Figure 6
Figure 6. The −12-kb enhancer requires C/EBPα and cross-interaction with the URE. (A) Venn diagram showing the global numbers of DHSs in macrophages and B cells. The condition for peak overlap was that center of the peak in the first dataset should lie within boundaries of a peak in the second dataset. (B) Numbers of PU.1-occupied DHSs in macrophages and B cells. Peak identification in panels A and B used a false discovery threshold of 0.1%. (C) Total number of PU.1-occupied DHSs unique to macrophages, unique to B cells, or common to both cell types found in promoter-proximal (within ± 500 bp from TSS) and distal (> 500 bp from TSS) genomic regions. (D) DNaseI in vivo footprinting of the −12-kb enhancer in C10 cells before (−E2) or after (+E2) 72 hours of β-estradiol induction, B cell, and macrophages. In vitro DNaseI-treated DNA served as a control, and a G-reaction was used to indicate genomic positions relative to the TSS. Samples were assayed with forward (left) and reverse (right) primers for the −12-kb enhancer, as well as with primers for the rDNA genes to control for equal digestion. Hyperreactivities are marked by closed circles and protections by open circles. (E) UCSC Genome browser image depicting C/EBPα ChIP-seq tag counts (normalized to 107 sequence tags) (log2) at the PU.1 locus in macrophages and B cells. (F) Luciferase assay in RAW264.7 macrophages stably transfected with the indicated constructs. Mutations in PU.1-binding motifs are indicated by blue or orange dots, respectively. Shown are the mean values ± SDs of 3 independent cell bulks for each construct. Activity of the wild-type construct was set as 100%. (G) ChIP-qPCRs at the −12-kb DHS in URE−/− and URE+/+ myeloid cell lines (see “Methods”) after retroviral transduction with a tamoxifen-responsive C/EBPα construct. Shown is PU.1 occupancy (black bars) and H3K9ac (gray bars) before and after 6 days of tamoxifen induction. (H) ChIP-qPCRs depicting H3K9ac levels at the exogenous −12-kb DHS of the indicated constructs in stably transfected RAW264.7 cells. Blue dots indicate mutation in PU.1 binding site; orange dots, mutation in C/EBP binding site. All values were normalized to integrated plasmid copy numbers.

The −12-kb enhancer requires C/EBPα and cross-interaction with the URE. (A) Venn diagram showing the global numbers of DHSs in macrophages and B cells. The condition for peak overlap was that center of the peak in the first dataset should lie within boundaries of a peak in the second dataset. (B) Numbers of PU.1-occupied DHSs in macrophages and B cells. Peak identification in panels A and B used a false discovery threshold of 0.1%. (C) Total number of PU.1-occupied DHSs unique to macrophages, unique to B cells, or common to both cell types found in promoter-proximal (within ± 500 bp from TSS) and distal (> 500 bp from TSS) genomic regions. (D) DNaseI in vivo footprinting of the −12-kb enhancer in C10 cells before (−E2) or after (+E2) 72 hours of β-estradiol induction, B cell, and macrophages. In vitro DNaseI-treated DNA served as a control, and a G-reaction was used to indicate genomic positions relative to the TSS. Samples were assayed with forward (left) and reverse (right) primers for the −12-kb enhancer, as well as with primers for the rDNA genes to control for equal digestion. Hyperreactivities are marked by closed circles and protections by open circles. (E) UCSC Genome browser image depicting C/EBPα ChIP-seq tag counts (normalized to 107 sequence tags) (log2) at the PU.1 locus in macrophages and B cells. (F) Luciferase assay in RAW264.7 macrophages stably transfected with the indicated constructs. Mutations in PU.1-binding motifs are indicated by blue or orange dots, respectively. Shown are the mean values ± SDs of 3 independent cell bulks for each construct. Activity of the wild-type construct was set as 100%. (G) ChIP-qPCRs at the −12-kb DHS in URE−/− and URE+/+ myeloid cell lines (see “Methods”) after retroviral transduction with a tamoxifen-responsive C/EBPα construct. Shown is PU.1 occupancy (black bars) and H3K9ac (gray bars) before and after 6 days of tamoxifen induction. (H) ChIP-qPCRs depicting H3K9ac levels at the exogenous −12-kb DHS of the indicated constructs in stably transfected RAW264.7 cells. Blue dots indicate mutation in PU.1 binding site; orange dots, mutation in C/EBP binding site. All values were normalized to integrated plasmid copy numbers.

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