gene (TTGGTGGTCGTGGTCAGT). The variability of the relative C and T peak heights suggests that there is variation in the degree of methylation of the CG dinucleotide in this regulatory element.

 
3.2 Expression of the β-MHC gene in leucocytes and atrial myocytes
To assay the steady-state prevalence of β-MHC mRNA we used RT-PCR to amplify β-MHC cDNA. After sequencing PCR products from atrial samples with fluorescent dideoxy terminators, we compared the sequence of the amplification bands in the region of exon 11 of β-MHC and {alpha}-MHC, where there are significant differences in nucleotide sequence. We established conclusively that the single ~550 bp product obtained after PCR of atrial samples was derived solely from the β-MHC cDNA, without contamination with {alpha}-MHC cDNA (data not shown).

As shown in Fig. 4, β-MHC cDNA was readily detectable after a single round of amplification in all atrial myocardial samples, but not in leucocyte cDNA samples, indicating that the steady-state prevalence of β-MHC transcripts in the latter was below the level of detection by this PCR method. The faint bands observed in the leucocyte lanes are non-specific amplification products that commonly arise in the absence of specific products.


Figure 4
View larger version (60K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Expression of the β-MHC gene in leucocytes and atrial myocytes. The steady-state prevalence of β-MHC mRNA was assessed using reverse transcription-PCR to amplify β-MHC cDNA. After electrophoretic separation of the PCR products a single major DNA band was seen in the atrial sample lanes. DNA sequencing established that it was derived from β-MHC cDNA. After a single round of amplification, β-MHC cDNA band was readily detectable in the atrial myocardial samples (H1 and H2), but not in leucocyte cDNA samples (L1 and L2), indicating that the steady-state prevalence of β-MHC mRNA transcripts in the latter was below the level of detection by this PCR method. The faint bands in the 2 leucocyte lanes are non-specific amplification products that arise in the absence of specific β-MHC cDNA amplification. M is the molecular weight ladder.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
Here we present evidence which indicates that methylation of regulatory elements within the 5' region of the β-MHC gene is inversely related to mRNA steady-state levels. Functional assays of β-MHC promoter/reporter plasmids containing progressive deletions of the 5' region of the gene have revealed that there are strong positive and negative elements between positions –340 and –200 upstream from the transcription start site [3]; some of these contain CG dinucleotides that are susceptible to methylation. We provide evidence that in atrial myocytes these sites are predominantly hypo-methylated and the gene is strongly expressed. In leucocytes, where ‘illegitimate’ gene transcription has only been observed by nested reverse transcription-PCR [12], and the protein is undetectable, the promoter elements are heavily methylated. Methylation is known to silence genes, and it is likely that these differences in expression are due to reduced transcription, rather than increased degradation. Furthermore, it is unlikely that {alpha}-MHC cDNA contributed significantly to the results shown in Fig. 4. To validate the reverse transcription-PCR data, we sequenced the ~550 bp amplification band obtained from the atrial samples. Although the sequences of {alpha}-MHC and β-MHC mRNAs are remarkably similar, there are significant base changes in the region of exon 11. These differences were sufficient to confirm that {alpha}-MHC cDNA was not detectable in the specific PCR bands observed in the atrial samples shown in Fig. 4, and demonstrate that the PCR primers used were specific for β-MHC cDNA under our amplification conditions.

After bisulphite conversion, we have observed some variability in the relative C and T peak heights of the electropherograms from different individuals at CG dinucleotides within the regulatory elements in atrial DNA samples (e.g. Fig. 3). There are 3 possible explanations for these observations. First, it may simply reflect technical artefact due to variability in the efficacy of bisulphite to convert unmethylated cytosines. We do not favour this possibility because we have found that all cytosines that do not occur in CG dinucleotides are consistently converted by this reagent in our protocol. Secondly, the difference may result from complete methylation of a site in some cells and complete demethylation in other myocytes, with the proportions of each type varying between individuals. This may be the case, at least to some extent, given that in atrial tissue there is marked cellular heterogeneity in the expression of each MHC; cells containing only {alpha}-MHC (presumably with heavy methylation of the β-MHC gene and complete repression of expression) can be found adjacent to cells expressing only β-MHC (containing hypo-methylated β-MHC promoters), whilst others contain both MHCs [13, 14]. Finally, it may be due to variable methylation of one or other allele within a single cell. All the myocardial samples analysed in these studies consisted of many thousands of myocytes, and it is not possible to distinguish between the latter two alternatives. We are in the process of refining techniques to assess differential expression of β-MHC alleles using single-cell PCR. However, at present it is not possible to quantify accurately the level of methylation in single-cell preparations.

There are several lines of evidence implicating methylation of critical regulatory regions in the control of expression of a number of genes. For instance, there is an inverse relationship between expression of the murine MyoD gene [15]and the avian myosin light chain MLC1f and MLC3f genes [16], and the level of methylation of promoter elements. Similar results have been obtained for a variety of non-myocyte genes [17–20]. Alteration of the methylation status of regulatory elements of various genes by in vitro methylation analysis [17–25]and by administration of the demethylating agents 5-aza-2'-deoxycytidine, 5-azacytidine or 3-deazaadenosine [5, 26–29]has been shown to have a profound effect on their expression. In addition, methylation of cytosine residues is thought to play a major role in producing hemizygous expression of imprinted and X-linked genes by permanently altering chromatin structure or by promoting attachment of methylcytosine binding proteins [30–32].

Our results therefore raise a number of interesting questions. First, the inverse relationship between the level of expression of the β-MHC gene and the degree of methylation of regulatory elements suggests that the latter may be involved in the dramatic changes in cardiac myosin isoforms observed during ontogeny, eventually leading to the establishment of the tissue specific pattern of expression of this gene in the adult. In rats and rabbits, β-MHC is more abundant than {alpha}-MHC in the late foetal ventricle. However, soon after birth the isomyosin profile begins to change to the {alpha}-MHC form. The rat ventricle then maintains the predominance of {alpha}-MHC, whilst the rabbit reverts back towards the β-MHC form. On the other hand, in the human ventricle β-MHC predominates at all stages of development, {alpha}-MHC constituting no more than 15% of the total myosin in the adult healthy left ventricle [33]. In human atria, {alpha}-MHC predominates, but β-MHC is present in substantial amounts and can be up-regulated further by pressure overload (see below).

Secondly, we speculate that during the process of cardiac myocyte hypertrophy changes in genomic DNA methylation may play a role in co-ordinating the programme of gene expression [1, 4]. Although a myosin isoform shift is not observed in the adult human ventricle in response to hypertrophic stimuli, atrial β-MHC expression increases further in proportion to the extent of atrial distension associated with valvular disease [14, 34]. When the rodent ventricles hypertrophy there is a ‘recapitulation’ of the foetal pattern of MHC gene expression which results in up-regulation of β-MHC gene and down-regulation of {alpha}-MHC expression, with replacement of {alpha}-MHC by β-MHC in the thick filaments [1]. We propose that in cardiac tissues these changes are accompanied by reciprocal alteration of promoter methylation, whilst in tissues that do not express β-MHC, such as peripheral blood leucocytes, the almost complete methylation of regulatory elements remains relatively fixed.

Finally, we raise the exciting possibility that ‘new’ cardiac myocytes could be generated by altering the phenotype of non-myocyte ventricular cells such as fibroblasts with agents which demethylate genomic DNA. There are already striking precedents for this. When undifferentiated mouse fibroblasts were treated for 24 h with 5-azacytidine, a cytosine analogue that inhibits DNA methyl-transferases and produces genomic demethylation, a small proportion of the cells differentiated into multinucleate striated muscle cells that twitched [35]. A similar chemical, 5-aza-2'-deoxycytidine, has also been shown to enhance the efficacy of the myogenic determination factor, MyoD, to induce expression of myosin in fibroblasts [36, 37]. We are now evaluating the efficacy of demethylating agents to change their phenotype of human cardiac fibroblasts into contractile myocytes. These studies are important in the context of myocardial diseases characterised by ventricular myocardial insufficiency, such as myocardial infarction and hypoplastic ventricle syndromes, where the restoration of ventricular function would have a major impact on prognosis.

In conclusion, we have demonstrated an inverse relationship between the level of expression of the β-MHC gene and the degree of methylation of 5' regulatory elements. We propose that methylation of promoter motifs plays a role in the processes determining tissue-specific expression, and perhaps also in the coordinated programme of gene expression that characterises cardiac myocyte hypertrophy.

Time for primary review 29 days.


   Acknowledgements
 
We thank the Hammersmith Hospital Special Trustees for financial support. Dr. Clifford was a Medical Research Council Clinical Training Fellow.


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

  1. Swynghedauw B. Developmental and functional adaptation of contractile proteins in cardiac and skeletal muscles. Physiol Rev (1986) 66:710–771.[Abstract/Free Full Text]
  2. Morkin E. Regulation of myosin heavy chain genes in the heart. Circulation (1993) 87:1451–1460.[Abstract/Free Full Text]
  3. Mably J.D., Liew C.-C. Factors involved in cardiogenesis and the regulation of cardiac-specific gene expression. Circ Res (1996) 79:4–13.[Abstract/Free Full Text]
  4. Schiaffino S., Reggiani C. Molecular diversity of myofibrillar proteins: gene regulation and functional significance. Physiol Rev (1996) 76:371–423.[Abstract/Free Full Text]
  5. Razin A., Cedar H. DNA methylation and gene expression. Microbiol Rev (1991) 55:451–458.[Abstract/Free Full Text]
  6. Kass S.U., Pruss D., Wolffe A.P. How does DNA methylation repress transcription? Trends Genet (1997) 13:444–449.[CrossRef][ISI][Medline]
  7. Donnelly L.E., Rendell N.B., Murray S., et al. Arginine-specific mono(ADP-ribosyl)transferase activity on the surface of human polymorphonuclear neutrophil leucocytes. Biochem J (1996) 315:635–641.[ISI][Medline]
  8. Brown L.A., Nunez D.J., Wilkins M.R. Differential regulation of natriuretic peptide receptor messenger RNAs during the development of cardiac hypertrophy in the rat. J Clin Invest (1993) 92:2702–2712.[ISI][Medline]
  9. Liew C.-C., Sole M.J., Yamauchi-Takihara K., et al. Complete sequence and organization of the human cardiac β-myosin heavy chain gene. Nucleic Acids Res (1990) 18:3647–3651.[Free Full Text]
  10. Frommer M., McDonald L.E., Millar D.S., et al. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci USA (1992) 89:1827–1831.[Abstract/Free Full Text]
  11. Clark SJ, Frommer M. Bisulphite genomic sequencing of methylated cytosines. In: Taylor GR, editor. Laboratory Methods for the Detection of Mutations and Polymorphisms. Boca Raton: CRC Press, 1997:151–162.
  12. Rosenzweig A., Watkins H., Hwang D.S., et al. Preclinical diagnosis of familial hypertrophic cardiomyopathy by genetic analysis of blood lymphocytes. N Engl J Med (1991) 325:1753–1760.[Abstract]
  13. Gregory P, Zak R. Cardiac myosin isoenzymes in disease. In: Spry CJF, editor. Immunology and Molecular Biology of Cardiovascular Diseases. Lancaster: MTP Press, 1987:21–38.
  14. Gorza L., Mercadier J.J., Schwartz K., Thornell L.E., Sartore S., Schiaffino S. Myosin types in the human heart. An immunofluorescence study of normal and hypertrophied atrial and ventricular myocardium. Circ Res (1984) 54:694–702.[Abstract/Free Full Text]
  15. Brunk B.P., Goldhamer D.J., Emerson C.P. Regulated demethylation of the myoD distal enhancer during skeletal myogenesis. Dev Biol (1996) 177:490–503.[CrossRef][ISI][Medline]
  16. Lamson G., Stockdale F.E. Developmental and muscle-specific changes in methylation of the myosin light chain LC1f and LC3f promoters during avian myogenesis. Dev Biol (1989) 132:62–68.[CrossRef][ISI][Medline]
  17. Benvenuto G., Carpentieri M.L., Salvatore P., Cindolo L., Bruni C.B., Chiariotti L. Cell-specific transcriptional regulation and reactivation of galectin-1 gene expression are controlled by DNA methylation of the promoter region. Mol Cell Biol (1996) 16:2736–2743.[Abstract]
  18. Yokomori N., Moore R., Negishi M. Sexually dimorphic DNA demethylation in the promoter of the Slp (sex-limited protein) gene in mouse liver. Proc Natl Acad Sci USA (1995) 92:1302–1306.[Abstract/Free Full Text]
  19. Yokomori N., Kobayashi R., Moore R., Sueyoshi T., Negishi M. A DNA methylation site in the male-specific P450 (Cyp 2d9) promoter and binding of the heteromeric transcription factor GABP. Mol Cell Biol (1995) 15:5355–5362.[Abstract]
  20. Ngo V., Gourdji D., Laverriere J.N. Site-specific methylation of the rat prolactin and growth hormone promoters correlates with gene expression. Mol Cell Biol (1996) 16:3245–3254.[Abstract]
  21. Gupta M.P., Gupta M., Zak R. An E-box/M-CAT hybrid motif and cognate binding protein(s) regulate the basal muscle-specific and cAMP-inducible expression of the rat cardiac alpha-myosin heavy chain gene. J Biol Chem (1994) 269:29677–29687.[Abstract/Free Full Text]
  22. Iguchi-Ariga S.M.M., Schaffner W. CpG methylation of the cAMP-response enhancer/promoter sequence TGACGTCA abolishes specific factor binding as well as transcriptional activation. Genes Dev (1989) 3:612–619.[Abstract/Free Full Text]
  23. Klages S., Mollers B., Renkawitz R. The involvement of demethylation in the myeloid-specific function of the mouse M lysozyme gene downstream enhancer. Nucl Acids Res (1992) 20:1925–1932.[Abstract/Free Full Text]
  24. Comb M., Goodman H.M. CpG methylation inhibits proenkephalin gene expression and binding of the transcription factor AP-2. Nucl Acids Res (1990) 18:3975–3982.[Abstract/Free Full Text]
  25. Kirillov A., Kistler B., Mostoslavsky R., Cedar H., Wirth T., Bergman Y. A role for nuclear NK-KB in B-cell-specific demethylation of the IgK locus. Nat Genet (1996) 13:435–441.[CrossRef][ISI][Medline]
  26. Jones P.A. Altering gene expression with 5-azacytidine. Cell (1985) 40:485–486.[CrossRef][ISI][Medline]
  27. Haaf T. The effects of 5-azacytidine and 5-azadeoxycytidine on chromosome structure and function: implications for methylation-associated cellular processes. Pharmacol Ther (1995) 65:19–46.[CrossRef][ISI][Medline]
  28. Chiang P.K. Conversion of 3T3-L1 fibroblasts to fat cells by an inhibitor of methylation: effect of 3-deazaadenosine. Science (1981) 211:1164–1166.[Abstract/Free Full Text]
  29. Chiang P.K., Burbelo P.D., Brugh S.A., Gordon R.K., Fukuda K., Yamada Y. Activation of collagen IV gene expression in F9 teratocarcarcinoma cells by 3-deazaadenosine analogs. Indirect inhibitors of methylation. J Biol Chem (1992) 267:4988–4991.[Abstract/Free Full Text]
  30. Boyes J., Bird A. Repression of genes by DNA methylation depends on CpG density and promoter strength: evidence for involvement of a methyl-CpG binding protein. EMBO J (1992) 11:327–333.[ISI][Medline]
  31. Boyes J., Bird A. DNA methylation inhibits transcription indirectly via a methyl-CpG binding protein. Cell (1991) 64:1123–1134.[CrossRef][ISI][Medline]
  32. Barlow D.P. Gametic imprinting in mammals. Science (1995) 270:1610–1613.[Abstract/Free Full Text]
  33. Mercadier J.J., Bouveret P., Gorza L., et al. Myosin isoenzymes in normal and hypertrophied human ventricular myocardium. Circ Res (1983) 53:52–62.[Abstract/Free Full Text]
  34. Mercadier J.J., de la Bastie D., Menasche P., et al. Alpha-myosin heavy chain isoform and atrial size in patients with various types of mitral valve dysfunction: a quantitative study. J Am Coll Cardiol (1987) 9:1024–1030.[Abstract]
  35. Constantinides P.G., Jones P.A., Gevers W. Functional striated muscle cells from non-myoblast precursors following 5-azacytidine treatment. Nature (1977) 267:364–366.[CrossRef][Medline]
  36. Chen J., Jones P.A. Potentiation of MyoD1 activity by 5-aza-2'-deoxycytidine. Cell Growth Differ (1990) 1:383–392.[Abstract]
  37. Boukamp P., Chen J., Gonzales F., Jones P.A., Fusenig N.E. Progressive stages of "transdifferentiation" from epidermal to mesenchymal phenotype induced by MyoD1 transfection, 5-aza-2'-deoxycytidine treatment, and selection for reduced cell attachment in the human keratinocyte line HaCaT. J Cell Biol (1992) 116:1257–1271.[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 Clifford, C.P.
Right arrow Articles by Nunez, D.J.R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Clifford, C.P.
Right arrow Articles by Nunez, D.J.R.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?