Integrated genome-wide chromatin occupancy and expression analyses identify key myeloid pro-differentiation transcription factors repressed by Myb

Abstract

To gain insight into the mechanisms by which the Myb transcription factor controls normal hematopoiesis and particularly, how it contributes to leukemogenesis, we mapped the genome-wide occupancy of Myb by chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-Seq) in ERMYB myeloid progenitor cells. By integrating the genome occupancy data with whole genome expression profiling data, we identified a Myb-regulated transcriptional program. Gene signatures for leukemia stem cells, normal hematopoietic stem/progenitor cells and myeloid development were overrepresented in 2368 Myb regulated genes. Of these, Myb bound directly near or within 793 genes. Myb directly activates some genes known critical in maintaining hematopoietic stem cells, such as Gfi1 and Cited2. Importantly, we also show that, despite being usually considered as a transactivator, Myb also functions to repress approximately half of its direct targets, including several key regulators of myeloid differentiation, such as Sfpi1 (also known as Pu.1), Runx1, Junb and Cebpb. Furthermore, our results demonstrate that interaction with p300, an established coactivator for Myb, is unexpectedly required for Myb-mediated transcriptional repression. We propose that the repression of the above mentioned key pro-differentiation factors may contribute essentially to Myb's ability to suppress differentiation and promote self-renewal, thus maintaining progenitor cells in an undifferentiated state and promoting leukemic transformation.

Figure 1.

Expression profiling analysis of Myb regulated genes. (A) Schematic diagram of the experimental design. (B) The ER-Myb fusion transcript is expressed at an almost identical level to endogenous Myb mRNA in c-Kit^high bone marrow progenitors. Expression was measured by qRT–PCR and normalized to Hprt. The level of the ER-Myb fusion was set to 1. Data are presented as mean ± SD. (C) Hierarchical clustering of normalized expression levels of class A Myb regulated genes, including 352 Myb-activated and 460 repressed genes. The heat map of relative log₂-transformed expression levels during the β-E₂ withdrawal and re-addition time course is shown; each row represents a single gene. Simplified arrow diagrams of corresponding kinetic profiles are shown next to each cluster tree. (D) The majority of candidate Myb regulated genes were validated in both ERMYB cells and Myb transduced primary bone marrow cells.

Figure 2.

Global identification of MBRs in vivo. (A) Both anti-ER antibody and anti-Myb sera worked in ChIP assays in ERMYB cells: the ER-Myb fusion protein occupied a known Myb binding site in Mpo promoter (Mpo -0.32 kb). B2m TSS region served as a negative control. (B) Representation of ChIP-Seq sequencing reads (raw data) across loci of two established Myb targets—Mpo and Bcl2. (Arrows) ChIP-Seq peak locations relative to TSS of the respective gene (kb). (C) Identified Myb motif shares the AACNG core binding consensus sequence with the previously reported Myb binding consensus in Transfac database. (D) Distribution of MBRs relative to the nearest TSS (kb). Regions 5′ to the TSS are indicated as negative values on the x-axis. (E) Distribution of MBRs relative to Refseq gene features.

Figure 3.

Validation of ChIP-Seq findings. (A) All 23 high-confidence MBRs were validated in ERMYB cells. Four of 5 MBRs (denoted by ^Δ) which just failed EdgeR P-value filter were also validated. Four irrelevant regions were included as negative controls. (B) Twenty-five of 27 MBRs validated using anti-ER antibody (A) were further validated using anti-Myb sera. Data are presented as mean ± SD. Background binding level represents (mean + 1.64 SD) of α-ER or α-Myb signals of four irrelevant control regions. ‘Asterisk’ denotes significant decreases in Myb occupancy upon β-E₂ withdrawal (Student’s t-test, P < 0.05). (C) Twenty of 21 conserved MBRs were further validated in HL-60 cells using pooled MYB antibodies. Background binding level represents (mean + 1.64 SD) of α-MYB signals of three control regions.

Figure 4.

Integration of expression profiling and genome occupancy data. (A) Direct Myb target genes were defined as genes bound and regulated by Myb, including 418 Myb-activated and 375 repressed targets. (B) Significant overlap between Myb bound genes, Myb regulated genes in ERMYB cells and differentially expressed genes in MYB^KD THP-1 cells.

Figure 5.

MBRs were enriched near Myb target genes. (A) Direct Myb targets (classes A and B) generally have significantly more MBRs than those Myb bound but not regulated or not expressed genes (class C). *P < 0.05 (Wilcoxon rank sum test). (B) MBRs are enriched in the proximity of TSS of Myb activated genes (classes A and B). Myb repressed genes appeared to have more MBRs at locations relatively distal to TSS. Average numbers of MBR per gene of each microarray class were plotted against their distances relative to the nearest TSS in 1 kb bins up to 20 kb away from TSS.

Figure 6.

p300 was required for both activation and repression by Myb. (A) p300 occupancy was detected at 25 of 27 MBRs and the occupancy generally decreased when Myb was inactivated by β-E₂ withdrawal. Data are presented as mean ± SD. IgG background represents the (mean + 1.64 SD) of IgG signals across all regions. ‘Asterisk’ denotes significant decrease in Myb occupancy upon β-E₂ withdrawal (Student’s t-test, P < 0.05). (B) L302A.CT3-Myb did not maintain the repression or activation of direct Myb target genes by CT3-Myb. Expression of 53 direct Myb target genes was shown for L302A.CT3-Myb transduced bone marrow cells (y-axis, log2 scale) relative to CT3-Myb transduced cells (set to 1). ‘Asterisk’ denotes significant difference between the two samples (Student’s t-test, P < 0.05).

Figure 7.

Myb controls a core transcriptional regulatory network. Transcriptional factors directly targeted by Myb were used to construct a network using IPA. The most interconnected factors were subsequently extracted to construct a core regulatory network. Ellipses with gray background represent Myb activated transcription factors, whereas open ellipses with white background represent ones repressed by Myb.