Epigenetic deregulation of lamina-associated domains in Hutchinson-Gilford progeria syndrome

Abstract

Background: Hutchinson-Gilford progeria syndrome (HGPS) is a progeroid disease characterized by the early onset of age-related phenotypes including arthritis, loss of body fat and hair, and atherosclerosis. Cells from affected individuals express a mutant version of the nuclear envelope protein lamin A (termed progerin) and have previously been shown to exhibit prominent histone modification changes.

Methods: Here, we analyze the possibility that epigenetic deregulation of lamina-associated domains (LADs) is involved in the molecular pathology of HGPS. To do so, we studied chromatin accessibility (Assay for Transposase-accessible Chromatin (ATAC)-see/-seq), DNA methylation profiles (Infinium MethylationEPIC BeadChips), and transcriptomes (RNA-seq) of nine primary HGPS fibroblast cell lines and six additional controls, two parental and four age-matched healthy fibroblast cell lines.

Results: Our ATAC-see/-seq data demonstrate that primary dermal fibroblasts from HGPS patients exhibit chromatin accessibility changes that are enriched in LADs. Infinium MethylationEPIC BeadChip profiling further reveals that DNA methylation alterations observed in HGPS fibroblasts are similarly enriched in LADs and different from those occurring during healthy aging and Werner syndrome (WS), another premature aging disease. Moreover, HGPS patients can be stratified into two different subgroups according to their DNA methylation profiles. Finally, we show that the epigenetic deregulation of LADs is associated with HGPS-specific gene expression changes.

Conclusions: Taken together, our results strongly implicate epigenetic deregulation of LADs as an important and previously unrecognized feature of HGPS, which contributes to disease-specific gene expression. Therefore, they not only add a new layer to the study of epigenetic changes in the progeroid syndrome, but also advance our understanding of the disease's pathology at the cellular level.

Keywords: Aging; Chromatin accessibility; DNA methylation; Epigenetics; Hutchinson-Gilford progeria syndrome; Lamina-associated domains (LADs).

Fig. 1

ATAC-see identifies chromatin accessibility changes in HGPS fibroblasts. a ATAC-see reveals loss of highly accessible chromatin foci in malformed HGPS nuclei. Scale bar = 10 μm. b Quantification of cells with three or more ATAC-see foci (P = 1.43e−2, unpaired t-test). Fifty nuclei were counted per sample (three replicates). c Correlation of the number of cells with three or more ATAC-see foci with the number of cells with malformed nuclei (HGPS: R² = 0.30). Malformed nuclei were quantified as in b

Fig. 2

ATAC-seq reveals an enrichment of chromatin accessibility changes in lamina-associated domains (LADs). a Regions gaining (n = 397) or losing (n = 148) accessibility in HGPS compared with controls (q < 0.05, Benjamini-Hochberg). b Distribution of ATAC-seq peaks across genes, promoters and enhancers. c Distribution of ATAC-seq peaks across lamin A-, lamin B-, and non-LAD-associated regions (*P < 0.05, Fisher’s exact test). d HOMER transcription factor motif enrichment analysis reveals that members of the AP1 family are highly enriched (q < 0.01, Benjamini-Hochberg) in differentially accessible regions. Motif densities were calculated using the HOMER motif density tool for the top de novo motifs.

Fig. 3

DNA methylation profiling in HGPS reveals two patient subgroups and lamina-associated domain (LAD)-enriched hypermethylation. a Scatter plot comparing the methylomes of HGPS and control fibroblasts. Differentially (P < 0.05, F-test) methylated probes are shown in blue. b Consensus clustering based on the 5000 most variable probe clusters between HGPS and control samples (1 = CpG islands, 2 = promoter, 3 = gene body, 4 = intergenic, 5 = lamin A-assoc., 6 = lamin B-assoc.). β values are colored from blue (β = 0) to red (β = 1). c Differential (β value) methylation of all (P < 2.20e−16), non-LAD- (P < 2.20e−16), lamin A LAD- (P < 2.20e−16), solo-WCGW HMD- (P = 4.42e−15), and solo-WCGW PMD- (P < 2.20e−16) associated probes (all: Welch’s two-sample t-test) with median indicated as a black line. HMD = highly methylated domain, PMD = partially methylated domain. d Enrichment of LAD-associated probes among all differentially (P < 0.05, F-test) methylated probes, as well as those hyper- and hypomethylated in HGPS samples (*P < 0.01, chi-squared test). Expected numbers were calculated based on the fraction of LAD-associated probes among all probes normalized to the number of differentially methylated probes. Sign = significantly. e Differential (β value) methylation of probes overlapping with different histone modifications. Median indicated as a black line. H3K4me1: P < 2.20e−16, H3K4me2: P < 2.20e−16, H3K4me3: P < 2.20e−16, H3K27ac: P < 2.20e−16, H3K36me3: P < 8.68e−15, H3K9me3: P < 2.20e−16, H3K27me3: P < 2.20e−16 (all: Welch’s two-sample t-test). Outliers were hidden for H3K4me3

Fig. 4

Epigenetic deregulation of lamina-associated domains (LADs) contributes to aberrant gene expression in HGPS. a 50 most differentially (q < 0.05, Benjamini-Hochberg) expressed genes in six HGPS vs. three control samples. Lowly expressed genes are shown in blue, highly expressed ones in red. FPKM = fragments per kilobase of transcript per million mapped reads. b Selection of Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and hallmark gene sets enriched (false discovery rate (FDR) q < 0.05) in HGPS fibroblasts. NES = normalized enrichment score. c TRANSFAC transcription factor (TF) network analysis of upstream factors controlling the observed expression changes. d Venn diagram showing numbers of genes overlapping between HGPS young (< 8 years) vs. control samples and HGPS old (> 8 years) vs. control samples, respectively. The GO processes characteristic of each comparison are given. e Median distance to the nearest LAD (lamin A and/or lamin B) for the indicated sets of genes. Numbers of genes in each group and Wilcoxon rank-sum test P values (with continuity correction) are given. *P = 2.25e−4. DNAm = DNA methylation

Fig. 5

Epigenetic deregulation is accompanied by intranuclear relocalization in HGPS. a Representative EDIL3 FISH images in HGPS and control nuclei. A telomeric probe (red) on Chr5 was used as a positive staining control. The distance from the FISH signal to the nuclear periphery was measured in the focal plane in cells exhibiting a clear biallelic signal. b, c Quantification of a for EDIL3 and IGFBP7 loci in two control and three HGPS cell lines for 60 cells per sample. EDIL3: P = 3.03e−08, Welch’s two-sample t-test. IGFBP7: P = 2.75e−13, Welch’s two-sample t-test

Fig. 6

Epigenetic deregulation of lamina-associated domains (LADs) in HGPS. Progerin-driven nuclear malformation in HGPS nuclei causes substantial, but potentially locally stochastic epigenetic reconfiguration of LAD-specific chromatin. As peripheral heterochromatin is diminished in these cells, many of the affected regions gain a more relaxed chromatin environment that is more permissive to the binding of transcription factors and might thus facilitate differential expression. In some cases, chromatin decondensation and disease-specific differential expression of formerly LAD-associated loci coincides with their relocalization within the nucleus, as detected in the case of EDIL3 and IGFBP7