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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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Transcriptional Network after RUNX1/ETO Depletion Is Enriched for C/EBPα Target Genes(A) The transcription-factor binding state for CEBPα, LMO2, PU.1, and RUNX1 after RUNX1/ETO knockdown is characterized by an overrepresentation of four dominant occupancy patterns. The number of peaks for all 15 factor combinations is shown on the left of the heatmap (red: bound, scored as 1; blue: not bound, scored as 0). Z scores on the right indicate the significance of deviation between observed and expected instances for all 15 binding patterns. Left: GSEAs of genes associated with the two most enriched dominant occupancy patterns (indicated by arrows) show highly significant enrichment of upregulated genes after RUNX1/ETO knockdown.(B) Genes associated with specific occupancy patterns that significantly change expression as measured by RNA-seq 4 days after RUNX1/ETO knockdown. The heatmap shows the RNA-seq overall fold change in Kasumi-1 cells 4 days after RUNX1/ETO knockdown.(C) GSEAs showing that genes associated with dominant occupancy patterns that are upregulated in Kasumi-1 cells behave similarly in patient cells.(D) Venn diagram depicting the number of genes bound by C/EBPα that are downregulated after RUNX1/ETO knockdown and show increased C/EBPα binding.See also Figure S6.
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Figure 6: Transcriptional Network after RUNX1/ETO Depletion Is Enriched for C/EBPα Target Genes(A) The transcription-factor binding state for CEBPα, LMO2, PU.1, and RUNX1 after RUNX1/ETO knockdown is characterized by an overrepresentation of four dominant occupancy patterns. The number of peaks for all 15 factor combinations is shown on the left of the heatmap (red: bound, scored as 1; blue: not bound, scored as 0). Z scores on the right indicate the significance of deviation between observed and expected instances for all 15 binding patterns. Left: GSEAs of genes associated with the two most enriched dominant occupancy patterns (indicated by arrows) show highly significant enrichment of upregulated genes after RUNX1/ETO knockdown.(B) Genes associated with specific occupancy patterns that significantly change expression as measured by RNA-seq 4 days after RUNX1/ETO knockdown. The heatmap shows the RNA-seq overall fold change in Kasumi-1 cells 4 days after RUNX1/ETO knockdown.(C) GSEAs showing that genes associated with dominant occupancy patterns that are upregulated in Kasumi-1 cells behave similarly in patient cells.(D) Venn diagram depicting the number of genes bound by C/EBPα that are downregulated after RUNX1/ETO knockdown and show increased C/EBPα binding.See also Figure S6.

Mentions: To test whether increased expression of C/EBPα was crucially involved in shifting the transcriptional network after RUNX1/ETO depletion, we defined overrepresented binding patterns for C/EBPα, PU.1, RUNX1, and LMO2 after RUNX1/ETO knockdown. Loss of RUNX1/ETO resulted in the formation of a transcriptional network dominated by C/EBPα-containing binding patterns, all of which were predominantly associated with upregulated genes in RUNX1/ETO-depleted Kasumi-1 and patient cells (Figures 6A–6C, S6A, and S6B; Table S3). Different patterns were again indicative of different classes of genes in terms of both GO and pathway analyses, with differentiation and signal transduction pathways being prominently featured (Figures S6C, S6D, and S7A). However, increased C/EBPα binding was also observed with a subset of genes that were downregulated (Figure 6D). Previous studies have shown that in addition to C/EBPα’s role in driving myeloid differentiation, low levels of C/EBPα are required for stem cell maintenance, as upregulation of C/EBPα represses genes required for stem-cell self-renewal (Zhang et al., 2004, 2013). Therefore, we identified genes that (1) were downregulated after RUNX1/ETO knockdown and (2) showed increased C/EBPα binding (a total of 145 genes met the latter criterion; Figure 6D). This category included stem cell genes such as ERG and CD34 (Figures S6F and S6G), as well as a large number of genes encoding for signaling molecules that are involved in regulating proliferation and differentiation, such as DUSP6 or PTK2 (Figure S6G).


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)

Transcriptional Network after RUNX1/ETO Depletion Is Enriched for C/EBPα Target Genes(A) The transcription-factor binding state for CEBPα, LMO2, PU.1, and RUNX1 after RUNX1/ETO knockdown is characterized by an overrepresentation of four dominant occupancy patterns. The number of peaks for all 15 factor combinations is shown on the left of the heatmap (red: bound, scored as 1; blue: not bound, scored as 0). Z scores on the right indicate the significance of deviation between observed and expected instances for all 15 binding patterns. Left: GSEAs of genes associated with the two most enriched dominant occupancy patterns (indicated by arrows) show highly significant enrichment of upregulated genes after RUNX1/ETO knockdown.(B) Genes associated with specific occupancy patterns that significantly change expression as measured by RNA-seq 4 days after RUNX1/ETO knockdown. The heatmap shows the RNA-seq overall fold change in Kasumi-1 cells 4 days after RUNX1/ETO knockdown.(C) GSEAs showing that genes associated with dominant occupancy patterns that are upregulated in Kasumi-1 cells behave similarly in patient cells.(D) Venn diagram depicting the number of genes bound by C/EBPα that are downregulated after RUNX1/ETO knockdown and show increased C/EBPα binding.See also Figure S6.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 6: Transcriptional Network after RUNX1/ETO Depletion Is Enriched for C/EBPα Target Genes(A) The transcription-factor binding state for CEBPα, LMO2, PU.1, and RUNX1 after RUNX1/ETO knockdown is characterized by an overrepresentation of four dominant occupancy patterns. The number of peaks for all 15 factor combinations is shown on the left of the heatmap (red: bound, scored as 1; blue: not bound, scored as 0). Z scores on the right indicate the significance of deviation between observed and expected instances for all 15 binding patterns. Left: GSEAs of genes associated with the two most enriched dominant occupancy patterns (indicated by arrows) show highly significant enrichment of upregulated genes after RUNX1/ETO knockdown.(B) Genes associated with specific occupancy patterns that significantly change expression as measured by RNA-seq 4 days after RUNX1/ETO knockdown. The heatmap shows the RNA-seq overall fold change in Kasumi-1 cells 4 days after RUNX1/ETO knockdown.(C) GSEAs showing that genes associated with dominant occupancy patterns that are upregulated in Kasumi-1 cells behave similarly in patient cells.(D) Venn diagram depicting the number of genes bound by C/EBPα that are downregulated after RUNX1/ETO knockdown and show increased C/EBPα binding.See also Figure S6.
Mentions: To test whether increased expression of C/EBPα was crucially involved in shifting the transcriptional network after RUNX1/ETO depletion, we defined overrepresented binding patterns for C/EBPα, PU.1, RUNX1, and LMO2 after RUNX1/ETO knockdown. Loss of RUNX1/ETO resulted in the formation of a transcriptional network dominated by C/EBPα-containing binding patterns, all of which were predominantly associated with upregulated genes in RUNX1/ETO-depleted Kasumi-1 and patient cells (Figures 6A–6C, S6A, and S6B; Table S3). Different patterns were again indicative of different classes of genes in terms of both GO and pathway analyses, with differentiation and signal transduction pathways being prominently featured (Figures S6C, S6D, and S7A). However, increased C/EBPα binding was also observed with a subset of genes that were downregulated (Figure 6D). Previous studies have shown that in addition to C/EBPα’s role in driving myeloid differentiation, low levels of C/EBPα are required for stem cell maintenance, as upregulation of C/EBPα represses genes required for stem-cell self-renewal (Zhang et al., 2004, 2013). Therefore, we identified genes that (1) were downregulated after RUNX1/ETO knockdown and (2) showed increased C/EBPα binding (a total of 145 genes met the latter criterion; Figure 6D). This category included stem cell genes such as ERG and CD34 (Figures S6F and S6G), as well as a large number of genes encoding for signaling molecules that are involved in regulating proliferation and differentiation, such as DUSP6 or PTK2 (Figure S6G).

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