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First published online 22 March 2005
doi: 10.1242/jcs.02291

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DNA CpG hypomethylation induces heterochromatin reorganization involving the histone variant macroH2A

Yinghong Ma¹, Stephanie B. Jacobs¹, Laurie Jackson-Grusby^2,*, Mary-Ann Mastrangelo^2,, José A. Torres-Betancourt¹, Rudolf Jaenisch^2,3 and Theodore P. Rasmussen^1,4,5,

¹ Center for Regenerative Biology, University of Connecticut, 1392 Storrs Road, Storrs, CT 06269-4243, USA
² Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142, USA
³ Department of Biology, MIT, Cambridge, MA 02139, USA
⁴ Department of Animal Science, University of Connecticut, Storrs, CT 06269-4040, USA
⁵ Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269-3042, USA

View larger version (55K): [in a new window]   Fig. 2 . Production of polyclonal antibodies against macroH2A. (A) MacroH2A consists of an N-terminal region (red) that is 64% identical to canonical H2As followed by a large C-terminal nonhistone domain (NHD) of unknown function (green). The dark green box labeled 1.2 indicates the position of one of two potential leucine zipper domains generated by alternative splicing. A PacI site was introduced into the macroH2A.2 cDNA at the junction of the histone and nonhistone domains (black triangle), allowing fusion of the nonhistone domain to GST. The GST-NHD fusion contains a thrombin cleavage site (open triangle) at the GST-NHD junction, allowing proteolytic cleavage to yield free NHD protein. (B) Recombinant NHD expression in bacterial cells. Lanes 1 and 2: Coomassie Blue-stained total bacterial protein from uninduced (lane 1) and IPTG-induced cells (lane 2) containing a GST-NHD expression plasmid. Lane 3: GST-NHD fusion protein purified from bacterial cells on a glutathione column eluted with reduced glutathione elution. Lanes 4 and 5: Purified NHD protein was eluted from a glutathione resin charged with GST-NHD by cleaving with thrombin. This allowed free NHD fusion protein to be eluted in two washes (samples of these supernatants are shown). (C) NHD protein from the same sample loaded in lane 4 was used to inoculate a rabbit that produced antibodies that recognize recombinant GST-NHD protein (lane 1) and endogenous macroH2A in total ES cell protein (lane 2) by immunoblotting.

View larger version (63K): [in a new window]   Fig. 1. Genomic methylation assays for repetitive sequences. Southern blots of total DNA extracted from wild-type (J1), rescued B9 cells (Dnmt1C/C cells containing a Dnmt1+ BAC) and Dnmt1C/C ES cells, cut with HpaII (methyl sensitive) and MspI (methyl insensitive) isoschizomeric restriction enzymes. (A) Southern analysis of equally loaded DNA using a probe that recognizes minor centromere repeats. (B) Identical Southern blot probed with pKS13b, which recognizes open reading frames of Line-1 repetitive transposable elements. (C) Identical Southern blot probed with mouse CoT1 DNA, which recognizes all highly repetitive sequences in the mouse genome.

View larger version (66K): [in a new window]   Fig. 3. Localization of macroH2A in wild-type and demethylated ES cells. (A) Representative ES cells subjected to macroH2A immunofluorescence (green) and DAPI staining of DNA (blue). J1 is a wild-type ES cell line. N/N, S/S and C/C denote ES cells with DnmtN/N, DnmtS/S and DnmtC/C genotypes, respectively. ES cell line B9 was derived by introduction of a Dnmt1 BAC into cell line into Dnmt1C/C cells resulting in partial remethylation of genomic CpG dinucleotides. (B) The same ES cell lines immunostained with CREST autoimmune serum, which recognizes centromeric kinetochore proteins including the histone H3 variant CENP-A. (C) Representative immunostainings of mitotic J1 and C/C ES cells using -macroH2A and CREST serum. Note that in J1, macroH2A and kinetochore immunostaining is distinct, while in C/C cells, immunostaining is nearly coincident.

View larger version (52K): [in a new window]   Fig. 4. Representative localization of macroH2A on mitotic chromosomes. Immunodetection of macroH2A distribution on individual metaphase chromosomes from (A) J1 (wild type) ES cells and (B) C/C (Dnmt1C/C) ES cells. Chromosomes were stained with DAPI (blue) to detect DNA, and with FITC (green) to detect -macroH2A antibody. Images in A and B had identical exposure times. Note enrichment of macroH2A immunostaining at telocentric centromeres in Dnmt1C/C cells. (C) Individual mitotic cells from J1 and C/C cultures were also subjected to combined immunofluorescence to detect macroH2A and kinetochore proteins (CREST).

View larger version (66K): [in a new window]   Fig. 5. Analysis of demethylation-induced chromatin remodeling in demethylated ES cells. (A) Outline of chromatin fractionation and analysis methods. Nuclei were isolated from ES cells and chromatin was digested in situ with micrococcal nuclease. Nucleosomes were then driven from nuclei by two successive hypotonic treatments (extractions), which liberate free nucleosomes into the supernatant. Thereafter, DNA and protein moieties of isolated nucleosomes were analyzed. (B) Micrococcal nuclease-digested DNA isolated from J1, B9 and C/C cells after initial and second hypotonic extractions. (C) Protein extracted from the same chromatin fractions shown in B detected by Coomassie Blue staining. (D) Western analysis of chromatin proteins in the same preparations shown in B and C. (Loading is identical to Coomassie Blue-stained gel in C.)

View larger version (35K): [in a new window]   Fig. 6. Western blot analysis of histone content in wild-type and demethylated ES cells. Equivalent amounts of cellular protein extracted from wild-type (J1) and Dnmt1C/C (c/c) ES cells were loaded onto SDS polyacrylamide gels and stained with Coomassie Blue to confirm equal loading. Equivalently loaded lanes were transferred to membranes for western blot analyses using antibodies specific for the N-terminal tail of histone H2B, macroH2A and modification-state-specific antibodies that recognize histone H3 trimethylated at lysine 27 (methyl-H3K27), lysine 9 (methyl-H3K9) or lysine 4 (methyl-H3K4).

View larger version (57K): [in a new window]   Fig. 7. Localization of post-translational methylation modifications of histone H3 in wild-type (J1) and Dnmt1C/C (c/c) ES cells. (A) Immunostaining with an antibody specific for histone H3 trimethylated at lysine 27. (B) Immunostaining with an antibody specific for histone H3 trimethylated at lysine 4. (C) Immunostaining with an antibody specific for histone H3 trimethylated at lysine 9.

View larger version (82K): [in a new window]   Fig. 8. Analysis of macroH2A distribution in T-antigen-transformed MEFs after conditional mutation of Dnmt1. (A,B) Male MEF line F11 was transfected with empty virus (A) or cre virus (B) to delete exons 4 and 5 of the Dnmt1 gene that encodes the methyltransferase core of the enzyme. Cells were fixed and subjected to combined immunofluorescence for macroH2A and kinetochore proteins (CREST) to mark chromocenters. (C,D) An independently derived cell line (F12) was subjected to similar mock (C) and cre-virus (D) transfections. F12 yielded results similar to those observed for F11. (E,F) A female MEF cell line (X17) was also subjected to mock (E) or cre-induced (F) loss of Dnmt1 function. Loss of Dnmt1 function in X17 and a second female cell line (X14, not shown) failed to perturb the localization of maroH2A.