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CpG methylation remodels chromatin structure in vitro1

https://doi.org/10.1006/jmbi.1997.0899Get rights and content

Abstract

One of the mechanisms proposed to explain how CpG methylation effects gene repression invokes a DNA methylation-determined chromatin structure. Previous work implied that this DNA modification does not influence nucleosome formation in vitro, thus current models propose that certain non-histone proteins or a preferential affinity by linker histones for methylated DNA may mediate changes in chromatin structure. We have reinvestigated whether CpG methylation alters the chromatin structure of reconstitutes comprising only core histones and DNA. We find that DNA methylation prevents the histone octamer from interacting with an otherwise high affinity positioning sequence in the promoter region of the chicken adult β-globin gene. This exclusion is attributed to methylation-determined changes in DNA structure within a triplet of CpG dinucleotides. In the affected nucleosome, this sequence motif is located 1.5 helical turns from the dyad axis and is oriented towards the histone core. These findings establish that DNA methylation does have the capacity to modulate chromatin structure directly, at its most fundamental level. Furthermore, our observations strongly suggest that a very limited number of nucleotides can make a decisive contribution to the translational positioning of nucleosomes.

Introduction

Nucleosomes can be directed to precise positions on DNA by signals in the sequence Simpson 1991, Thoma 1992, Wolffe 1994. For example, histone octamers reconstituted onto the promoter of the chicken adult β-globin gene, adopt characteristic translational positions with respect to the underlying DNA Yenidunya et al 1994, Davey et al 1995. By modulating the accessibility of promoter elements, these nucleosomes may participate in the developmental regulation of the gene McGhee et al 1981, Buckle et al 1991. In a wider context, nucleosome positioning signals may also participate in the folding of the β-globin gene region into an inactive higher order chromatin structure (Davey et al., 1995).

In vivo, nucleosome positioning signals can be modulated by non-histone proteins and, perhaps, by the modifications to the DNA and histones that often accompany changes in gene activity (Simpson, 1991). One such modification is DNA methylation, which occurs in vertebrates at the cytosine of the dinucleotide CpG. CpG methylation has been studied intensively because of its association with transcriptional repression (Razin & Cedar, 1991). This association is not necessarily a direct one as the methylation of CpG dinucleotides within certain transcription factor binding sequences, including that of SP1, does not prevent their binding (Harrington et al., 1988). Moreover, many genes, including the human β-globin (Yisraeli et al., 1988) and γ-globin genes (Murray & Grosveld, 1987) and the herpes simplex virus (HSV) thymidine kinase gene Keshet et al 1985, Graessmann et al 1994, are repressed in vivo when prior in vitro methylation is targeted to areas outside of transcription factor binding sites, indeed outside of the promoter region.

Expression variants of the human MGMT gene demonstrate a graded correlation between the extent of CpG methylation found within the promoter and its inaccessibility, which in turn correlate inversely with the degree of expression of the gene Costello et al 1994a, Costello et al 1994b. Observations of this type suggest that DNA methylation could cause repression by effecting changes in chromatin structure (Lewis & Bird, 1991). This hypothesis is supported by observations of the chromatin structure adopted when DNA, methylated in vitro, is introduced into cells. Keshet et al. (1986) demonstrated that when CpG-methylated, DNA integrated into the mouse L cell genome adopted a chromatin structure which exhibited a variety of nuclease-resistant features characteristic of inactive chromatin. Furthermore, Buschhausen et al. (1987) found that if methylation was to repress expression of the HSV thymidine kinase gene, assembly into chromatin, either in vivo or through reconstitution prior to microinjection, was essential. It has also been shown that the repressive chromatin structure adopted in vivo by in vitro-methylated patches of DNA can spread to unmethylated regions of the plasmid (Kass et al., 1993), supporting the hypothesis that in a CpG-rich environment, promoter methylation may not be a prerequisite for repression Bryans et al 1992, Hug et al 1996.

Methylation-associated changes in chromatin structure may be mediated by the non-histone proteins which bind specifically to methylated DNA Meehan et al 1992, Tate and Bird 1993; the association of MeCP2 with methylated heterochromatin could implicate a role for one such methyl-binding protein in modulating chromatin structure Lewis et al 1992, Nan et al 1996. Alternatively, the histone proteins could interact differently with methylated DNA per se. The preferential condensation of methylated chromatin into a repressive higher order structure is an attractive possibility first suggested by the finding that in bulk chromatin, methylated DNA is preferentially located in nucleosomes Razin and Cedar 1977, Solage and Cedar 1978 and, more specifically, in H1-containing nucleosomes (Ball et al., 1983). Although widely tested in vitro, no consensus has yet been reached as to whether H1 displays a preference for methylated naked DNA Campoy et al 1995, McArthur and Thomas 1996. Surprisingly few DNA types and precisely positioned nucleosomes have been examined for an influence of DNA methylation on the core histone octamer-DNA interaction. Nevertheless, these reconstitution studies, specifically carried out in vitro in the absence of non-histone proteins, have established the belief that methylation has very little or no effect on the capacity of the histone octamer to interact with DNA Felsenfeld et al 1982, Nickol et al 1982, Drew and McCall 1987, Englander et al 1993, Nightingale and Wolffe 1995.

In this context, we have investigated the promoter region of the chicken adult β-globin gene, which is both CpG-rich and exhibits a characteristic nucleosome positioning pattern directed by the DNA sequence. Our findings establish that CpG methylation can have a decisive influence on histone-DNA interactions and indicate that a very small focus of methylation, when appropriately located, is all that is required to disrupt important determinants of a nucleosome positioning signal.

Section snippets

Mapping of core particle boundaries identifies a DNA methylation-dependent change in nucleosome positioning

To investigate whether CpG methylation of DNA influences its capacity to interact with the histone proteins, we selected a DNA sequence which directs nucleosome positioning to a distinct and characteristic pattern and is abundant in CpG dinucleotides. These features are inherent to the promoter region of the chicken adult β-globin gene, as summarised for our plasmid pCBALe in Figure 1.

Unmethylated starting material, obtained by isolating pCBALe from a non-methylating strain of Escherichia coli,

Discussion

This study has addressed whether CpG methylation can influence chromatin structure at its most fundamental and ubiquitous level, that is, in terms of the interaction between the core histone octamer and the DNA wrapped around it. The complex pattern of translational positioning directed by the CpG-rich sequence of the chicken adult β-globin gene promoter has been compared for CpG-methylated and unmethylated DNA. Using three approaches, we have shown that site 5A, one of the four strong

Plasmid template

Plasmid pCBALe (Davey et al., 1995) comprises a 606 bp PvuII fragment of the chicken adult β-globin gene (−406 to +200, relative to the cap site) cloned into the EcoRV site of pBluescript KS-(Stratagene). pCBALe DNA was isolated from Escherichia coli JM110, a dam dcm strain; restriction with DpnI, DpnII, and ScrFI compared to an NciI control confirmed the absence of methylation. Single-stranded pCBALe DNA was isolated from E. coli strain DH11S (Lin et al., 1992).

Methylation of plasmid DNA

pCBALe was linearised with XmnI

Acknowledgements

We are indebted to Mike Calderwood, Nick Gilbert, Vicki Milner-Williams and Judith Widdowson for their participation in the original undertaking of this investigation. We thank Dr Richard Meehan for critical review of the manuscript. This work was supported by a Wellcome Trust Project grant to J.A. (043728) and a Wellcome Trust Senior Fellowship to S.P. (045117).

References (69)

  • I. Keshet et al.

    DNA methylation affects the formation of active chromatin

    Cell

    (1986)
  • J. Klysik et al.

    Effects of 5-cytosine methylation on the B-Z transition in DNA restriction fragments and recombinant plasmids

    J. Mol. Biol.

    (1983)
  • J. Lewis et al.

    DNA methylation and chromatin structure

    FEBS Letters

    (1991)
  • J.D. Lewis et al.

    Purification, sequence and cellular localisation of a novel chromosomal protein that binds to methylated DNA

    Cell

    (1992)
  • J.D. McGhee et al.

    A 20 base-pair region at the 5′ end of the chicken adult β-globin gene is accessible to nuclease digestion

    Cell

    (1981)
  • B.H.M. Mooers et al.

    Alternating and non-alternating dG-dC hexanucleoties crystallise as canonical A-DNA

    J. Mol. Biol.

    (1995)
  • K. Nightingale et al.

    Methylation at CpG sequences does not influence histone H1 binding to a nucleosome including a Xenopus borealis 5 S rRNA gene

    J. Biol. Chem.

    (1995)
  • M. Reitman et al.

    Primary sequence, evolution, and repetitive elements of the Gallus galus (chicken) β-globin cluster

    Genomics

    (1993)
  • R.T. Simpson

    Nucleosome positioningoccurrence, mechanisms, and functional consequences

    Prog. Nucl. Acid Res. Mol. Biol.

    (1991)
  • E.M. Southern

    Detection of specific sequences among DNA fragments separated by gel electrophoresis

    J. Mol. Biol.

    (1975)
  • P.H. Tate et al.

    Effects of DNA methylation on DNA-binding proteins and gene expression

    Curr. Opin. Genet. Dev.

    (1993)
  • F. Thoma

    Nucleosome positioning

    Biochim. Biophys. Acta

    (1992)
  • A.P. Wolffe

    Nucleosome positioning and modificationchromatin structures that potentiate transcription

    Trends Biochem. Sci.

    (1994)
  • A. Yenidunya et al.

    Nucleosome positioning on chicken and human globin gene promoters in vitro

    J. Mol. Biol.

    (1994)
  • G. Arents et al.

    Topography of the histone octamer surfacerepeating structural motifs utilised in the docking of nucleosomal DNA

    Proc. Natl Acad. Sci. USA

    (1993)
  • F.M. Ausubel et al.

    Current Protocols in Molecular Biology

    (1995)
  • D.J. Ball et al.

    5-Methylcytosine is localised in nucleosomes that contain histone H1

    Proc. Natl Acad. Sci. USA

    (1983)
  • M. Behe et al.

    Effects of methylation on a synthetic polynucleotidethe B-Z transition in poly(dG-m5dC)·(dG-m5dC)

    Proc. Natl Acad. Sci. USA

    (1981)
  • R. Buckle et al.

    The promoter and enhancer of the inactive chicken β-globin gene contain precisely positioned nucleosomes

    Nucl. Acids Res.

    (1991)
  • G. Buschhausen et al.

    Chromatin structure is required to block transcription of the methylated herpes simplex virus thymidine kinase gene

    Proc. Natl Acad. Sci. USA

    (1987)
  • A. Caplan et al.

    Perturbation of chromatin structure in the region of the adult beta-globin gene in chicken erythrocyte chromatin

    J. Mol. Biol.

    (1987)
  • J.M. Casasnova et al.

    Supercoiled induced transition to the Z-DNA conformation affects the ability of a d(CG/GC)12 sequence to be organised into nucleosome-cores

    Nucl. Acids Res.

    (1987)
  • J.F. Costello et al.

    Methylation-related chromatin structure is associated with exclusion of transcription factors from the suppressed expression of O-6-methylguanine DNA methyltransferase gene in human glioma cell lines

    Mol. Cell. Biol.

    (1994)
  • C. Davey et al.

    Periodicity of strong nucleosome positioning sites around the chicken adult β-globin gene may encode regularly spaced chromatin

    Proc. Natl Acad. Sci. USA

    (1995)
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