1. Volume 56, Issue 6, Pages 749-762 (December 2014)
A Negative Feedback Loop of Transcription Factors Specifies Alternative Dendritic Cell Chromatin States 
Chamutal Bornstein, Deborah Winter, Zohar Barnett-Itzhaki, Eyal David, Sabah Kadri, Manuel Garber, Ido Amit 
Molecular Cell 
Volume 56, Issue 6, Pages (December 2014)
DOI: /j.molcel
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2. Molecular Cell 2014 56, 749-762DOI: (10.1016/j.molcel.2014.10.014)
Copyright © 2014 Elsevier Inc. Terms and Conditions

3. Figure 1 Gene Expression Dynamics in moDC and pDC
(A) qPCR analysis for a representative set of moDC-specific genes (top) and pDC-specific markers (bottom) of untreated moDC and pDC. Values are represented as fold expression to the corresponding value of moDC.
(B) qPCR analysis for a representative set of inflammatory genes (left) and antiviral genes (right) of stimulated moDC and pDC with 100 ng/ml LPS or 5 μM CPG-A for 0, 2, 4, and 6 hr. Values represented as fold expression to the corresponding value at t = 0. Data are mean ± SD from triplicate experiments.
(C) Clustered heatmap of genome-wide expression of moDC and pDC at 0, 1, 2, 4, and 6 hr following stimulation (100 ng/ml LPS, 100 ng/ml PAM3CSK [PAM], 5 μM CpG A, 5 μM CpG B). Clusters represent moDC-specific genes (I), pDC-specific genes (IV), and moDC-specific inflammatory-response genes; upregulated following LPS and PAM stimuli (II and III), pDC-specific antiviral genes; upregulated following CPG-A stimulus (VII). Gene annotation enrichment analysis is detailed in Table S3. Selected genes are highlighted on the right. See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
pDC and moDC Are Distinguished by Thousands of Differential Enhancers
(A) A volcano plot depicting differentially expressed genes in pDC and moDC. The pDC and moDC specific genes (Experimental Procedures; Table S1) are superimposed in purple and orange, respectively. Some of the characteristic pDC and moDC genes are indicated. p values are calculated using a Student’s t test.
(B) Venn diagram showing differential and common H3k4me1 peaks between pDC and moDC (Experimental Procedures).
(C) Representative Integrative Genomics Viewer (IGV) tracks in moDC-specific genes (F10, Tlr4, and Tlr2) and pDC-specific genes (Ly6d, Siglech, and Flt3) loci showing ChIP-Seq for H3K4me1 and H3K27ac modifications, as well as ATAC-seq signal in moDCs (orange), pDCs (purple), and in vivo pDCs (dark purple).
(D) Global quantification of H3K4me1 signal intensity (cumulative distribution) in differentially expressed genes (Experimental Procedures; Figure 2A; Table S1) of moDCs (orange) and pDCs (purple). Differences between each pair of samples are significant with p < 10−50 (Kolmogorov-Smirnov test). See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3 Irf8 Binding Is Enriched in pDC-Specific Enhancers
(A) qPCR analysis of the TFs Pu.1, Cebpb, and Irf8 in untreated moDCs and pDCs. Expression values are represented as fold expression to the corresponding value of moDC, and are mean ± SD from triplicate experiments.
(B) Representative IGV tracks in moDC-specific genes (F10, Cxcl2, and Tlr2) and pDC-specific gene (Ly6d, Flt3, and Siglech) loci for H3K4me1 modification and TFs binding in moDCs (orange) and pDCs (purple).
(C) Percentage of Pu.1, Cebpb, and Irf8 binding in cell-specific enhancers (H3K4me1 peaks). Enrichments in binding of Irf8 in pDCs, Cebpb in moDCs, and Pu.1 in both cell-type-specific enhancers are significant (hypergeometric test, p < 10−10).
(D) Percentage of Irf8, Cebpb, and Pu.1 binding associated to cell-specific gene regions. Enrichments of binding of Irf8 in pDC-specific genes, Cebpb in moDC-specific genes, and Pu.1 in both cell types are significant with hypergeometric test, p < 10−5.
(E) Venn diagram showing unique and overlapping binding of Pu.1, Cebpb, and Irf8 (hypergeometric test, p < 10−50). Some of the characteristic pDC and moDC genes are indicated. See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
The Balance between Cebpb and Irf8 Expression Regulates moDC and pDC States
RNAi perturbation and overexpression assays for Cebpb and Irf8 were performed followed by gene expression measurements.
(A and B) DCs were perturbed with control (sh-control) or Irf8 (sh-Irf8) in pDCs (A) or Cebpb (sh-Cebpb) in moDCs (B). Following knockdown, cells were either treated or not treated (NT) with 100 ng/ml LPS for 4 hr. qPCR analysis for cell-specific markers is shown. Values are represented as fold expression relative to the untreated sh-control sample, and are mean ± SD from triplicate experiments.
(C) Irf8 overexpression in moDCs was performed by addition of doxycycline (dox) to dox-Irf8-infected moDCs after final moDC differentiation. Shown are qPCR analysis for cell-specific markers with (Irf8) or without dox (control), either not treated (NT) or following 100 ng/ml LPS stimulation for 4 hr.
(D) Cebpb overexpression in pDC was performed by infection of Cebpb gene fused to estrogen receptor (Cebpb-ER). Activation of Cebpb-ER protein was induced by 4-hydroxytamoxifen (4OHT) after final pDC differentiation. qPCR analysis was done for cell-specific markers with (Cebpb) or without 4OHT (control). Cells were either not treated (NT) or stimulated with 100 ng/ml LPS for 4 hr.
(E) Volcano plot depicting gene expression following Irf8 knockdown (sh-Irf8) in pDCs (left) and Cebpb knockdown (sh-Cebpb) in moDCs (right). The pDC- and moDC-specific genes (Experimental Procedures; Table S1) are superimposed in purple and orange, respectively. Some of the characteristic pDC and moDC genes are indicated. p values are calculated using a Student’s t test.
(F) Unsupervised NMF analysis (Experimental Procedures) for two classes was carried out for genome-wide expression of control pDCs and moDCs as well as RNAi and overexpression perturbations listed above. First component (right) marks the “moDC state,” second component (left) marks the “pDC state.” See also Figures S4 and S7.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
Balance between Cebpb and Irf8 Regulates the moDC and pDC Enhancer Landscapes
ChIP-seq for H3K4me1 was performed for control (sh-control), Cebpb knockdown (sh-Cebpb), or Irf8 knockdown (sh-Irf8) in moDCs and pDCs, respectively.
(A) Representative IGV tracks of moDC-specific gene (F10 and Cxcl2) and pDC-specific gene (Ly6d and Siglech) loci showing H3K4me1 modification in control (blue) or Irf8 knockdown (red) in pDC.
(B) Representative IGV tracks of moDC-specific gene (F10 and Cxcl2) and pDC-specific gene (Ly6d and Siglech) loci showing H3K4me1 signal in control (blue) or Cebpb knockdown (red) in moDC.
(C) Global quantification of H3K4me1 signal distribution in cell type specific genes (Experimental Procedures; Table S1) in control (sh-control) (blue) and Irf8 knockdown (sh-Irf8) (red) in pDCs. Differences are significant with Kolmogorov-Smirnov test p < 10−16.
(D) Global quantification of H3K4me1 signal distribution in cell type specific genes (Experimental Procedures; Table S1) in control (sh-control) pDCs (blue) and Cebpb knockdown (sh-Cebpb) (red) in moDCs. Differences are significant with Kolmogorov-Smirnov test p < 10−6. See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
A Double-Negative Feedback Loop of Irf8 and Cebpb Establishes the Differential Chromatin States of moDC and pDC
(A and B) qPCR analysis for the Irf8 (A) or Cebpb (B) genes in control (sh-control) or Cebpb knockdown (sh-Cebpb) in moDCs, and of control (sh-control) or Irf8 knockdown (sh-Irf8) in pDCs (B) either stimulated or not treated (NT) with 100 ng/ml LPS for 4 hr.
(C) qPCR analysis for the Cebpb gene in control or dox-induced Irf8 overexpression moDC, either not treated (NT) or stimulated with 100 ng/ml LPS for 4 hr.
(D) qPCR analysis for Irf8 gene in control or 4OHT activated Cebpb-ER in pDC, either not treated (NT) or stimulated with 100 ng/ml LPS for 4 hr. All qPCR values are represented as fold expression to the corresponding value of control sample, and are mean ± SD from triplicate experiments.
(E) IGV tracks in Cebpb and Irf8 genes loci showing biological replicates of Cebpb and Irf8 TFs binding in moDCs (orange) and pDCs (purple). Rectangles mark binding region for each TF.
(F and G) pDCs were infected with either control or Cebpb-ER constructs. Infected cells were either untreated or treated with 4-hydroxytamoxifen (4OHT) constitutively or pulsed with 4OHT followed by washing and addition of fresh media. Cell protein extracts were analyzed by western blot with the indicated antibodies (F). Gene expression levels were assessed by qPCR for both not treated (NT) and stimulated with 100 ng/ml LPS for 4 hr (G).
(H and I) moDC were infected with dox-inducible Irf8 fused to HA tag (Irf8-HA). Infected cells were either untreated or treated with doxycycline (dox) constitutively or pulsed with dox followed by washing and addition of fresh media. Cell protein extracts were analyzed by western blot with the indicated antibodies (H). Gene expression levels were assessed by qPCR for both not treated (NT) and stimulated with 100 ng/ml LPS for 4 hr (I). See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7
A Negative Feedback Loop of Cebpb and Irf8 Specifies Alternative Fate
(A) A model depicting the double negative feedback loop of Irf8 and Cebpb and its impact on the differential chromatin states of moDC and pDC.
(B) Relative mRNA expression of Cebpb and Irf8 during hematopoietic differentiation. Dashed arrows mark ex vivo moDCs and pDCs. Expression of Cebpb and Irf8 is relative to ex vivo differentiated moDCs and pDCs, respectively.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions