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

Zhao L, Glazov EA, Pattabiraman DR, Al-Owaidi F, Zhang P, Brown MA, Leo PJ, Gonda TJ - Nucleic Acids Res. (2011)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: The University of Queensland Diamantina Institute, Brisbane, Queensland 4102, Australia.

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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3113568&req=5

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.

Mentions: For ChIP-Seq, biological duplicate ChIP samples were prepared using ER-10 antibody from ERMYB cells cultured in the presence of β-E2 (+β-E2 or ‘activated’-Myb) or deprived of β-E2 for 6 h (−β-E2 or ‘inactivated’-Myb). The employment of the ER antibody rather than Myb antibody raised the potential concern that a subset of identified Myb binding regions (MBRs) might be occupied by ERα rather than the Myb fusion protein. However, our results indicate this is unlikely to be the case. Almost all examined MBRs which were validated using ER antibody have also been validated using mixed anti-Myb sera. Furthermore, almost all of a set of conserved MBRs have been validated in human HL-60 cells using pooled MYB antibodies (Figure 3C). Moreover, the canonical ER binding motif was not detected in the de novo motif discovery analysis (Supplementary Figure S2C). Similar concerns about the Myb regulated genes identified by expression profiling in ERMYB cells are allayed by the fact that very few of these showed significant expression changes in response to β-E2 in a cell line derived by transformation with non-ER-fused, constitutively activate CT3-Myb (A69 cells; Supplementary Table S2). Moreover, our Myb-regulated gene set showed no significant overlap with the well-established estrogen-response signature (data not shown), and furthermore, the majority of a set of identified Myb regulated genes have been validated independently in Myb transduced primary bone marrow cells (Figure 1D and Supplementary Table S4).Figure 1.


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

Zhao L, Glazov EA, Pattabiraman DR, Al-Owaidi F, Zhang P, Brown MA, Leo PJ, Gonda TJ - Nucleic Acids Res. (2011)

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113568&req=5

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.
Mentions: For ChIP-Seq, biological duplicate ChIP samples were prepared using ER-10 antibody from ERMYB cells cultured in the presence of β-E2 (+β-E2 or ‘activated’-Myb) or deprived of β-E2 for 6 h (−β-E2 or ‘inactivated’-Myb). The employment of the ER antibody rather than Myb antibody raised the potential concern that a subset of identified Myb binding regions (MBRs) might be occupied by ERα rather than the Myb fusion protein. However, our results indicate this is unlikely to be the case. Almost all examined MBRs which were validated using ER antibody have also been validated using mixed anti-Myb sera. Furthermore, almost all of a set of conserved MBRs have been validated in human HL-60 cells using pooled MYB antibodies (Figure 3C). Moreover, the canonical ER binding motif was not detected in the de novo motif discovery analysis (Supplementary Figure S2C). Similar concerns about the Myb regulated genes identified by expression profiling in ERMYB cells are allayed by the fact that very few of these showed significant expression changes in response to β-E2 in a cell line derived by transformation with non-ER-fused, constitutively activate CT3-Myb (A69 cells; Supplementary Table S2). Moreover, our Myb-regulated gene set showed no significant overlap with the well-established estrogen-response signature (data not shown), and furthermore, the majority of a set of identified Myb regulated genes have been validated independently in Myb transduced primary bone marrow cells (Figure 1D and Supplementary Table S4).Figure 1.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: The University of Queensland Diamantina Institute, Brisbane, Queensland 4102, Australia.

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.

Show MeSH
Related in: MedlinePlus