Fig 1.
Identification of c-Myb footprints.
(A) Workflow for identification of c-Myb footprints. (B) A pie chart representing the number of c-Myb footprints identified compared to the total number of footprints in K562 cells. (C) An illustration from Motiflab showing a c-Myb motif located at the start of the first intron of the gene FKBP5 that overlaps with a DNase I footprint with a high conservation value in K562 cells, illustration modified. The coordinates for the c-Myb footprint is shown above the illustration, and to the right are the signal intensity for the DNase I datasets, in addition to a conservation score. (D) The binding motif enriched in c-Myb footprints in K562 cells. (E) Graph of the presence of c-Myb footprints and the distances to the 100 most regulated genes upon KD in K562 cells (dots) or a selection of 100 random genes (squares), an average of ten repetitions. Zero base pair indicates that the c-Myb footprints are found inside the gene body (F) A c-Myb footprint at the TSS of GRSF1 gene mapped in all six cell-types analysed. Coordinates for c-Myb footprint are shown above, and to the left are the signal intensity for DNase I datasets. (G) Position of c-Myb footprints, and random selections of DNase I footprints and c-Myb motifs, respectively, around ENSEMBL annotated TSS in K562 cells. (H) Distribution of c-Myb footprints at annotated genes, promoters and intergenic regions in K562 cells. *Overlapping significantly higher with c-Myb footprints than expected by random sampling of K562 DNase I footprints (p' < 5x10-2).
Fig 2.
c-Myb enhances transcription from genomic elements with c-Myb footprints.
Luciferase-based reporter assay to study the responsiveness of genomic regions, with one or more c-MYB footprints mapped. (A) Representative Immunoblot of CV1 cells transfected with reporter plasmid and-/+ c-Myb. (B) Map of the pGL4.26 vector used for the luciferase assays. The grey box illustrates the genomic fragment containing a c-Myb footprint or control region. The black box illustrates the minimal promoter. (C) Positive control with three MREs. (D-E) Negative controls, (F-K) genomic loci identified to contain c-Myb footprints. The upper panels show the genomic region in the UCSC browser (hg19) presenting DNase I signals, c-Myb footprints and oligos for selected region. The coordinates for the c-Myb footprint are shown above the illustration. The values are the average from three independent experiments-/+ SEM.
Fig 3.
Validation of c-Myb footprints by DAMID.
(A) Expression of the Flag-c-Myb-Dam construct. K562 cells were transfected with a plasmid encoding the c-Myb-Dam together with the activator plasmid pVgRXR and treated with 2 uM of Ponasterone A. Expression of the fusion construct was detected by immunoblotting against Flag-tag. (B-J) Association of the control Dam and c-Myb-Dam at specific loci containing one or more c-Myb footprints quantified with qPCR and normalised to Dam. The upper panels show the genomic region in the UCSC browser (hg19) presenting DNase I signals, c-Myb footprints and oligos for qPCR. The coordinates for the c-Myb footprint are shown above the illustration. The values represent the average from two independent cell lines-/+ SEM.
Fig 4.
c-Myb footprints co-localises with active histone marks and co-regulatory TFs at c-Myb-regulated genes.
(A-D) The number of c-Myb footprints (grey), which co-localize with ChIP-seq peaks for the active histone marks H3K4me3, H3K4me1, H3K9ac, (green) and the repressive mark H3K27me3 (red) in K562 cells. *Significantly different than expected from random sampling K562 DNase I footprints (p', > 4x10-4). (E) Suggested co-regulatory TFs for c-Myb in K562 cells. Green and red denotes factors in the positive and negative set respectively. Next to each factor, the normalised ratio for the co-localisation with c-Myb footprints at positively or negatively regulated genes is displayed. The distance between each factor and c-Myb is calculated as a measure of the normalized ratio, as described in Methods.
Fig 5.
c-Myb controls differentiation and cell development.
(A) Gain and loss of c-Myb footprints between CD34+ cells and CD20+ and Th1 cells, respectively. To the right are the top enriched functions for genes nearby c-Myb footprints specific for either CD20+ cells or Th1 cells as compared to CD34+ cells. For the full list of enriched functions, see S9 Fig. (B) Functional analysis of c-Myb footprints in all six cell-types. The functional analysis was made with GREAT [61].