Most GWAS hits lie in noncoding regions of the genome, making it hard to figure out what they do. To address this, researchers led by Christopher Glass at the University of California, San Diego, analyzed epigenetic data from purified populations of neurons, astrocytes, oligodendrocytes, and microglia from the young human cortex. They catalogued which regulatory regions were active in each cell type, and then looked for associations with known risk loci for neurological and psychiatric diseases. In the November 14 Science, they report that while risk variants for psychiatric diseases mostly lie in neuron-specific regulatory regions, Alzheimer’s risk variants are concentrated in microglial enhancers. In other words, many polymorphisms associated with AD affect gene expression only in microglia. The findings add to recent data suggesting that these cells drive Alzheimer’s pathogenesis.
- Scientists map enhancer-promoter interactions in four cell types of healthy brain.
- This atlas places most AD risk variants in microglial-specific enhancers.
- For example, the causal risk variant for BIN1 affects its expression only in microglia.
“This is giant,” said Oleg Butovsky at Brigham and Women’s Hospital in Boston. “They use cutting-edge technology to study human data, and open up a door to understanding how microglia initiate Alzheimer’s disease.”
In addition to these Alzheimer’s findings, the dataset could enable researchers to generate and test hypotheses for neurological disease in general, Glass noted. “This is the first atlas of genetic regulatory elements specific for the major cell types of the human brain,” he told Alzforum. Alison Goate at the Icahn School of Medicine, Mount Sinai, New York, agreed. “This provides an important resource for people working on brain disorders,” she said.
Microglia and Alzheimer’s. AD variants from two GWASs are associated almost exclusively with enhancers in microglia (left two columns, darker blue), while variants associated with psychiatric disorders and behavioral traits cluster strongly in neuronal regulatory regions (center and right column groupings). [Courtesy of Nott et al., Science/AAAS.]
Goate previously examined how AD risk variants affect gene expression, but had to use monocytes and macrophages as a proxy for microglia, because epigenetic data on these brain cells were not yet available. She found that AD risk factors were preferentially located in myeloid-specific enhancers, in agreement with the new findings (Aug 2019 news). For their part, Glass and colleagues previously isolated microglia from healthy human brain and published transcriptomic, but not epigenetic, data on these cells (Jun 2017 news).
For the current study, first authors Alexi Nott, Inge Holtman, and Nicole Coufal purified the four major brain cell types from cortical tissue samples taken from six young people aged 4–18 who were undergoing surgery to prevent future epileptic seizures. The researchers used chromatin immunoprecipitation and sequencing (ChIP-seq) to find active enhancer and promoter regions in each cell population. Active enhancers are marked by acetylation of histone H3K27, and promoters by trimethylation of H3K4 (Creyghton et al., 2010; Heintzman et al., 2007). The scientists found that all four brain-cell types shared a similar set of active promoters, but had distinct sets of active enhancers.
When transcription factor complexes assemble at active enhancers, the DNA strand loops to bind nearby promoters, initiating transcription of those genes. To find enhancer-promoter pairings in each cell type, the authors cross-linked DNA strands that were in proximity and sequenced those regions. They found 219,509 unique interactions. These clustered by cell type, defining a distinct set of enhancers that controlled the genes characteristic of each cell population.
Distant Pairings Modifications to histone proteins (green and red dots) mark active enhancers, where a transcription complex (colored circles) assembles on a chromosome. DNA then loops over (arrow) to contact an active promoter (gray rectangle) and initiate transcription. [Courtesy of Calo and Wysocka, 2013.]
With this information in hand, the researchers examined disease-linked GWAS variants. Most polymorphisms associated with psychiatric disorders and behavioral traits such as autism, schizophrenia, neuroticism, and risk behavior were located in neuronal enhancers and promoters. A few of them appeared in glial promoters as well. For Alzheimer’s disease, on the other hand, GWAS risk variants were enriched only in microglial enhancers (see image above).
These microglial enhancers regulated numerous genes associated with AD, such as ABCA7, SORL1, and TREM2. The genes formed a protein interaction network centered around ApoE, the main risk factor for late-onset AD (see image below).
A detailed analysis of microglial enhancer-promoter interactions turned up some surprises. For example, AD risk variants in the SLC24A4 locus did not affect transcription of that gene, but instead mapped to promoters that controlled expression of the genes ATXN3, TRIP11, and CPSF2. This information will guide research on the functional effects of risk variants, Glass noted.
Hello Again. Alzheimer’s risk genes expressed in microglia, which have been identified in one (gray), two (green), or three (yellow) GWAS studies, and several genes with possible causal variants identified in this study (diamonds). They encode proteins that form an interaction network. [Courtesy of Nott et al., Science/AAAS.]
Although GWAS risk SNPs associate with disease, they are not necessarily the causal variant. To look for them, the authors used “fine mapping” methods pioneered by geneticists at the University of California, Los Angeles. These methods leverage the strength of disease associations to pinpoint the most likely causal variant within a locus (Kichaev et al., 2014). In 13 AD loci, the authors identified a risk variant with a high likelihood of being causal. Eight of these occurred in microglial-specific enhancers that regulated gene promoters.
One of them, the rs6733839 SNP in the BIN1 locus, had a 97 percent chance of being the causal variant. This SNP is notable for conferring a high risk of AD, second only to the ApoE4 allele among common GWAS hits. The SNP lies in a microglial-specific enhancer that binds the BIN1 promoter. The authors confirmed this microglial specificity in cultured human neurons, astrocytes, and microglia derived from induced pluripotent stem cells. Deletion of the enhancer region abolished BIN1 expression in microglia, but not in the other two cell types. The findings suggest that rs6733839 exerts its effects only through microglia. Glass presented some of these BIN1 data at a 2018 Keystone conference (Jul 2018 conference news).
Glass’ epigenetic data are broadly similar to Goate’s findings from myeloid cells. “We predicted that the AD risk would be largely microglial, and they demonstrate that in this dataset,” Goate told Alzforum. The next step will be to examine how risk and protective alleles alter microglial function, and if there are common patterns, she said.
Meanwhile, Glass is interested in how disease alters epigenetic interactions in brain cells. He noted that many AD risk alleles did not map to any enhancers or promoters in his study. One possibility is that they are expressed in other brain cell types, such as endothelial cells. It could also be that some enhancers become active in the context of disease, and all the cells in this study were isolated from brains of young people without Alzheimer’s. Glass and colleagues are now developing protocols for analyzing cells from postmortem brain samples taken from people who died of various neurodegenerative diseases. “I think that will give us a tremendous amount of information about pathological signaling pathways in the brain,” Glass said.
Butovsky believes the new data reinforce other recent findings suggesting that ApoE wreaks its havoc mostly through microglia (Sep 2017 news; Oct 2019 news). “We need to focus on microglial biology in Alzheimer’s. This is the primary target for therapy right now,” he said.—Madolyn Bowman Rogers
- AD Genetic Risk Tied to Changes in Microglial Gene Expression
- What Makes a Microglia? Tales from the Transcriptome
- A Delicate Frontier: Human Microglia Focus of Attention at Keystone
- ApoE and Trem2 Flip a Microglial Switch in Neurodegenerative Disease
- In Tauopathy, ApoE Destroys Neurons Via Microglia
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