Figure 4
Figure 4. Synergism between PU.1 gene distal cis elements. (A) The top part shows a MVista representation of sequence conservation across ∼ 50 kb of the region of mouse chromosome 2 that harbors the PU.1 locus. The conservation panels correspond to, from top to bottom, mouse/human, mouse/dog, and mouse/opossum alignments. The conservation plots show regions with ≥ 50% of conservation (indicated on the y-axis). Exons are shown in blue, noncoding conserved regions in pink. Positions of nonconserved sequence homology regions are indicated. Precise locations of the previously unidentified homology regions are −12 kb = 12.3 kb, −10 kb = 10.5 kb, −9 kb = 9.5 kb, and −8 kb = 7.8 kb upstream of the PU.1 TSS. The bottom part shows a University of California Santa Cruz (UCSC) genome browser screenshot of DNaseI hypersensitivity site sequencing profiles across the mouse PU.1 locus in bone marrow–derived macrophages and splenic B cells. Relative enrichments are indicated at the y-axes; ++ and + on the right indicates strong or intermediate PU.1 expression, respectively. (B) ChIP-chip mapping of H3K9ac across the murine PU.1 locus with the use of custom-designed genomic tiling arrays. The y-axis represents the log 2 enrichment of ChIPed DNA over input DNA, whereas the x-axis depicts 50-kb genomic sequence spanning the mouse PU.1 locus. At the top, positions of the indicated DHSs and the covered genes are marked. The genomic coordinates (in base pairs) of mouse chromosome 2 covered on the array are shown at the bottom. (C) Raw p300 ChIP-sequencing data across the murine PU.1 locus in bone marrow–derived macrophages showing p300 occupancy exclusively at the indicated DHSs. (D) Luciferase activity assay of RAW264.7 macrophages stably transfected with the indicated constructs. Multiple independent clones were assayed for each construct, and luciferase activity was normalized to the plasmid copy number as determined by Southern blotting. Shown are the means ± SDs of ≥ 6 independent clones for each construct. The average activity of the PU.1 promoter only construct was set to 1. The inlet shows a higher magnification of the values of the indicated constructs; n.s. indicates not significant. (E) Luciferase activity assay of Namalwa B cells stably transfected with the indicated constructs. Shown are the mean ± SD of 3 independent cell pools for each construct.

Synergism between PU.1 gene distal cis elements. (A) The top part shows a MVista representation of sequence conservation across ∼ 50 kb of the region of mouse chromosome 2 that harbors the PU.1 locus. The conservation panels correspond to, from top to bottom, mouse/human, mouse/dog, and mouse/opossum alignments. The conservation plots show regions with ≥ 50% of conservation (indicated on the y-axis). Exons are shown in blue, noncoding conserved regions in pink. Positions of nonconserved sequence homology regions are indicated. Precise locations of the previously unidentified homology regions are −12 kb = 12.3 kb, −10 kb = 10.5 kb, −9 kb = 9.5 kb, and −8 kb = 7.8 kb upstream of the PU.1 TSS. The bottom part shows a University of California Santa Cruz (UCSC) genome browser screenshot of DNaseI hypersensitivity site sequencing profiles across the mouse PU.1 locus in bone marrow–derived macrophages and splenic B cells. Relative enrichments are indicated at the y-axes; ++ and + on the right indicates strong or intermediate PU.1 expression, respectively. (B) ChIP-chip mapping of H3K9ac across the murine PU.1 locus with the use of custom-designed genomic tiling arrays. The y-axis represents the log 2 enrichment of ChIPed DNA over input DNA, whereas the x-axis depicts 50-kb genomic sequence spanning the mouse PU.1 locus. At the top, positions of the indicated DHSs and the covered genes are marked. The genomic coordinates (in base pairs) of mouse chromosome 2 covered on the array are shown at the bottom. (C) Raw p300 ChIP-sequencing data across the murine PU.1 locus in bone marrow–derived macrophages showing p300 occupancy exclusively at the indicated DHSs. (D) Luciferase activity assay of RAW264.7 macrophages stably transfected with the indicated constructs. Multiple independent clones were assayed for each construct, and luciferase activity was normalized to the plasmid copy number as determined by Southern blotting. Shown are the means ± SDs of ≥ 6 independent clones for each construct. The average activity of the PU.1 promoter only construct was set to 1. The inlet shows a higher magnification of the values of the indicated constructs; n.s. indicates not significant. (E) Luciferase activity assay of Namalwa B cells stably transfected with the indicated constructs. Shown are the mean ± SD of 3 independent cell pools for each construct.

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