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DNA Cpg Hypomethylation Induces Heterochromatin Reorganization Involving the Histone Variant Macroh2a

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DNA Cpg Hypomethylation Induces Heterochromatin Reorganization Involving the Histone Variant Macroh2a Research Article 1607 DNA CpG hypomethylation induces heterochromatin reorganization involving the histone variant macroH2A Yinghong Ma1, Stephanie B. Jacobs1, Laurie Jackson-Grusby2,*, Mary-Ann Mastrangelo2,‡, José A. Torres-Betancourt1, Rudolf Jaenisch2,3 and Theodore P. Rasmussen1,4,5,§ 1Center for Regenerative Biology, University of Connecticut, 1392 Storrs Road, Storrs, CT 06269-4243, USA 2Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142, USA 3Department of Biology, MIT, Cambridge, MA 02139, USA 4Department of Animal Science, University of Connecticut, Storrs, CT 06269-4040, USA 5Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269-3042, USA *Present address: Boston Children’s Hospital, Pathology Department, 300 Longwood Ave, Boston, MA 02115, USA ‡Present address: Section of Infectious Diseases, Department of Medicine, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA §Author for correspondence (e-mail: [email protected]) Accepted 26 January 2005 Journal of Cell Science 118, 1607-1616 Published by The Company of Biologists 2005 doi:10.1242/jcs.02291 Summary In mammalian heterochromatin, cytosine bases of CpG immunostaining in Dnmt1–/– cells becomes coincident with dinucleotides are symmetrically modified by methylation. chromocenters detected by HP1 content. MacroH2A, but Patterns of CpG methylation are maintained by the not HP1, is enriched in nuclease-resistant chromatin action of Dnmt1, the mammalian maintenance cytosine fractions extracted from Dnmt1–/– cells. Normal methyltransferase enzyme. We genetically manipulated the localization of macroH2A was restored upon levels of CpG methylation and found that extensive reintroduction of a Dnmt1 transgene into Dnmt1–/– cells. chromatin alterations occur in pericentric MacroH2A localization was also affected in T-antigen- heterochromatin. Homozygous mutations in Dnmt1 cause transformed fibroblasts subjected to the conditional severe hypomethylation of pericentric heterochromatin mutation of Dnmt1. Together, these results suggest that and concomitant chromatin reorganization involving the pericentric heterochromatin can be maintained in the histone variant macroH2A. Demethylation-induced absence of CpG methylation, but in a significantly altered alterations in macroH2A localization occur in both configuration. interphase and mitotic embryonic stem (ES) cells. Heterochromatin protein 1 (HP1) marks interphase Key words: chromatin, methylation, Dnmt1, MacroH2A, centromere, Journal of Cell Science pericentric heterochromatin (chromocenters). MacroH2A chromocenter Introduction nucleosomes assembled at centromeres (Palmer et al., 1987). The genomes of mammals are organized as chromatin, which Pericentric nucleosomes also contain a high content of histone is a complex and dynamic assemblage of DNA and proteins. H3 that is post-translationally modified by methylation at Transcriptionally silent DNA can be roughly divided into two lysine residue 9 (me-H3-K9), a modification that is associated functional compartments – constitutive and facultative with transcriptional inactivity (Lachner et al., 2001; Maison et heterochromatin. Constitutive heterochromatin contains DNA al., 2002; Rea et al., 2000). The me-H3-K9 modification serves in a state of obligate transcriptional silence and includes as a cis-acting binding site for heterochromatin protein 1 (HP1) (Bannister et al., 2001; Jacobs and Khorasanizadeh, 2002; elements such as centromeres, pericentric DNA, telomeres Lachner et al., 2001), an interaction that serves to sequester and silenced mobile elements (LINE-1 elements, SINE interphase centromeres in discrete foci (chromocenters). In elements, disabled retroviruses, etc.). In contrast, facultative addition, regions in and around mammalian centromeres are heterochromatin is conditionally silenced. Facultative subject to heavy DNA methylation at CpG dinucleotides. In heterochromatin includes genes subject to tissue-specific short, constitutive heterochromatin contains a distinct silencing, inactive X chromosomes of female eutherian molecular signature consisting of heavy methylation of CpG mammals, imprinted genes and perinucleolar DNA. Though dinucleotides coupled with chromatin enriched in CENP-A, both classes of heterochromatin are organized into me-H3-K9 and concomitant association with HP1. nucleosomes, a growing body of evidence reveals that Facultative heterochromatin such as that of the inactive X constitutive and facultative heterochromatin differ chromosome (Xi) also contains a distinct molecular signature. substantially in their content of variant histones and post- Though numerous reports show that CpG islands of silenced translational histone modifications. X-linked genes are highly methylated, the overall levels of Constitutive heterochromatin is assembled upon mammalian methylation on Xi appear to be reduced relative to the active centromeres and surrounding pericentric sequences. The X chromosome (Xa) and autosomes (Viegas-Pequignot et al., histone H3 variant CENP-A replaces histone H3 in 1988). In addition, the chromatin of Xi is enriched in histone 1608 Journal of Cell Science 118 (8) H3 methylated at lysine 27 (me-H3K27) (Plath et al., 2003; Southern blot analysis Silva et al., 2003) and macroH2A, a variant of canonical Southern blotting was performed by separating 10 µg of genomic histone H2A, which contains a large C-terminal extension DNA (digested to completion with indicated restriction enzymes) per (Costanzi and Pehrson, 1998; Pehrson and Fried, 1992). On a lane on 1% agarose gels. DNA was then transferred to Genescreen molecular level, MacroH2A interferes with NF-kappaB PlusTM nylon membrane (Dupont-NEN). Blots were hybridized with transcription factor binding and SWI/SNF chromatin probes derived from radiolabeled extension of random hexamers remodeling (Angelov et al., 2003). Furthermore, the C- annealed to the purified inserts of plasmids pMR150 (mouse terminal nonhistone domain (NHD) of macroH2A may be centromeric repeats) or pKS13b (Line-1 elements), or CoT1 DNA (Gibco; containing total mouse repetitive DNA). involved in ADP-ribosylation of chromatin with potential implications for transcriptional silencing (Ladurner, 2003). The crystal structure of the AF1521 protein from Antibody development Archaeoglobus fulgidus was recently determined (Allen et al., Polyclonal antibodies directed against macroH2A were prepared by 2003). This protein is similar to the NHD of macroH2A and cloning a cDNA fragment corresponding to the complete nonhistone probably binds nucleic acids, suggesting a possible role for domain (NHD) portion of a macroH2A1.2 cDNA (Rasmussen et al., macroH2A in chromatin condensation. 1999) into the plasmid pGEX-3T (Frorath et al., 1992). To do this, a Methylation of CpG dinucleotides in mice is maintained by unique PacI site was introduced at the junction of the histone and NHD coding regions in the macroH2A1.2 cDNA (Rasmussen et al., the action of Dnmt1, a maintenance DNA methyltransferase 1999), and the NHD coding region was fused to GST in a vector with that converts hemimethylated CpG dinucleotides to a thrombin cleavage site between the GST and NHD moieties (Fig. symmetrically methylated dinucleotides (Pradhan et al., 1999). 2A). Recombinant GST/NHD fusion protein was expressed in E. coli Loss of Dnmt1 results in embryonic lethality, but strain DH5α and GST-NHD fusion protein was then eluted with undifferentiated embryonic stem (ES) cells lacking Dnmt1 reduced glutathione. Alternatively, pure NHD was released from the function are fully viable (Li et al., 1992). Since ES cells are column by thrombin digestion and used to immunize a New Zealand viable in the absence of CpG demethylation, we used these white rabbit (Covance). Serum samples were assayed by western cells to investigate the molecular composition of demethylated blotting and IgG was purified from reactive serum on protein A- heterochromatin. We show that loss of DNA methylation agarose beads (Sigma). Alternatively, antibody was affinity-purified causes a significant reorganization of constitutive from serum using an affinity column assembled by covalently attaching pure NHD protein to cyanogen bromide-activated Sepharose heterochromatin involving the histone variant macroH2A. This CL-4B (Sigma). Affinity-purified antibody was eluted from this finding raises the intriguing possibility that mammalian matrix with low pH, and the eluted antibody was dialyzed in PBS. heterochromatin adopts a default state, reminiscent of The resulting antibody recognized endogenous macroH2A in total facultative heterochromatin, in response to DNA cellular protein from embryonic stem cells. In addition, the antibody demethylation. detected inactive X chromosomes in female fibroblasts, and centrosomal-associated macroH2A in undifferentiated ES cells (data not shown). These are both established patterns of macroH2A localization as determined with an independently derived antibody Materials and Methods (Costanzi and Pehrson, 1998; Rasmussen et al., 2000). Journal of Cell Science Cell culture Male ES cell lines with genotypes Dnmt1N/N, Dnmt1S/S and Dnmt1C/C were used. The N allele of Dnmt1 consists of a targeted deletion in Cell biological methods the N-terminal coding region of Dnmt1 that causes dramatic reduction Immunofluorescence and western blotting were performed using α- of CpG methylation (Li et al., 1992). The S allele of Dnmt1 disrupts macroH2A (above), α-HP1 (Serotec, cat. no. MAC353) and CREST the
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