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Identification of a dynamic core transcriptional network in t(8;21) AML that regulates differentiation block and self-renewal.

Ptasinska A, Assi SA, Martinez-Soria N, Imperato MR, Piper J, Cauchy P, Pickin A, James SR, Hoogenkamp M, Williamson D, Wu M, Tenen DG, Ott S, Westhead DR, Cockerill PN, Heidenreich O, Bonifer C - Cell Rep (2014)

Bottom Line: We show that the transcriptional program underlying leukemic propagation is regulated by a dynamic equilibrium between RUNX1/ETO and RUNX1 complexes, which bind to identical DNA sites in a mutually exclusive fashion.Perturbation of this equilibrium in t(8;21) cells by RUNX1/ETO depletion leads to a global redistribution of transcription factor complexes within preexisting open chromatin, resulting in the formation of a transcriptional network that drives myeloid differentiation.Our work demonstrates on a genome-wide level that the extent of impaired myeloid differentiation in t(8;21) is controlled by the dynamic balance between RUNX1/ETO and RUNX1 activities through the repression of transcription factors that drive differentiation.

View Article: PubMed Central - PubMed

Affiliation: School of Cancer Sciences, College of Medicine and Dentistry, University of Birmingham, Birmingham B15 2TT, UK.

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Loss of RUNX1/ETO Triggers C/EBPα-Driven Reorganization of the Leukemic Transcriptional Network(A) RUNX1/ETO and CEBPA mRNA expression levels in Kasumi-1 cells 72 hr after electroporation with the indicated siRNAs. siRE, RUNX1/ETO siRNA; siCEBPA, C/EBPα siRNA; siMM, mismatch control siRNA. Results represent the mean ± SEM of five independent experiments. *p < 0.05; ns, not significant by paired Student’s t test.(B) Western blot indicating RUNX1/ETO and C/EBPα protein expression levels in single- and double-knockdown cells as indicated. An antibody against H3 was used as control. Mock, no siRNA.(C) mRNA levels of MS4A3, NKG7, and RNASE2 72 hr after electroporation with the indicated siRNAs. Results represent the mean ± SEM of five independent experiments. *p < 0.05, **p < 0.01 by paired Student’s t test.(D) UCSC genome browser screenshot showing the binding pattern of RUNX1/ETO, C/EBPα, and DHSs at the MS4A3 locus in Kasumi-1 cells treated for 48 hr with mismatch control siRNA (siMM) and with RUNX1/ETO siRNA (siRE).(E) Model of RUNX1/ETO-mediated control of leukemic transcription. The competitive equilibrium in locus occupation between RUNX1/ETO and RUNX1 complexes drives leukemic self-renewal. Depletion of RUNX1/ETO increases the levels and DNA binding of its direct target gene, C/EBPα, which together with other differentiation genes reinstalls a transcriptional program that promotes myeloid differentiation.See also Figure S7.
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Figure 7: Loss of RUNX1/ETO Triggers C/EBPα-Driven Reorganization of the Leukemic Transcriptional Network(A) RUNX1/ETO and CEBPA mRNA expression levels in Kasumi-1 cells 72 hr after electroporation with the indicated siRNAs. siRE, RUNX1/ETO siRNA; siCEBPA, C/EBPα siRNA; siMM, mismatch control siRNA. Results represent the mean ± SEM of five independent experiments. *p < 0.05; ns, not significant by paired Student’s t test.(B) Western blot indicating RUNX1/ETO and C/EBPα protein expression levels in single- and double-knockdown cells as indicated. An antibody against H3 was used as control. Mock, no siRNA.(C) mRNA levels of MS4A3, NKG7, and RNASE2 72 hr after electroporation with the indicated siRNAs. Results represent the mean ± SEM of five independent experiments. *p < 0.05, **p < 0.01 by paired Student’s t test.(D) UCSC genome browser screenshot showing the binding pattern of RUNX1/ETO, C/EBPα, and DHSs at the MS4A3 locus in Kasumi-1 cells treated for 48 hr with mismatch control siRNA (siMM) and with RUNX1/ETO siRNA (siRE).(E) Model of RUNX1/ETO-mediated control of leukemic transcription. The competitive equilibrium in locus occupation between RUNX1/ETO and RUNX1 complexes drives leukemic self-renewal. Depletion of RUNX1/ETO increases the levels and DNA binding of its direct target gene, C/EBPα, which together with other differentiation genes reinstalls a transcriptional program that promotes myeloid differentiation.See also Figure S7.

Mentions: We next evaluated whether C/EBPα was required for the upregulation of repressed RUNX1/ETO target genes. For this purpose, we depleted RUNX1/ETO with and without a concomitant C/EBPα knockdown. Knockdown of RUNX1/ETO led to a 2-fold increase in C/EBPα expression (Figures 3A, 7A, and 7B) and increases in expression of the direct RUNX1/ETO target genes, including MS4A3, NKG7, and RNASE2, which all show increased C/EBPα binding upon RUNX1/ETO depletion (Figures 7C and 7D; data not shown). Codepletion of C/EBPα diminished the induction of the three target genes in both Kasumi-1 and SKNO-1 cells (Figures 7D and S7B–S7D). These data indicate that derepression of C/EBPα caused by RUNX1/ETO depletion is required for the full upregulation of a number of RUNX1/ETO target genes. However, we cannot rule out a similar function for other C/EBP members and in particular C/EBPδ and C/ EBPε, which are both upregulated upon RUNX1/ETO knockdown (Figure 5B and data not shown). Nevertheless, our data confirm that C/EBPα plays an important role in orchestrating a transcriptional network that drives myeloid differentiation downstream of the original RUNX1/ETO network (Figure 7E).


Identification of a dynamic core transcriptional network in t(8;21) AML that regulates differentiation block and self-renewal.

Ptasinska A, Assi SA, Martinez-Soria N, Imperato MR, Piper J, Cauchy P, Pickin A, James SR, Hoogenkamp M, Williamson D, Wu M, Tenen DG, Ott S, Westhead DR, Cockerill PN, Heidenreich O, Bonifer C - Cell Rep (2014)

Loss of RUNX1/ETO Triggers C/EBPα-Driven Reorganization of the Leukemic Transcriptional Network(A) RUNX1/ETO and CEBPA mRNA expression levels in Kasumi-1 cells 72 hr after electroporation with the indicated siRNAs. siRE, RUNX1/ETO siRNA; siCEBPA, C/EBPα siRNA; siMM, mismatch control siRNA. Results represent the mean ± SEM of five independent experiments. *p < 0.05; ns, not significant by paired Student’s t test.(B) Western blot indicating RUNX1/ETO and C/EBPα protein expression levels in single- and double-knockdown cells as indicated. An antibody against H3 was used as control. Mock, no siRNA.(C) mRNA levels of MS4A3, NKG7, and RNASE2 72 hr after electroporation with the indicated siRNAs. Results represent the mean ± SEM of five independent experiments. *p < 0.05, **p < 0.01 by paired Student’s t test.(D) UCSC genome browser screenshot showing the binding pattern of RUNX1/ETO, C/EBPα, and DHSs at the MS4A3 locus in Kasumi-1 cells treated for 48 hr with mismatch control siRNA (siMM) and with RUNX1/ETO siRNA (siRE).(E) Model of RUNX1/ETO-mediated control of leukemic transcription. The competitive equilibrium in locus occupation between RUNX1/ETO and RUNX1 complexes drives leukemic self-renewal. Depletion of RUNX1/ETO increases the levels and DNA binding of its direct target gene, C/EBPα, which together with other differentiation genes reinstalls a transcriptional program that promotes myeloid differentiation.See also Figure S7.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 7: Loss of RUNX1/ETO Triggers C/EBPα-Driven Reorganization of the Leukemic Transcriptional Network(A) RUNX1/ETO and CEBPA mRNA expression levels in Kasumi-1 cells 72 hr after electroporation with the indicated siRNAs. siRE, RUNX1/ETO siRNA; siCEBPA, C/EBPα siRNA; siMM, mismatch control siRNA. Results represent the mean ± SEM of five independent experiments. *p < 0.05; ns, not significant by paired Student’s t test.(B) Western blot indicating RUNX1/ETO and C/EBPα protein expression levels in single- and double-knockdown cells as indicated. An antibody against H3 was used as control. Mock, no siRNA.(C) mRNA levels of MS4A3, NKG7, and RNASE2 72 hr after electroporation with the indicated siRNAs. Results represent the mean ± SEM of five independent experiments. *p < 0.05, **p < 0.01 by paired Student’s t test.(D) UCSC genome browser screenshot showing the binding pattern of RUNX1/ETO, C/EBPα, and DHSs at the MS4A3 locus in Kasumi-1 cells treated for 48 hr with mismatch control siRNA (siMM) and with RUNX1/ETO siRNA (siRE).(E) Model of RUNX1/ETO-mediated control of leukemic transcription. The competitive equilibrium in locus occupation between RUNX1/ETO and RUNX1 complexes drives leukemic self-renewal. Depletion of RUNX1/ETO increases the levels and DNA binding of its direct target gene, C/EBPα, which together with other differentiation genes reinstalls a transcriptional program that promotes myeloid differentiation.See also Figure S7.
Mentions: We next evaluated whether C/EBPα was required for the upregulation of repressed RUNX1/ETO target genes. For this purpose, we depleted RUNX1/ETO with and without a concomitant C/EBPα knockdown. Knockdown of RUNX1/ETO led to a 2-fold increase in C/EBPα expression (Figures 3A, 7A, and 7B) and increases in expression of the direct RUNX1/ETO target genes, including MS4A3, NKG7, and RNASE2, which all show increased C/EBPα binding upon RUNX1/ETO depletion (Figures 7C and 7D; data not shown). Codepletion of C/EBPα diminished the induction of the three target genes in both Kasumi-1 and SKNO-1 cells (Figures 7D and S7B–S7D). These data indicate that derepression of C/EBPα caused by RUNX1/ETO depletion is required for the full upregulation of a number of RUNX1/ETO target genes. However, we cannot rule out a similar function for other C/EBP members and in particular C/EBPδ and C/ EBPε, which are both upregulated upon RUNX1/ETO knockdown (Figure 5B and data not shown). Nevertheless, our data confirm that C/EBPα plays an important role in orchestrating a transcriptional network that drives myeloid differentiation downstream of the original RUNX1/ETO network (Figure 7E).

Bottom Line: We show that the transcriptional program underlying leukemic propagation is regulated by a dynamic equilibrium between RUNX1/ETO and RUNX1 complexes, which bind to identical DNA sites in a mutually exclusive fashion.Perturbation of this equilibrium in t(8;21) cells by RUNX1/ETO depletion leads to a global redistribution of transcription factor complexes within preexisting open chromatin, resulting in the formation of a transcriptional network that drives myeloid differentiation.Our work demonstrates on a genome-wide level that the extent of impaired myeloid differentiation in t(8;21) is controlled by the dynamic balance between RUNX1/ETO and RUNX1 activities through the repression of transcription factors that drive differentiation.

View Article: PubMed Central - PubMed

Affiliation: School of Cancer Sciences, College of Medicine and Dentistry, University of Birmingham, Birmingham B15 2TT, UK.

Show MeSH
Related in: MedlinePlus