An environment-dependent transcriptional network specifies human microglia identity

Abstract

Microglia play essential roles in central nervous system (CNS) homeostasis and influence diverse aspects of neuronal function. However, the transcriptional mechanisms that specify human microglia phenotypes are largely unknown. We examined the transcriptomes and epigenetic landscapes of human microglia isolated from surgically resected brain tissue ex vivo and after transition to an in vitro environment. Transfer to a tissue culture environment resulted in rapid and extensive down-regulation of microglia-specific genes that were induced in primitive mouse macrophages after migration into the fetal brain. Substantial subsets of these genes exhibited altered expression in neurodegenerative and behavioral diseases and were associated with noncoding risk variants. These findings reveal an environment-dependent transcriptional network specifying microglia-specific programs of gene expression and facilitate efforts to understand the roles of microglia in human brain diseases.

Figure 1. Human microglia transcriptomes

(A) Heat map of mRNA expression values determined in microglia isolated from 19 individuals and in cortex from 5 of these individuals. 881 genes exhibiting > 10-fold higher average expression in microglia compared to cortex are indicated at the top of the heat map. (B) Individual variation in gene expression of the 30 most highly expressed genes in human microglia. (C) Enrichment of the 881 microglia-enriched genes upregulated in HIV-associated neurocognitive disorder (HAND) in centrum semiovale (CS) (48), upregulated in schizophrenia (SCZ) in the anterior prefrontal cortex (PFC) (49), positively correlated with Braak stage of AD in PFC (50), upregulated or downregulated in PD in cortex (51), upregulated in frontotemporal lobar degeneration (FTLD) in frontal cortex (52), or upregulated in AD in the CA1 region of the brain (53). (D) Pie chart depicting the relative expression of genes associated with AD risk variants in brain cortex and microglia. (E) Combined bar graphs and dot plots of the TPM expression levels of the 21 most highly expressed genes associated with AD risk variants in microglia and cortex. Red stars indicate significant differential expression (> 2-fold, FDR 0.05).

Figure 2. Comparison of human and mouse microglia transcriptomes

(A) Scatter plot depicting mRNA expression levels of mouse and human genes with one-to-one orthologs, highlighting significantly differentially expressed genes in human (blue) and mouse microglia (green) (> 10-fold, FDR 0.05). (B) Identification of a mouse microglia gene signature consisting of 900 mRNAs expressed > 10-fold higher in microglia (FDR 0.05) compared to the average expression values of macrophages from skin, lung, liver, and kidney. (C) Functional annotations of the microglia gene signature. (D) Scatter plot depicting mRNAs expression levels of human and mouse microglia with orthologs intersected with the microglia gene signature expression profile from (B). Genes with conserved expression are depicted in red, genes more highly expressed in human are in blue, and genes more highly expressed in mice are in green. (E) Pie chart showing relative expression of genes associated with AD risk alleles in human and mouse microglia. (F) Combined bar graphs and dot plots illustrating the expression of the top 21 most abundant genes associated with AD risk variants in human and mouse microglia (average TPM-rank ordered). Red stars indicate significant differential expression (> 2-fold, FDR 0.05).

Figure 3. Active genomic regulatory regions of human microglia

(A) ATAC-seq and ChIP-seq data sets for PU.1, H3K27ac and H3K4me2 in the vicinity of the BIN1 gene locus. Each color represents a different individual. rs6733839 is a significant risk allele associated with AD and is located within a region highly bound by PU.1. (B) Motifs enriched at distal ATAC-seq peaks associated with H3K4me2 in human and mouse microglia. (C) Heat map of mRNA expression values of most highly expressed transcription factors recognizing motifs shown in panel B. (D) Heat map of mRNA expression values of transcription factors associated with super-enhancers in human and mouse microglia. (E) Scatter plot depicting mRNA expression values of orthologous genes encoding transcription factors in human and mouse microglia relative to overall transcriptome profiles (gray dots). Orthologous transcription factors similarly expressed are denoted in black, and transcription factors more highly expressed (> 10-fold change, FDR 0.05) in human are colored in blue, and those more elevated in mice are colored in green. (F) Scatter plot of H3K27ac tag counts at human and mouse promoters. Promoters associated with RNA transcripts expressed 10-fold higher in human microglia are colored in blue and promoters associated with RNA transcripts expressed 10-fold higher in mouse microglia are colored in green.

Figure 4. Influence of tissue culture environment on microglia gene expression

(A) Comparison of gene expression of ex vivo microglia from two individuals (panel i), effect of transition to an in vitro environment in presence of IL-34 for 7 days on microglia from each individual (panels ii and iii), and comparison of in vitro gene expression profiles (panel iv). (B) Scatter plot illustrating effects of culture environment on the conserved microglial gene signature (red) in human microglia. (C) Pie chart of the genes associated with AD risk variants that are expressed in ex vivo or in vitro microglia above a log₂ (TPM +1) value of 2. Genes more highly expressed ex vivo are in blue and gene more highly expressed in vitro are in purple. (D) Combined bar graphs and dot plots of the 21 most expressed genes, indicating effects of in vitro culture on expression of genes associated with AD risk alleles. Red stars indicate significant differential expression (> 2-fold, FDR 0.05). (E) Heat map illustrating changes in microglia gene expression 6, 24 h and 7 days after transfer to culture environment.

Figure 5. Environment-dependent enhancer landscapes

(A) Heat map illustrating the effect of culture environment on human and mouse microglia mRNA expression of transcription factors recognizing motifs highly enriched ex vivo. (B) Heat map illustrating the effect of culture environment on human and mouse microglia on mRNA expression of transcription factors associated with super-enhancers ex vivo. (C) UCSC browser visualization of regulatory elements near genes SPI1 and P2RY12, and the SALL1 SE in human microglia. Top panels display regions of accessible chromatin in microglia ex vivo, as defined by ATAC-seq. Bottom panels display H3K27ac abundance ex vivo (blue) and in vitro (yellow). (D) Fractions of ATAC-seq + H3K27ac regions in human microglia exhibiting similar, gained or reduced H3K27ac signal after transfer to a culture environment for 7 days. (E) Venn diagram of super-enhancers identified in ex vivo and in vitro human microglia based on H3K27ac signal. (F) Motifs enrichment at distal accessible chromatin regions in ex vivo human microglia defined by ATAC-seq that are associated with 2-fold loss of H3K27ac signals following maintenance in culture environment for 7 days.

Figure 6. A network of developmentally programmed and environment-dependent transcription factors establishes microglia identity and function

(A) Heat map displaying changes in mRNA expression of key microglia transcription as a function of development of the mouse brain. (B) Venn diagram illustrating significant overlap of mouse microglia genes repressed (> 2-fold, FDR 0.05) in culture and genes that increased (> 2-fold, FDR 0.05) in expression during brain development. p-value of the overlap is provided (Fisher’s exact test). (C) Venn diagram illustrating significant overlap of mouse microglia genes repressed (> 2-fold, FDR 0.05) in culture after 7 days and genes maintained/induced (> 2-fold, FDR 0.05) by chronic stimulation with Tgf-β1. p-value of the overlap is provided (Fisher’s exact test). (D) Pie chart of microglia-enriched genes compared to brain cortex overlapping with differentially expressed genes in neurodegenerative or behavioral diseases from Fig. 1C color-coded according to whether they are increased, decreased or unchanged after transfer to a tissue culture environment. (E) Diagram illustrating main features of transcription factor network regulating microglia cell identity and functions.