Transcription factors (TFs) control cell identity and function. How their activity is altered during healthy aging is critical for an improved understanding of aging and disease risk, yet relatively little is known about such changes at cell-type resolution. Here we present and validate a TF activity estimation method for single cells from the hematopoietic system that is based on TF regulons, and apply it to a mouse single-cell RNA-sequencing atlas, to infer age-associated differentiation activity changes in the immune cells of different organs. This revealed an age-associated signature of macrophage dedifferentiation, which is shared across tissue types, and aggravated in tumor-associated macrophages. By extending the analysis to all major cell types, we reveal cell-type and tissue-type-independent age-associated alterations to regulatory factors controlling antigen processing, inflammation, collagen processing and circadian rhythm, that are implicated in age-related diseases. Finally, our study highlights the limitations of using TF expression to infer age-associated changes, underscoring the need to use regulatory activity inference methods.
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A meta-analysis of immune-cell fractions at high resolution reveals novel associations with common phenotypes and health outcomes
Genome Medicine Open Access 31 July 2023
Cell-attribute aware community detection improves differential abundance testing from single-cell RNA-Seq data
Nature Communications Open Access 05 June 2023
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Data analyzed in this manuscript is publicly available from the GEO (www.ncbi.nlm.nih.gov/geo/) under accession numbers GSE56046 (DNAm data) and GSE130973 (skin aging scRNA-seq dataset), from EBI ArrayExpress (https://www.ebi.ac.uk/arrayexpress) under accession numbers E-MTAB-6149 and E-MTAB-6653 (lung cancer scRNA-seq dataset). FACS-sorted expression data from purified blood cell subtypes are available from https://haemosphere.org/datasets/show. The lung cancer bulk RNA-seq dataset is available from the TCGA data portal (http://tcgaportal.org/). The TMS scRNA-seq data are available from https://doi.org/10.6084/m9.figshare.8273102.v2. The regulons for the 328 hematopoietic TFs and their cell-specific lineage information are provided in Supplementary Data 1. The DOROTHEA regulons are available from https://saezlab.github.io/dorothea. ChIP–seq data were downloaded from the ChIP–seq Atlas (https://chip-atlas.org/). Processed data are available from the corresponding author upon reasonable request. Source data are provided with this paper.
SCIRA functions for estimating TFA are available from the scira R package (http://github.com/aet21/scira/). We also provide an R markdown file and associated data objects from the figshare repository (https://figshare.com/articles/software/R-markdown_file_and_data_objects_for_estimating_TFA_in_TMS_lung-tissue_scRNA-seq_dataset/17167085) that illustrate in a few examples how to estimate TFA and how to correlate it with age in the TMS dataset.
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This work was supported by National Science Foundation of China grants (31771464, 32170652 and 31970632). We thank the TMS consortium for making their data open access and to everyone else who shares data in public repositories.
The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. The authors declare no competing interests.
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Extended Data Fig. 1 Comparison of validation accuracies between SCIRA and Dorothea and validation of mouse hematopoietic TF-regulons.
a) Barplots compare validation accuracies of estimated regulatory activities for blood-cell-lineage specific TF-regulons, obtained using the TF-regulons from SCIRA or those from the DOROTHEA database, as indicated. Top-panel is for mouse RNA-Seq ImmGen dataset which profiled purified blood cell subtypes from the various lineages as shown. Lower-panel is for the mouse ImmGen2 dataset. B = B-cell-lineage, DC = dendritic cell, Mon/Mac=Monocyte/Macrophage lineage, MPP = multipotent progenitor, Neu=Neutrophil, NK = Natural Killer, RPP = Restricted potency progenitor, T = T-cell lineage, All=accuracy obtained over all lineages. b) Diffusion maps on the left display diffusion components DC1 & DC2, of the transcription factor activity (TFA) matrix defined over the hematopoietic TFs and 4142 human bone marrow cells from Setty et al, with cells colored by cell-type as annotated in Setty et al (HSC = hematopoietic stem cell, Pre=precursor/multipotent progenitor, Mono=monocytes, DC = dendritic cells, Ery=Erythroid cells, top panel), and with cells colored by estimated diffusion pseudotime (DPT, lower panel). TFA was estimated using our SCIRA-derived TF-regulons. Tip-points are marked in red, displaying the starting branch point in the lower left corner, and the two terminal branch points representing the endpoints of the erythroid (green dashed line) and myeloid (red dashed line) branches. The diffusion maps to the right have cells colored by the estimated TFA of TFs that are known to be specific for HSCs (HOXB5), monocytes (CEBPB), erythrocytes (GATA1) and dendritic cells (IRF8) (top-panel). Lower panel displays the TFA of these same TFs as a function of diffusion pseudotime along their respective branches.
a) Heatmap panels display the age-associated patterns of differentiation activity (TFA) for lineage-specific TFs in different tissues and in corresponding cell-types of that lineage. Clockwise, panels represent the patterns for T-cell, B-cell and NK lineages. Each heatmap displays the signed statistical significance level as indicated, where the P-value is derived from the t-statistic of a regression of TFA against age, adjusting for sex. b) Violin plot displaying estimated TFA vs age-group for Lef1 in lung Cd4 + T-cells. Pearson Correlation Coefficient (PCC) and associated P-value are shown. c) Scatterplot of the t-statistics of association of TFA with age for T-cell specific TFs as derived in CD4 + T-cells from lung (x-axis) vs. T-cells from spleen (y-axis). TFs significant in both tissues are colored. Pearson Correlation Coefficient and P-value between the two tissues is given. d) Violin plot displaying estimated TFA vs age-group for Spib in lung B-cells. Pearson Correlation Coefficient (PCC) and associated P-value are shown.
Heatmap displays age-associated differentiation activity (TFA) changes for neutrophils from lung and spleen, as inferred from the Tabula Muris Senis dataset. Scatterplot displays the corresponding t-statistics of association of TFA with age. TFs colored in red are significant in both tissues. Pearson Correlation Coefficient (PCC) and associated two-sided P-value between lung and spleen is given.
Extended Data Fig. 4 Age-associated DNA methylation at macrophage-specific TFs in purified monocyte samples.
For 4 macrophage-specific TFs that exhibit dysregulation of regulatory activity with age in monocytes/macrophages, as inferred from the TMS 10X scRNA-seq dataset, we display the signed statistical significance (y-axis) of age-association of Illumina 450k DNA methylation probesm as derived from the 1200 purified monocyte sample set from Reynolds et al. In the y-axis we display the sign of the age-associated t-statistic times the negative log10[Q-value], where Q-value is the FDR-estimate. X-axis labels the genomic position of the probes. Probes in red are significantly associated with age (Q < 0.05).
Extended Data Fig. 5 Validation of hematopoetic TF regulons in human datasets and TFA patterns of macrophage-specific TFs in lung adenocarcinoma.
a-b) Validation of hematopoetic TF regulons in human datasets. a) Barplots displaying the validation accuracy of blood cell-type specific SCIRA TF-regulons in 3 independent human FACS mRNA expression datasets (Ebert, Schultze & de Graf) from the Haemopedia resource. Accuracy was estimated as the fraction of cell-type specific TFs whose regulons predicted a significantly higher TFA in the corresponding blood cell types compared to all other cell-types, as assessed using a one-sided t-test (P < 0.05). Only cell-types with at least 5 samples were included in this analysis. Total number of cell-sorted samples per dataset were: n = 211 (Ebert), n = 384 (Schultze), n = 42 (de Graf). b) Left panel: tSNE-diagram of a 10X scRNA-Seq peripheral blood mononuclear cell (PBMCs) dataset from Zheng et al. Right panel: Barplot displaying the validation accuracy of blood cell type specific TF-regulons derived from cell-sorted bulk expression data from Haemopedia in the scRNA-Seq PBMC data from Zheng et al. c-e) Pattern of TFA of macrophage-specific TFs in lung adenocarcinoma TCGA set. c) Color bars displaying the t-statistics (t) and P-values (P) between the TFA of 11 macrophage-specific TFs and normal-cancer status using only paired samples (n = 45 pairs). Thus, cyan indicates lower TFA in tumor vs normal. d) As a), but now adjusted for macrophage marker (LYZ & CD14) expression. e) Kaplan Meier overall survival curves for all primary LUAD samples, with samples stratified into low, middle and high tertiles according to IRF2 TFA activity. Hazard Ratio and chi-square test P-value derive from a Cox-regression of IRF2 TFA (treated as continuous variable).
Extended Data Fig. 6 Regulon size comparison between DOROTHEA and SCIRA and their influence on age-associations.
a) Violin plots compare the regulon sizes of the DOROTHEA regulons vs the hematopoietic TF regulons built with SCIRA. The number of TF-regulons in each group is displayed below. Scatterplots to the right plot the regulon size (x-axis) vs. the fraction of regulon genes where the interaction between TF and gene is positive (fPOS, y-axis). b) Panels display scatterplots of statistical significance (-log10P, y-axis) vs AUC (x-axis) derived from a two-sided Wilcoxon-test comparing the regulon size of DOROTHEA TF-regulons that were significantly associated with age (Linear model P < 0.001) vs the size of TF-regulons that were not associated with age, as assessed in cell-types within the given tissue. Each datapoint represents one distinct cell-type within the tissue. The green dashed-line indicates the line AUC = 0.5. Points to the right (AUC > 0.5) are cell-types for which the regulon sizes of age-associated TF-regulons were higher compared to non age-associated ones, with the y-axis indicating statistical significance level. c) Boxplots display the SCIRA TF regulon size vs a binary variable indicating whether the TFA derived from the TF-regulon was significantly associated with age (SigTF) or not (NonSigTF), as assessed using a P < 0.001 threshold. The number of TF-regulons in each category is given below each boxplot. The P-value comparing the regulon sizes between the significant and non-significant regulons is indicated within each panel and is derived from a two-tailed Wilcoxon rank sum test. The bar within each boxplot represents the median, the box itself the interquartile range (IQR), and whiskers extend to 1.5 times the IQR.
Heatmap displays the signed significance of the age-associated t-statistics of TFA with age for 6 selected TFs across 75 cell-types and 11 tissue types as indicated. The 6 TFs were selected on the basis of displaying highly significant skews towards either increased or decreased regulatory activity with age.
Extended Data Fig. 8 Rfx5 activity changes in heart cells and inverse association with Col1a2 expression.
a) Violin plots display the regulatory activity (TFA) of Rfx5 against age [months] for 4 cell-types in heart, as indicated. The number of cells at each timepoint is indicated above violin plot. P-value shown is from a linear regression of TFA against age, adjusting for sex. b) Heatmap of significance statistics for Rfx5 TFA against age (including Col1a2 in its regulon), Rfx5 TFA against age not including Col1a2 in its regulon (‘no Col1a2’) and for Col1a2 expression against age.
Extended Data Fig. 9 Validation of Nfkb1 increased activity in T-cells using CD4 + T-cell NFKB1 ChIP-Seq targets (5 kb).
Left panels: Genome plots display the signed statistical significance of NFKB1 target expression with age, as assessed in T-cells from the given tissue-type. Gene targets within 5 kb of the NFKB1 ChIP-Seq peak are shown with the color indicating increased (magenta) or decreased (cyan) mRNA expression with age. Alternating grey/black bars indicate successive mouse chromosomes. Right panels: Violin plots of the corresponding Spearman Rank Correlation Coefficients (SCC), with the P-value derived from a one-tailed t-test, testing for the null that the average SCC = 0. The number of ChIP-Seq targets is 126. Similar results are seen when defining NFKB1 ChIP-Seq targets as having a peak within 10 kb and 1 kb of the gene.
Extended Data Fig. 10 Association of antigen processing machinery (APM) and Rfx5 regulatory activity with age.
a) Boxplots display the Spearman rank correlation coefficient (SCC) between the gene-expression of APM genes (n = 16) with age, stratified according to tissue and cell-type. Boxes shown in magenta (cyan) indicate cell-types where Rfx5 activity increased (decreased) significantly with age (P < 0.05). P-values above boxplots derive from a two-tailed t-test, testing if the average SCC values are significantly different from zero. P-values in red are those with P < 0.05. Number of datapoints in each boxplot is 16. The bar within each boxplot represents the median, the box itself the interquartile range (IQR), and whiskers extend to 1.5 times the IQR. b) A scatterplot of the average SCC with age (x-axis) against the t-statistic of association between Rfx5 regulatory activity and age (y-axis), with each datapoint representing one tissue-cell-type pair (as in a)). Datapoints highlighted in red indicate consistent significant associations, those in blue indicate inconsistent associations. Overall Spearman Correlation Coefficient (SCC) and two-sided P-value are given.
Statistical source data (enrichment table for hematopoietic TFs with ChIP–seq data, with columns labeling the TF, number of regulon genes, number of direct targets, AUC and associated one-sided P value)
Statistical source data (validation accuracies summarized by cell lineage, tissue and technology type, and method.
Statistical source data (significance levels, that is, sign(t-statistic) × −log10(P value) of age associations for macrophage-specific TFs and macrophage/monocyte populations in various tissues).
Statistical source data (TFA values for the 11 macrophage-specific TFs and all lung macrophages, with cells labeled by barcode and normal/tumor status.
Statistical source data (significance levels, that is, sign(t-statistic) × −log10(P value) of age associations for 168 selected TFs and all cell types considered).
Statistical source data (TFA values for DOROTHEA TFs across keratinocyte cells, cells labeled by barcode and age group).
Statistical source data (z-statistics of differential TFA and DE for ARNTL).
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Maity, A.K., Hu, X., Zhu, T. et al. Inference of age-associated transcription factor regulatory activity changes in single cells. Nat Aging 2, 548–561 (2022). https://doi.org/10.1038/s43587-022-00233-9
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