Histone modifications depict an aberrantly heterochromatinized FMR1 gene in fragile x syndrome

Abstract

Fragile X syndrome is caused by an expansion of a polymorphic CGG triplet repeat that results in silencing of FMR1 expression. This expansion triggers methylation of FMR1's CpG island, hypoacetylation of associated histones, and chromatin condensation, all characteristics of a transcriptionally inactive gene. Here, we show that there is a graded spectrum of histone H4 acetylation that is proportional to CGG repeat length and that correlates with responsiveness of the gene to DNA demethylation but not with chromatin condensation. We also identify alterations in patient cells of two recently identified histone H3 modifications: methylation of histone H3 at lysine 4 and methylation of histone H3 at lysine 9, which are marks for euchromatin and heterochromatin, respectively. In fragile X cells, there is a decrease in methylation of histone H3 at lysine 4 with a large increase in methylation at lysine 9, a change that is consistent with the model of FMR1's switch from euchromatin to heterochromatin in the disease state. The high level of histone H3 methylation at lysine 9 may account for the failure of H3 to be acetylated after treatment of fragile X cells with inhibitors of histone deacetylases, a treatment that fully restores acetylation to histone H4. Using 5-aza-2'-deoxycytidine, we show that DNA methylation is tightly coupled to the histone modifications associated with euchromatin but not to the heterochromatic mark of methylation of histone H3 at lysine 9, consistent with recent findings that this histone modification may direct DNA methylation. Despite the drug-induced accumulation of mRNA in patient cells to 35% of the wild-type level, FMR1 protein remained undetectable. The identification of intermediates in the heterochromatinization of FMR1 has enabled us to begin to dissect the epigenetics of silencing of a disease-related gene in its natural chromosomal context.

Figure 1

Fragile X cell lines treated with azadC. A, Light cycler RT-PCR quantitation of FMR1 transcript following azadC treatment of fragile X cell lines harboring 230 CGGs, 410 CGGs, or 530 CGGs. The amount of FMR1 transcript in 100 ng of total RNA (expressed as percent of transcript from a normal cell line) is plotted against the time of azadC treatment. B, RT-PCR and western blot analysis of azadC-treated normal control cells and fragile X cells with either 230 CGGs or 530 CGGs. Cells were treated with azadC for the indicated times, and samples were collected for conventional RT-PCR and western blot analysis. In the western analysis, the indicated amounts of total cell lysate were loaded on the gel. A cross-reacting band of unknown identity is observed when large amounts of protein (60 μg) from all cell lines, regardless of treatment, are loaded in a lane.

Figure 2

Histone H3 and histone H4 acetylation at FMR1 in fragile X cell lines harboring 230 CGGs, 410 CGGs, or 530 CGGs. A, A representative multiplex PCR analysis of DNAs immunoprecipitated with either anti-acetyl histone H3 or anti-acetyl histone H4 antibodies from a normal cell line or three fragile X cell lines. The top band is specific for the constitutively active, X-linked glucose 6-phosphate dehydrogenase (G6PD) gene and the bottom band specific for FMR1. B, Quantitation of the level of histone H3 or H4 acetylation associated with FMR1 in the normal and the three fragile X cell lines. Amount of FMR1 DNA immunoprecipitated, relative to the internal control G6PD DNA, is averaged from three independent experiments. Error bars = 1 SD.

Figure 3

Nuclease accessibility analysis of FMR1 DNA associated with various levels of acetylated histone H4. A, Map of the 5′ end of FMR1, showing the location of the relevant restriction sites, the transcriptional start site, and the CGG repeat tract. B, Southern analysis of FMR1 DNA recovered after nuclease accessibility analysis from a normal cell line (lanes 1–4), the 230-CGG repeat fragile X cell line (lanes 5–8), the 530-CGG repeat fragile X cell line (lanes 9–12), and the 530-CGG repeat cell line treated with 330 nM TSA for 24 h (lanes 13–16).

Figure 4

ChIP analysis of histone H4 and H3 acetylation, H3 methylation at lysine 9 and histone H3 methylation at lysine 4 in a normal and a fragile X cell line carrying a 530-CGG repeat allele without HDAC inhibitor treatment (lanes 1 and 2) and the fragile X cell line treated with 330 nM TSA or 10 mM sodium butyrate for 24 h (lanes 3 and 4). The antibodies used and the lysine modifications they are directed against are indicated for each panel.

Figure 5

DNA methylation, ChIP, and RT-PCR analysis of FMR1 after addition and withdrawal of azadC. The fragile X cell line harboring 530 CGG repeats was treated with 1 μM azadC for 5 d (shaded area), followed by removal of the drug from the media. The treated cells continued to be passaged for an additional 30 d. Histone H3 and H4 acetylation is plotted as a percentage of untreated normal, the amount of demethylation is plotted as percent of FMR1 DNAs cleaved with the methyl-sensitive restriction enzyme BssHII, and the amount of FMR1 transcript is plotted as the percent of FMR1 transcript found in untreated normal cells.

Figure 6

DNA methylation, RT-PCR, and ChIP analysis of FMR1 during the course of azadC treatment. The fragile X cell line carrying 410 CGG repeats was treated with 1 μM azadC for 16 d. Equal numbers of cells were harvested at the indicated times for DNA, RNA, and chromatin analysis. DNA demethylation is expressed as the sum of intensities of the digested bands divided by the sum of the intensities of the digested and undigested bands. Histone methylation at lysine 9 during the course of azadC treatment is plotted as percent of untreated for three independent experiments.