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, 43 (20), 9680-93

Epigenetic Program and Transcription Factor Circuitry of Dendritic Cell Development

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Epigenetic Program and Transcription Factor Circuitry of Dendritic Cell Development

Qiong Lin et al. Nucleic Acids Res.

Abstract

Dendritic cells (DC) are professional antigen presenting cells that develop from hematopoietic stem cells through successive steps of lineage commitment and differentiation. Multipotent progenitors (MPP) are committed to DC restricted common DC progenitors (CDP), which differentiate into specific DC subsets, classical DC (cDC) and plasmacytoid DC (pDC). To determine epigenetic states and regulatory circuitries during DC differentiation, we measured consecutive changes of genome-wide gene expression, histone modification and transcription factor occupancy during the sequel MPP-CDP-cDC/pDC. Specific histone marks in CDP reveal a DC-primed epigenetic signature, which is maintained and reinforced during DC differentiation. Epigenetic marks and transcription factor PU.1 occupancy increasingly coincide upon DC differentiation. By integrating PU.1 occupancy and gene expression we devised a transcription factor regulatory circuitry for DC commitment and subset specification. The circuitry provides the transcription factor hierarchy that drives the sequel MPP-CDP-cDC/pDC, including Irf4, Irf8, Tcf4, Spib and Stat factors. The circuitry also includes feedback loops inferred for individual or multiple factors, which stabilize distinct stages of DC development and DC subsets. In summary, here we describe the basic regulatory circuitry of transcription factors that drives DC development.

Figures

Figure 1.
Figure 1.
Gene expression, H3K4me1, H3K4me3, H3K27me3 and PU.1 occupancy in DC development. (A) Schematic representation of DC commitment from MPP to CDP and DC subset specification from CDP to cDC or pDC. The number of differentially expressed genes between a pair of cell states is given in red. (B) Differentially expressed genes were clustered according to their expression in MPP/CDP, MPP, CDP, pan-DC, cDC and pDC as indicated. Heat map representation of gene specific mRNA expression (red, high expression; blue, low expression) and the respective H3K4me1, H3K4me3, H3K27me3 and PU.1 occupancy is shown (dark colors indicate high occupancy; light or white colors indicate low or no occupancy). H3K4me3 and H3K27me3 signals at promoter regions (TSS±1kb); H3K4me1 and PU.1 signals at distal regions/enhancers (TSS±50kb). Key regulatory factors are listed. (C) For each of the six clusters, mRNA expression, H3K4me1, H3K4me3, H3K27me3 and PU.1 occupancy were calculated using the geometric mean of the levels of respective genes and are shown in heat map format. Color code as in (B). (D) Occupancy for H3K4me1, H3K4me3, H3K27me3, PU.1 and mRNA profile (log2 expression) of Flt3 gene in MPP, CDP, cDC and pDC. Promoter and enhancer regions with PU.1 binding are indicated (gray box).
Figure 2.
Figure 2.
Histone modifications and PU.1 binding dynamics of key DC transcriptional regulators during DC development. Occupancy for H3K4me1, H3K4me3, H3K27me3 and PU.1 and mRNA expression (log2 expression) of DC progenitor genes (Gfi1 and Cebpa), cDC genes (Id2, Batf3) and pDC genes (Tcf4, Spib, Irf7r and Irf1) in MPP, CDP, cDC and pDC. Irf8, a central DC transcription factor, is also shown. Arrow indicates the direction of transcription.
Figure 3.
Figure 3.
PU.1 transcription factor binding in DC development. (A) Principal component analysis of genome-wide PU.1 binding profiles in MPP, erythroid progenitor cells (EP), T/B lymphoid cells (double negative T cells, DN; pro B cells, ProB) and DC progenitors and subsets (CDP, cDC and pDC; GM-CSF derived DC, GM-DC). The DC cluster is highlighted. (B) PU.1 binding peaks occurring in enhancers or active promoters in MPP, CDP, cDC and pDC are shown. Regions were defined as active promoters if marked with both H3K4me1 and H3K4me3, and enhancers if marked only with H3K4me1. (C) Differential PU.1 peaks between MPP versus CDP (left) and cDC versus pDC (right) are depicted in blue and red as indicated. Non-differential peaks are colored in gray. De-novo PU.1 motifs calculated for cell type-specific peaks and the classical PU.1 motif (UP00085; (53)) are shown. (D) The proportion of PU.1 target genes with differential PU.1 peaks close to differentially regulated genes in progenitors (MPP versus CDP) and DC subsets (cDC versus pDC) are shown in percent (filled bars). The percentage of PU.1 targets in all genes was used as background control (open bars). The Fisher's exact test was employed to calculate the enrichment of PU.1 targets.
Figure 4.
Figure 4.
Identification of lineage-specific transcription factor regulatory networks. (A) Heat map depicts the enrichment of transcription factor motifs (row) in MPP, CDP, cDC and pDC (column). P values are plotted and color-coded using a continuous spectrum from gray (p value > 0.05) to blue (p value < 0.05). Twenty transcription factors that are considered as potential PU.1 co-binding partners are labeled in red. Clusters I to VI are indicated. (B) PU.1 and Irf8 occupancy of Id2 and Irf1 loci are shown for MPP, CDP, cDC and pDC. Differential PU.1 peaks and Irf8 peaks are highlighted by gray boxes. Green lines indicate transcription factor binding sites (TFBS) predicted inside differential PU.1 peak regions. Selected TFBS are labeled by gene symbol.
Figure 5.
Figure 5.
Construction and validation of stage-specific transcription factor regulatory networks. (A) Transcription factor regulatory networks of MPP, CDP, cDC and pDC. Nodes represent identified PU.1 co-binding partners from Figure 4A that are active (red) or inactive (gray) in MPP, CDP, cDC and pDC. Selected key DC regulators from the literature (white) were included in the analysis (,–45). A directed edge from transcription factor a to target gene b (black) indicates the association with gene activation, i.e., (i) the transcription factor is enriched in the respective cell type, (ii) the target gene is differentially expressed during DC commitment (MPP versus CDP) or DC subset specification (cDC versus pDC) and (iii) there is a transcription factor binding site at a differential PU.1 peak of the target gene. A self-loop edge (gray box) indicates an auto-regulatory feedback loop. The gray edges show regulatory links predicted in at least one of the networks. (B) The density plot of predicted Irf8 target genes shows higher gene expression in Irf8+/+ CDP than in Irf8−/− CDP. (C) Irf8 target genes in CDP of (A) are validated by comparing gene expression data of Irf8−/− and Irf8+/+ CDP, CMP and MDP (GSE34915, GSE45467). Red and blue, higher or lower gene expression, respectively, in Irf8+/+ than Irf8−/− cells. (D) The density plot of predicted Stat3/Stat5 target genes in Stat5-ER DC progenitors shows an increase of gene expression in response to 8h taxmoxifen (tmx) treatment. (E) Stat3/Stat5 target genes in CDP and cDC of (A) are validated using conditional Stat5-ER expression for 8, 16 and 24 hours. Red and blue, higher and lower gene expression in response to tamoxifen activated Stat5-ER, respectively. P values were calculated using Wilcoxon rank sum test.
Figure 6.
Figure 6.
The regulatory circuitry of DC development. The network illustration depicts the organization of the integrated DC regulatory circuitry. Nodes represent key regulatory factors involved in DC development. A directed edge from factor a to factor b indicates an active function (green) or inhibition function (red) of factor a to factor b. Dotted edges represent additional interactions obtained from the literature (,–45). The pDC sub-network of Irf1, Ets1, Spib and Tcf4 is shown (gray box).
Figure 7.
Figure 7.
Sub-network of Irf4 and Spib by ChIP-seq analysis. (A) PU.1, Irf8 and Stat3/Stat5 are predicted to impact on Irf4 in cDC. The auto-regulatory loop of Irf4 is indicated (see network in Figure 6). (B) Occupancy of PU.1, Irf4, Irf8 and Stat3 in the enhancer region (H3K4me1) of Irf4 gene in cDC (highlighted in gray box) verify the predicted transcription factor binding sites (green bars). The ChIP-seq data of Irf4, Irf8 and Stat3 in cDC are from GSE36104, GSE53311 and GSE27161. (C) PU.1, Irf8 and Tcf4 are predicted to impact on Spib in pDC. The auto-regulatory loop of Spib is also indicated. (D) Occupancy of PU.1, Irf8 in Spib enhancer region (H3K4me1) in pDC (highlighted in gray box) is in line with the predicted co-binding of Irf8 and PU.1 (green bars). (E) The PU.1 peak in mouse pDC is mapped to human genome using UCSC liftOver tool and shown as blue bar. The PU.1 peak collocates with Tcf4 in human pDC (highlighted in gray box) and predicted transcription factor binding sites for PU.1, Tcf4, Spib, Irf1 and Irf8 (green bars). The ChIP-seq data of Irf8 and Tcf4 in pDC are from GSE62702 and GSE43876.

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