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. 2010 May 28;38(4):576-89.
doi: 10.1016/j.molcel.2010.05.004.

Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities

Sven Heinz  1 , Christopher Benner, Nathanael Spann, Eric Bertolino, Yin C Lin, Peter Laslo, Jason X Cheng, Cornelis Murre, Harinder Singh, Christopher K Glass
Affiliations

Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities

Sven Heinz et al. Mol Cell. .

Abstract

Genome-scale studies have revealed extensive, cell type-specific colocalization of transcription factors, but the mechanisms underlying this phenomenon remain poorly understood. Here, we demonstrate in macrophages and B cells that collaborative interactions of the common factor PU.1 with small sets of macrophage- or B cell lineage-determining transcription factors establish cell-specific binding sites that are associated with the majority of promoter-distal H3K4me1-marked genomic regions. PU.1 binding initiates nucleosome remodeling, followed by H3K4 monomethylation at large numbers of genomic regions associated with both broadly and specifically expressed genes. These locations serve as beacons for additional factors, exemplified by liver X receptors, which drive both cell-specific gene expression and signal-dependent responses. Together with analyses of transcription factor binding and H3K4me1 patterns in other cell types, these studies suggest that simple combinations of lineage-determining transcription factors can specify the genomic sites ultimately responsible for both cell identity and cell type-specific responses to diverse signaling inputs.

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Figures

Figure 1
Figure 1. Identification and functional analysis of PU.1 binding sites in primary macrophages and splenic B cells
(A) Simplified scheme for macrophage and B cell differentiation from hematopoietic stem cells, indicating genetically defined factors specific for B cells (blue) and macrophages (red) at the developmental transitions most severely affected by loss of a given factor. (B) UCSC Genome Browser image depicting PU.1 ChIP-Seq tags at the macrophage-specific Csf1r gene or the B cell-specific Irf4 gene. Dashed gray lines indicate an estimated 0.1% false discovery rate (FDR). Input DNA signal is shown 10-fold magnified. (C) PU.1-bound genomic sites are visualized by their respective normalized PU.1 ChIP-Seq tag counts (log2) within 200 bp of a given peak in macrophages and B cells. The coordinates of peaks a, b and c from panel 1B are indicated. Peak positions within 500 bp of a Refseq transcription start site are colored green. Jitter was added to the normalized tag counts to visualize otherwise overlapping data points. (D) Total number of common and cell-type specific PU.1-bound regions found in both promoter-proximal and distal genomic regions. Cell type-specificity was assigned to regions with 4-fold greater normalized tag counts in one cell type relative to the other. (E) Total number of genes with the specified number of PU.1 binding sites near their promoters. Genes were divided into subgroups of expressed and non-expressed genes based on their absolute normalized expression levels as measured by microarray hybridization (threshold: log2(microarray signal) = 6.5). (F) Distribution of the gene expression values for the combined subgroups of genes in the vicinity of the given number of peaks depicted in 1E. (G) Relationship between differential PU.1 binding and differential gene expression between macrophages and B cells. Subsets of PU.1 binding sites defined by their distance to the nearest TSS were sorted according to their difference in normalized tag counts between cell types. The moving average of the difference in gene expression values of the gene with the nearest TSS is reported relative to differential binding. The Pearson’s correlation coefficient for each group is reported in the insert (all p-values < 10−100).
Figure 2
Figure 2. Transcription factors co-localize with PU.1 binding sites in macrophages and B cells
(A) Sequence logos corresponding to enriched sequence elements identified by de novo motif analysis of promoter-proximal or inter-/intragenic PU.1 binding sites. Motifs at PU.1 sites common to macrophages and B cells were identified by comparing common peaks to randomly selected genomic regions. Motifs enriched at PU.1 sites specific to macrophages or B cells were discovered by directly comparing the sets of peaks that were exclusive to each cell type. Motif frequencies in different subsets of PU.1 peak regions are reported in Table S2. All motifs are enriched over background with p values < 10−100. (B) Frequencies of discovered motifs in PU.1 peaks (±100 bp) as a function of differential binding of PU.1 in macrophages and B cells. Expected motif frequencies based on 100k random genomic regions are reported in the legend. (C) Context-specific frequencies of AP-1, C/EBP, OCT, and E2A motifs in the vicinity of the central PU.1 motif found in PU.1 binding sites. Moving averages of motif frequencies within a 23 bp window are centered on distal macrophage-specific PU.1 sites (red), distal B cell-specific PU.1 binding sites (blue), and common distal PU.1 binding sites (gray) (>500 bp from the nearest TSS). (D) Differential PU.1 occupancy of PU.1 peaks as in Figure 1C; PU.1 peaks are colored orange if a C/EBPβ peak is located within 100 bp of the PU.1 peak. (E and F) Sequence logos are shown for the most highly enriched sequence motifs in C/EBPβ and Oct-2 binding sites, respectively. The fraction of peaks containing at least one instance of each motif within 100 bp of the peak center is given to the right of the motif with the expected frequency of the motif in 50k random regions given in parentheses.
Figure 3
Figure 3. Co-operative interactions between PU.1 and cell type-restricted transcription factors define the macrophage and B cell-specific cistromes
(A) Motifs in the vicinity of gained PU.1 sites during B cell development. Regions of 200 bp, centered on ChIP-Seq PU.1 peaks in the respective cell types were classified according to presence or absence of a given motif. Displayed is the ratio of the fraction of gained peaks (>3-fold tag count change) containing the motif relative to the fraction of unchanged peaks (<2-fold tag count change) containing the motif. Absolute peak numbers are provided in Supplemental Table S3. (B) Same plot as in Figure 1C, with PU.1 peaks seen specifically enriched in Rag1−/− vs. EBF−/−cells colored in red. (C) UCSC browser image of the PU.1 binding pattern at the IgK 3’ enhancer region. Top to bottom: B cells, Rag1−/− pro-B cells, E2A−/− CLP/pre-pro-B cells reconstituted with a conditional E2A (E47-ER) or a transactivation domain-deficient E2A (bHLH-ER) 6 h after tamoxifen treatment and E2A−/− CLP/pre-pro-B cells, as well as B cell ChIP input. The PU.1 site at the Il7r promoter is shown as a control for a PU.1 binding site that is invariant between the different cell types. (D) Top motif in the vicinity of PU.1 binding sites gained following reconstitution with full length E2A compared to constitutively bound PU.1 sites identified by de novo motif analysis. The fraction of PU.1 peaks containing at least one instance of the motif within 100 bp of the peak center in gained and constitutively bound peaks is given to the right and in parentheses, respectively. (E) UCSC browser tracks of PU.1 and C/EBPβ binding at the CD14 locus in primary macrophages, PU.1−/− cells and PUER cells without and 1 h after tamoxifen treatment. Top to bottom: PU.1 in macrophages, PUER cells after or without 1 h tamoxifen treatment and PU.1−/− cells (blue - dark to light), and C/EBPβ in PU.1−/− cells, PUER cells without or after 1 h tamoxifen treatment, and macrophages (brown to red). (F) Top motif enriched in the vicinity of gained C/EBPβ sites following activation of the PU.1-ER fusion protein compared to the constitutively bound sites identified by de novo motif analysis. The fraction of C/EBPβ peaks containing at least one instance of the motif within 100 bp of the peak center in gained and constitutively bound peaks is given to the right and in parentheses, respectively.
Figure 4
Figure 4. PU.1 and cooperating factors are required for histone modifications associated with enhancers
(A) Same plot of PU.1 peaks as in 1C, peaks are colored red if the surrounding region (±500 bp) contains more than 4-fold more H3K4me1 tags in macrophages than in B cells. Peak positions fulfilling the same criteria for B cells versus macrophages are colored blue. (B) Cell type-matched cumulative normalized H3K4me1 ChIP-Seq and input sequencing tag counts per base pair are shown around distal peak positions (>3 kb from a TSS) of PU.1 (B cells and macrophages), C/EBPβ (macrophages), Oct-2 (B cells). (C) Temporal and spatial relationships of PU.1, C/EBPβ and H3K4me1 in PUER cells at different time points. Six kb-wide regions centered on genomic sites that gained PU.1 > 6-fold after 1 h tamoxifen were clustered according to their PU.1, C/EBPβ and H3K4me1 ChIP-Seq profiles at 0 h, 1 h or 24 h of tamoxifen treatment. Shown are representative sections (10 % of each group) of the resulting heat map. (D) Average nucleosome positions centered on induced PU.1 peaks before and after 1 h tamoxifen treatment as defined by MNase-Seq. (E) Sequence motifs associated with promoter-distal H3K4me1-marked 1 kb regions in macrophages. The top 4 motif results from de novo motif analysis are shown. The fraction of H3K4me1 marked regions containing at least one instance of each motif within 500 bp of the peak center is given to the right of the motif with the expected frequency of the motif in random regions in parentheses. (F) Pie chart depicting the overlap of PU.1 and C/EBPβ within 1 kb of focal H3K4me1 ChIP-Seq peaks. (G) Association of p300 with PU.1 and C/EBPβ co-bound sites. Percentages of transcription factor-bound regions co-bound by p300 are given. Absolute numbers were: PU.1 only, 21223 total, Co-bound by p300: 1921; C/EBPβ only, 20481 total, 1274 with p300; PU.1 & C/EBPβ, 13874 total, 4230 with p300. Peak positions for p300 were determined by analysis of p300 ChIP-Seq data for resting bone marrow-derived macrophages (Ghisletti et al., 2010).
Figure 5
Figure 5. PU.1 establishes part of the LXRβ cistrome and H3K4me1 pattern around LXRβ binding sites in macrophages, but not vice versa
(A) Genomic annotation of high confidence LXRβ binding sites defined by ChIP-Seq of biotin-tagged LXRβ in RAW264.7 murine macrophages. (B) De novo motif analysis of LXR-bound regions in macrophages. The fraction of peaks containing at least one instance of each motif within 100 bp of the peak center is given to the right of the motif with the expected frequency of the motif in random regions in parentheses. (C) Macrophage and B cell PU.1 binding as in Figure 1C; PU.1 peak positions are colored green if an LXRβ peak is located within 100 bp of a PU.1 peak. (D) UCSC browser image of the LXR target gene locus ABCG1 depicting coordinate binding of LXR and PU.1, together with the associated H3K4me1 signature. The 2.5-fold magnified BirA control track denotes sequencing of a pulldown from formaldehyde-fixed, BirAtransgenic RAW264.7 devoid of BLRP-tagged proteins. (E) Cumulative H3K4me1 levels in PU.1−/− myeloid progenitors (PU.1−/−), PUER cells treated for 24 h with tamoxifen (PUER 24 h) or mouse liver as control (liver H3K4me1 ChIP-Seq data from (Robertson et al., 2008)) around LXRβ peak positions defined in RAW264.7 macrophages. (F) LXR binding at the indicated loci in PU.1−/− cells vs. PUER cells treated with tamoxifen for 24 h as compared by ChIP-qPCR. Values represent fold enrichment over ChIP with IgG control antibody. (G) Cumulative H3K4me1 levels in bone marrow macrophages derived from LXRα−/−/ LXRβ−/−mice (LXR DKO BMDM), wild type mice (WT BMDM) or liver (Robertson et al., 2008) as non-macrophage control, around LXRβ peak positions defined in RAW264.7 macrophages. (H) Scatter plot depicting PU.1 ChIP-Seq tag counts (log2) within 200 bp of combined genomic PU.1 peak positions defined in bone marrow-derived macrophages from wild type mice (WT MФ) or LXRα−/−/ LXRβ−/− mice (LXR DKO MФ). Genomic PU.1 peaks within 100 bp of an LXRβ peak in RAW264.7 macrophages are colored red.
Figure 6
Figure 6. PU.1 is necessary for LXR- and TLR4-dependent gene expression in macrophages
(A) Relative enhancer activity of indicated LXR-bound regions inserted upstream of a minimal TK promoter driving luciferase expression in RAW264.7 macrophages. Data are presented as average fold change (±SEM) comparing 24 h treatment with 1 µM GW3965 vs. vehicle (DMSO) of three independent experiments performed in triplicate. (B) PUER cells were treated with tamoxifen or vehicle (EtOH) for 24 h, then with 1 µM GW3965 or vehicle (DMSO) for an additional 24 h. Average mRNA expression fold changes (±SEM) of the indicated genes as determined by quantitative real-time RT-PCR (qRT-PCR) comparing GW3965 versus vehicle in two independent experiments are shown, all expression changes were statistically significant (p<0.05, t-test). (C) Overlap of vicinal PU.1, C/EBPβ and H3K4me1 peaks in resting macrophages associated with genes that are induced more than 3-fold by KLA at 6 h. (D) PUER cells were treated with vehicle (DMSO) or tamoxifen for 24 h, then with vehicle (PBS) or KLA for 6 h. Messenger RNA levels for the indicated TLR4-responsive genes were determined by qRT-PCR and displayed as a heat map with absolute expression values ranging from low (blue) to high (red) compared to the mean level of expression for each gene. Asterisks denote genes whose KLA response is PU.1-dependent. (E) Schematic depicting the proposed model.
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