PMC

(A) Fetal livers were isolated from E13.5–14 day old embryos from WT (URE^+/+), heterozygous (URE^+/−), and homozygous (URE^−/−) animals. Distinct erythroid populations were identified by staining with TER119 and CD71, with representative stainings shown. The percent of R1 cells present from two animals from each genotype was averaged and is displayed as a bar graph. * t-test<0.05, ** t-test<0.01 compared to URE^+/+. (B) Apoptosis was analyzed by Annexin-V staining of R1 cells. The percentage of Annexin-V positive cells is shown as an average of two animals from each genotype. *t-test<0.05 compared to URE^+/+.

(A) ChIP-Seq peaks were identified as described in . A point represents the log₂ of the number of sequence reads from ES-EP and MEL cells contained in a peak. Each peak was assigned to one of two categories based on the ratio of the number of reads in the two cell types. Peaks within a ratio ≤5 are classified as shared between both cell types (represented in light blue), whereas peaks with a ratio >5 are enriched in a cell specific manner (represented in red for ES-EP or dark blue for MEL cells). (B) Venn diagrams illustrate the number of peaks (left) or genes with a peak within 2 kb of TSSs (right) in ES-EP and MEL cells (colors as A).

(A) and (B) IPA analysis was performed as described in on PU.1 target genes in both ES-EP and MEL cells. The PI3K/Akt and ERK/MAPK signaling pathways were identified as being significantly over-represented among those genes. The left part of each panel depicts all of the components of the indicated IPA pathway. Components denoted with a filled yellow circle indicate that the gene for that component is associated with a PU.1 ChIP-Seq peak in both ES-EP and MEL cells. To the right of each pathway is shown the results of gene expression analysis of the indicated genes in early erythroid progenitors derived from wild-type embryos relative to PU.1 low embryos.

(A) and (B) Signal tracks of PU.1 ChIP-Seq data from MEL cells (blue) and ES-EP (red) in the vicinity of the genes depicted schematically (black) using IGB. Myb, Fli1, Gfi1b, and EpoR are transcribed from the negative strand (right to left), whereas Sfpi1 (PU.1) and Klf1 are expressed in the sense direction (left to right). Input DNA controls are also shown for both cell types. (C) and (D) qChIP validations of PU.1 occupancy at the regions highlighted in yellow in (A) and (B). qChIP was performed as described in with chromatin from MEL cells (left) and ES-EP (right) with primers described in . A HA antibody was used as an isotype control. Standard deviations were calculated from triplicate PCR reactions. Similar results were obtained with at least two independent chromatin preparations.

(A) Sample signal tracks of PU.1 ChIP-Seq data from MEL cells and ES-EP are shown for an ∼500 kb region of chromosome 15 in the Integrated Genome Browser (IGB) (Affymetrix), with the y-axis representing the number of reads. Input DNA controls are also shown for both cell types. (B) Sequence logos for the enriched motifs within PU.1 ChIP-Seq peaks from MEL (left) and ES-EP (right) cells, derived from MEME motif analysis (see ). (C) The distance between each PU.1 ChIP-Seq peak and the TSS within 2 kb was computed and the results are binned and plotted for MEL (top left) and ES-EP (bottom left). Peaks were further annotated by their genomic locations with respect to current gene annotation (right) and classified as proximal promoter (+/−2 kb of TSS), 3′ end of gene (+/−2 kb of TES), gene body (between +2 kb of TSS and −2 kb of TES), miRNA promoters, or otherwise intergenic regions.

(A) PU.1 target genes with a 2-fold expression difference (as measured by Affymetrix gene array) between MEL and ES-EP cells were identified. The log₂ of the number of PU.1 ChIP-Seq reads within peaks of these genes' promoters are computed and plotted here. Blue circles represent genes for which expression is higher in MEL cells, while red circles represent genes for which expression is higher in ES-EP. The diameter of each circle is proportional to the degree of difference in gene expression between the two cell types. (B) The expression levels of genes with and without PU.1 bound at proximal promoters in MEL and ES-EP are shown as boxplots, with the lower and upper sides of the box representing the lower and upper quartiles, respectively, and the line in the box representing the median expression value. (C) The log₂ratio of the fold change in gene expression from early erythroid progenitors derived from wild-type embryos compared with PU.1 low embryos for genes bound by PU.1 in MEL (left) or ES-EP (right) are plotted as a function of the number of reads associated with the peak(s) corresponding to each gene. The positions of the values for Klf1, EpoR, Gfi1b, Myb, Fli1, and Sfpi1 in the plots are labeled.

(A) qChIP was performed for PU.1 as described in with chromatin from 32D cells using primers described in . An HA antibody was used as an isotype control. The same primers were used to show that PU.1 binds close to these genes in MEL cells and ES-EP (). The myogenin gene serves as a negative control. Standard deviations were calculated from triplicate PCR reactions. Similar results were obtained with at least two independent chromatin preparations. (B) Expression data from PU.1^−/− cells stimulated to differentiate into macrophages by the restoration of PU.1 and HSC from PU.1 knockdown mice were used to compare with PU.1 dependent gene regulation in early erythroid progenitors from PU.1 knockdown mice. Of the PU.1 bound genes in MEL and ES-EP that displayed a ≥1.5-fold change in expression, 308 were upregulated and 209 were downregulated. 162 and 265 of these PU.1 dependent genes were annotated in the other 2 datasets. Venn diagrams display the comparisons between the three datasets of these upregulated genes (left) and downregulated genes (right).