Conserved epigenomic signals in mice and humans reveal immune basis of Alzheimer's disease

Abstract

Alzheimer's disease (AD) is a severe age-related neurodegenerative disorder characterized by accumulation of amyloid-β plaques and neurofibrillary tangles, synaptic and neuronal loss, and cognitive decline. Several genes have been implicated in AD, but chromatin state alterations during neurodegeneration remain uncharacterized. Here we profile transcriptional and chromatin state dynamics across early and late pathology in the hippocampus of an inducible mouse model of AD-like neurodegeneration. We find a coordinated downregulation of synaptic plasticity genes and regulatory regions, and upregulation of immune response genes and regulatory regions, which are targeted by factors that belong to the ETS family of transcriptional regulators, including PU.1. Human regions orthologous to increasing-level enhancers show immune-cell-specific enhancer signatures as well as immune cell expression quantitative trait loci, while decreasing-level enhancer orthologues show fetal-brain-specific enhancer activity. Notably, AD-associated genetic variants are specifically enriched in increasing-level enhancer orthologues, implicating immune processes in AD predisposition. Indeed, increasing enhancers overlap known AD loci lacking protein-altering variants, and implicate additional loci that do not reach genome-wide significance. Our results reveal new insights into the mechanisms of neurodegeneration and establish the mouse as a useful model for functional studies of AD regulatory regions.

Extended Data Figure 1. Epigenomic and transcriptomic profiling of a mouse model of AD

a, Experimental design and progression pathology in the CK-p25 mice. b, Gene expression and histone modification levels at the SPI1 locus at 6 weeks of inducible p25 overexpression. Profiled are histone marks associated with repression (blue); histone marks associated with enhancers (orange); histone marks associated with promoters (red); histone marks associated with gene bodies (green); RNA-seq (black).

Extended Data Figure 2. Differential microglia-specific gene expression changes in the CK-p25 mice

RT-qPCR of selected microglia markers and immune response genes shows upregulation of gene expression in FAC-sorted CD11b+ CD45^Low microglia from 2 week induced CK-p25 mice (red bars) relative to respective controls (black bars). Actb (b-actin) was used as a negative control. Values were normalized to Cd11b expression (n=3, *P < 0.05, two-tailed t-test); NS, non-significant.

Extended Data Figure 3. Chromatin state conservation

a, Combinatorial patterns of seven histone modifications profiled were used to define promoter (1-3; A, active; D, downstream; U, upstream), gene body (4-6; tx, transcribed, 3P, 3 prime), enhancer (7-9; G, genic, 1 = strong, 2 = weak), bivalent (10), repressed Polycomb (11), heterochromatin (12), and low signal (13-14) chromatin states. Darker blue indicates a higher enrichment of the measured histone mark (x axis) to be found in a particular state (y-axis). b, Promoter, enhancer, and repressed chromatin states in mouse hippocampus (rows), as profiled in this study, align to matching chromatin states in human (columns), as profiled by the Roadmap Epigenomics Consortium. Shading indicates enrichment relative to human chromatin state abundance (columns). The number of regions overlapping is shown in each cell of the heatmap.

Extended Data Figure 4. Differential gene expression and histone mark levels at regulatory regions in CK-p25 mice

a–e, Shown are six distinct classes of differentially modified regions: transient (early) increase (pink) or decrease (light blue), consistent increase (red) or decrease (blue), and late (6 wk) increase (dark red) or decrease (navy blue). The heatmap shows the log fold change relative to 2 week controls for a, gene expression; b, H3K4me3 peaks at “TSS (transcription start site)” chromatin state; c, H3K27ac peaks at enhancer chromatin state; d, H3K27me3 peaks overlapping the Polycomb repressed chromatin state; e, H3K9me3 peaks overlapping the heterochromatin chromatin state. Numbers denote peaks falling into each category.

Extended Data Figure 5. Relationship between changes of gene expression and regulatory regions in CK-p25 mice

For each class of gene expression change in the CK-p25 model (x axis), overlap with different histone modifications is shown (y axis) for a, H3K4me3 at promoters; b, H3K27ac at enhancers; c, H3K27me3 at Polycomb repressed regions. Histone modifications were mapped to the nearest transcription start site (Supplementary Table 3) to show the enrichment of the changing regulatory regions relative to those that are stable in CK-p25. The significance is calculated based on the hypergeometric P value of the overlap.

Extended Data Figure 6. Enrichment of immune cell eQTLs in increasing mouse enhancers

Enrichment of eQTL SNP (y axis; −log₁₀(binomial P < 10⁻⁴)) in monocytes and CD4⁺ (ref. 25,26) is compared to the orthologous regions of CK-p25 affected enhancers relative to enhancers whose levels do not change.

Extended Data Figure 7. Weak enrichment of AD GWAS SNPs at differential CK-p25 promoters

Enrichment of AD-associated SNPs (y-axis, binomial P value) in human regions orthologous to different classes of mouse promoters

Extended Data Figure 8. Enrichment of tissue-specific enhancer annotations from the Roadmap Epigenomics Consortium for AD-associated SNPs and mouse enhancers

Enrichment of AD-associated SNPs (y-axis, permutation P value) in tissue-specific enhancer annotations from the Roadmap Epigenomics Consortium (points), relative to their enrichment for a, increased-level and b, decreased-level (colors of different classes along y-axis) of orthologous enhancer regions in the mouse AD model (x-axis, hypergeometric P value). Linear regression trend line and R², based on Pearson correlation is shown.

Extended Data Figure 9. Cell type composition

a, For each class of gene expression change (x-axis), shown is the enrichment of cell-type-specific gene markers from published datasets^–. The macrophage and monocyte categories are computed relative to microglia^,. The enrichment is calculated relative to the genes that do not change in expression level in the CK-p25 mice. Cells in the heatmap are labeled based on the −log₁₀(P value) (hypergeometric t-test). Cases where no genes overlapped are shown in grey. b, Summary of a, showing the inferred change in cell type composition across time.

Figure 1. Conserved gene expression changes between mouse and human AD are associated with immune and neuronal functions

a, Six distinct temporal classes of differentially expressed genes are denoted, transient (early) increase (pink) or decrease (light blue), consistent increase (red) or decrease (blue), and late (6 wk) increase (dark red) or decrease (navy blue). Expression is shown relative to the mean of three replicates at 2 week control (CK) mice. Shown are the most significant distinct biological process Gene Ontology categories in each class of differentially regulated genes (asterisk denotes enrichment of hypergeometric P < 0.01). Grey boxes indicate no overlapping genes. b, T-statistic identifying the bias of each differentially regulated class of genes in AD cases relative to controls; negative t denotes lower expression in AD, positive t denotes higher expression in AD. c, Enrichment of Gene Ontology categories for differentially expressed genes between AD cases and controls in human. Enrichment of each Gene Ontology category examined in the gene expression analysis was calculated for d, H3K4me3 promoters (red) and e, H3K27ac enhancers (yellow). Asterisk denotes categories with a binomial P < 0.01. Enrichment of regulatory motifs within changing f, promoters (top) and g, enhancers (bottom) in the mouse AD model. Overlap of changing h, promoters (top) and i, enhancers (bottom) with regions shown to be bound by immune (orange) and neuronal (purple) transcriptional factors (TF) and co-factors profiled using ChIP-seq in mouse immune and neuronal tissues^–.

Figure 2. AD GWAS loci are preferentially enriched in increasing enhancer orthologs with immune function

a, Enrichment (y-axis) of changing mouse AD enhancer orthologs, with a focus on consistently increasing (red) category of enhancers, in 127 cell and tissue types profiled by the Roadmap Epigenomics Consortium (columns). Roadmap samples are grouped into fetal brain (purple), adult brain (green), immune/blood cell types (orange), and all other (grey). b, Cell-type-specific fold luciferase reporter expression change relative to control (ctrl) for selected increasing enhancer regions in BV-2 microglia (orange) vs N2A neurons (purple) (n=3, *P < 0.05, two-tailed t-test). c, Enrichment of AD-associated SNPs (y-axis, binomial P value) in human regions orthologous to the mouse enhancers. d,e, Enrichment of AD-associated SNPs (y-axis, permutation P value) in tissue-specific enhancer annotations from the Roadmap Epigenomics Consortium (points), relative to their enrichment for d, consistently increasing and e, consistently decreasing orthologous enhancer regions in the mouse AD model (x-axis, hypergeometric P value). Linear regression trend line and R², based on Pearson correlation, is shown.

Figure 3. Increasing enhancer orthologs help interpret AD-associated noncoding loci

Overlap of disease-associated SNPs (top) with increasing enhancers (2nd row, red) and immune enhancers in human (CD14⁺ primary cells) is shown for genome-wide significant (INPP5D and CELF1 (containing the SPI1 gene); a and b) and below-significance (ABCA1; c) AD GWAS loci. Roadmap chromatin state annotations for immune cells (CD14⁺ primary; E029), hippocampus (E071), and fetal brain (E81), with colors as shown in the key. Light red highlight denotes increasing enhancer regions tested in luciferase assay. kb, kilobases; Mb, megabases. d, AD associated SNP rs1377416 amplifies in vitro luciferase activity of putative enhancer region 38,313–37,359 base pairs (bp) upstream of SPI1 (PU.1) gene in BV-2 cells. n=3, P < 0.0001, one-way analysis of variance (ANOVA); **P < 0.01, Tukey’s multiple comparison post-hoc test. NS, nonsignificant.