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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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Specific Transcription-Factor Binding Patterns in t(8;21) Cells Correlate with the Response to RUNX1/ETO KnockdownGenes bound by RUNX1/ETO are preferentially upregulated, whereas genes not bound by RUNX1/ETO are preferentially downregulated.(A) Analysis of combinatorial binding identifies prevalent patterns in Kasumi-1 cells. The numbers of peaks are shown on the left of the heatmap for 61 factor-binding combinations (red: bound, scored as 1; blue: not bound, scored as 0 with the order of factors as depicted on top of the heatmap). Z scores on the right indicate the significance of deviation between observed and expected instances for all 61 combinatorial binding patterns. We identified 11 overrepresented binding patterns, which we analyzed further when each was associated with more than 100 genes. GSEA of selected large groups of genes (indicated by arrows) shows a highly significant enrichment of genes upregulated (upper left) or downregulated (lower left) after 4 days of RUNX1/ETO knockdown.(B) Heatmap showing the RNA-seq overall fold change in Kasumi-1 cells 4 days after RUNX1/ETO knockdown.(C) GSEA plots showing enrichment for up- or downregulated genes associated with dominant binding patterns in patient cells subjected to RUNX1/ETO knockdown, demonstrating that changes in gene expression were concordant between Kasumi-1 and patient cells after RUNX1/ETO knockdown. Note that in patient cells, RUNX1/ETO was only depleted for 48 hr and it takes about 4 days for the majority of genes to be downregulated (Ptasinska et al., 2012), thus explaining the lower p value seen with downregulated genes.See also Figure S4.
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Figure 4: Specific Transcription-Factor Binding Patterns in t(8;21) Cells Correlate with the Response to RUNX1/ETO KnockdownGenes bound by RUNX1/ETO are preferentially upregulated, whereas genes not bound by RUNX1/ETO are preferentially downregulated.(A) Analysis of combinatorial binding identifies prevalent patterns in Kasumi-1 cells. The numbers of peaks are shown on the left of the heatmap for 61 factor-binding combinations (red: bound, scored as 1; blue: not bound, scored as 0 with the order of factors as depicted on top of the heatmap). Z scores on the right indicate the significance of deviation between observed and expected instances for all 61 combinatorial binding patterns. We identified 11 overrepresented binding patterns, which we analyzed further when each was associated with more than 100 genes. GSEA of selected large groups of genes (indicated by arrows) shows a highly significant enrichment of genes upregulated (upper left) or downregulated (lower left) after 4 days of RUNX1/ETO knockdown.(B) Heatmap showing the RNA-seq overall fold change in Kasumi-1 cells 4 days after RUNX1/ETO knockdown.(C) GSEA plots showing enrichment for up- or downregulated genes associated with dominant binding patterns in patient cells subjected to RUNX1/ETO knockdown, demonstrating that changes in gene expression were concordant between Kasumi-1 and patient cells after RUNX1/ETO knockdown. Note that in patient cells, RUNX1/ETO was only depleted for 48 hr and it takes about 4 days for the majority of genes to be downregulated (Ptasinska et al., 2012), thus explaining the lower p value seen with downregulated genes.See also Figure S4.

Mentions: We next analyzed our ChIP-seq data sets to identify the core transcriptional network that characterizes the cellular identity of t(8;21) cells by determining overrepresented combinatorial binding patterns for the transcription factors RUNX1/ETO, C/EBPα, HEB, LMO2, PU.1, and RUNX1 (Tijssen et al., 2011). ChIP sequences in RUNX1/ETO-positive cells were enriched for just 11 of the 63 possible different binding patterns, which included six significantly enriched combinatorial patterns containing RUNX1/ETO and five patterns that did not (Figure 4A, marked by asterisks). Two possible binding patterns (111010 and 110011) were not observed. We then associated such elements with the nearest genes and performed a gene set enrichment analysis (GSEA) using gene-expression data sets derived from a time course of RUNX1/ETO knockdown in two different t(8;21) cell lines (Figures S4A and S4B; Ptasinska et al., 2012). In addition, we compared these gene signatures with a RNA-seq-based gene-expression data set derived from a 4-day RUNX1/ETO knockdown in Kasumi-1 cells (Figures 4B and S4C). This analysis demonstrated that all overrepresented RUNX1/ETO-containing binding patterns were associated with the upregulation of gene expression upon knockdown (Figure 4B, red asterisks), whereas loci that do not bind RUNX1/ETO were enriched in genes that were downregulated after RUNX1/ETO knockdown (green asterisks). The very same genes behaved similarly when assayed after knockdown of RUNX1/ETO in patient cells, confirming the similarity between cell lines and primary cells (Figure 4C).


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)

Specific Transcription-Factor Binding Patterns in t(8;21) Cells Correlate with the Response to RUNX1/ETO KnockdownGenes bound by RUNX1/ETO are preferentially upregulated, whereas genes not bound by RUNX1/ETO are preferentially downregulated.(A) Analysis of combinatorial binding identifies prevalent patterns in Kasumi-1 cells. The numbers of peaks are shown on the left of the heatmap for 61 factor-binding combinations (red: bound, scored as 1; blue: not bound, scored as 0 with the order of factors as depicted on top of the heatmap). Z scores on the right indicate the significance of deviation between observed and expected instances for all 61 combinatorial binding patterns. We identified 11 overrepresented binding patterns, which we analyzed further when each was associated with more than 100 genes. GSEA of selected large groups of genes (indicated by arrows) shows a highly significant enrichment of genes upregulated (upper left) or downregulated (lower left) after 4 days of RUNX1/ETO knockdown.(B) Heatmap showing the RNA-seq overall fold change in Kasumi-1 cells 4 days after RUNX1/ETO knockdown.(C) GSEA plots showing enrichment for up- or downregulated genes associated with dominant binding patterns in patient cells subjected to RUNX1/ETO knockdown, demonstrating that changes in gene expression were concordant between Kasumi-1 and patient cells after RUNX1/ETO knockdown. Note that in patient cells, RUNX1/ETO was only depleted for 48 hr and it takes about 4 days for the majority of genes to be downregulated (Ptasinska et al., 2012), thus explaining the lower p value seen with downregulated genes.See also Figure S4.
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Figure 4: Specific Transcription-Factor Binding Patterns in t(8;21) Cells Correlate with the Response to RUNX1/ETO KnockdownGenes bound by RUNX1/ETO are preferentially upregulated, whereas genes not bound by RUNX1/ETO are preferentially downregulated.(A) Analysis of combinatorial binding identifies prevalent patterns in Kasumi-1 cells. The numbers of peaks are shown on the left of the heatmap for 61 factor-binding combinations (red: bound, scored as 1; blue: not bound, scored as 0 with the order of factors as depicted on top of the heatmap). Z scores on the right indicate the significance of deviation between observed and expected instances for all 61 combinatorial binding patterns. We identified 11 overrepresented binding patterns, which we analyzed further when each was associated with more than 100 genes. GSEA of selected large groups of genes (indicated by arrows) shows a highly significant enrichment of genes upregulated (upper left) or downregulated (lower left) after 4 days of RUNX1/ETO knockdown.(B) Heatmap showing the RNA-seq overall fold change in Kasumi-1 cells 4 days after RUNX1/ETO knockdown.(C) GSEA plots showing enrichment for up- or downregulated genes associated with dominant binding patterns in patient cells subjected to RUNX1/ETO knockdown, demonstrating that changes in gene expression were concordant between Kasumi-1 and patient cells after RUNX1/ETO knockdown. Note that in patient cells, RUNX1/ETO was only depleted for 48 hr and it takes about 4 days for the majority of genes to be downregulated (Ptasinska et al., 2012), thus explaining the lower p value seen with downregulated genes.See also Figure S4.
Mentions: We next analyzed our ChIP-seq data sets to identify the core transcriptional network that characterizes the cellular identity of t(8;21) cells by determining overrepresented combinatorial binding patterns for the transcription factors RUNX1/ETO, C/EBPα, HEB, LMO2, PU.1, and RUNX1 (Tijssen et al., 2011). ChIP sequences in RUNX1/ETO-positive cells were enriched for just 11 of the 63 possible different binding patterns, which included six significantly enriched combinatorial patterns containing RUNX1/ETO and five patterns that did not (Figure 4A, marked by asterisks). Two possible binding patterns (111010 and 110011) were not observed. We then associated such elements with the nearest genes and performed a gene set enrichment analysis (GSEA) using gene-expression data sets derived from a time course of RUNX1/ETO knockdown in two different t(8;21) cell lines (Figures S4A and S4B; Ptasinska et al., 2012). In addition, we compared these gene signatures with a RNA-seq-based gene-expression data set derived from a 4-day RUNX1/ETO knockdown in Kasumi-1 cells (Figures 4B and S4C). This analysis demonstrated that all overrepresented RUNX1/ETO-containing binding patterns were associated with the upregulation of gene expression upon knockdown (Figure 4B, red asterisks), whereas loci that do not bind RUNX1/ETO were enriched in genes that were downregulated after RUNX1/ETO knockdown (green asterisks). The very same genes behaved similarly when assayed after knockdown of RUNX1/ETO in patient cells, confirming the similarity between cell lines and primary cells (Figure 4C).

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