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RUNX1-EVI1 blocks RUNX1 and EVI1 driven cell fate
Taken together, our data show that RUNX1-EVI1 expression is incompatible with multipotent precursor development, and when induced in precursors, leads to cell cycle arrest and an increase in apoptosis.
RUNX1-EVI1 induction alters gene expression in
a differentiation-stage dependent and independent fashion
We next wanted to understand the molecular basis of the observed phenotypes. To this end, we sorted HE1, HE2 and HP cells on the basis of their surface marker phe- notypes, as described in Figure 1A and D and performed RNA-seq on the resulting matched cell populations. Biological duplicates were well correlated (Online Supplementary Figure S3A) and the average was used for further analysis. Hierarchical clustering of these datasets (Figure 3A) showed that the overall gene expression pat- terns in the different cell types were preserved in the pres- ence of RUNX1-EVI1, but with genes being de-regulated at every stage, particularly in HE2 cells (Online Supplementary Figure S3B, Supplementary Tables S1 to S3). A subset of these genes were validated by quantitative reverse transcriptase polymerase chain reaction (qRT- PCR) (Online Supplementary Figure S3C) and the HP gene expression changes, being the target cell for the leukemic transformation, were compared to two previously pub- lished t(3;21) patient RNA-seq datasets (Online Supplementary Figure S3D). This analysis showed that those genes which are specific to t(3;21) patients as com- pared to healthy CD34+ cells were upregulated following induction of RUNX1-EVI1 in HP, with genes such as Cdh5, Hes1, Maff and Arhgef12 being overexpressed in both patients and HP expressing RUNX1-EVI1.
The differentiation of blood cells in the in vitro differen- tiation system is not entirely synchronous, therefore RUNX1-EVI1 induction occurs in different cell types rep- resenting a differentiation trajectory. Many changes nor- mally seen within the differentiation process were main- tained after induction. For example, genes which were up- or downregulated during the transition from HE1 to HE2 or HE2 to HP continued to be up- or downregulated (Figure 3B; Online Supplementary Tables S4 and S5), includ- ing those essential for these transitions such as Tek and Gfi1b. However, a subset of genes failed to be up- or downregulated to the extent it normally should. For exam- ple, some regulators of the MAPK pathway including Mapk3 and Dusp6 were downregulated during the transi- tion from HE2 to HP more than they should be following RUNX1-EVI1 induction. Alongside these developmental changes, a core set of genes were upregulated at least 2- fold in two, or all three cell types (Figures 3C and D), including Dusp5, Cdkn1c and Pdgfa. Cdkn1c is a negative regulator of the cell cycle and its universal upregulation may underpin the cell cycle arrest (Figures 2B and C) and the de-regulation of multiple cell cycle associated genes (Online Supplementary Figure S3E).
We also examined how stage-specific gene expression changes related to the differentiation program using known marker genes. In HE cells, the expression of the vascular/smooth muscle program was deregulated (Figure 3E). The smooth muscle genes Acta2, Tagin, Cnn1 and the genes encoding the cardiac regulator TBX20 and homeo- box protein Nkx-2.5 were further downregulated in HE1, but were then upregulated when RUNX1-EVI1 was induced in HE2. When specifically examining hematopoi-
etic lineage gene signatures, we did not see a downregula- tion of myeloid or erythroid genes as expected from the colony forming assays. Indeed, we found a widespread, albeit modest (>1.5-fold), increase in expression of genes related to a multipotent progenitor identity with the con- comitant expression of a multi-lineage gene expression program consisting of myeloid, lymphoid and megakary- ocyte/erythroid genes (Figure 3E; Online Supplementary Figure S3C). Taken together, these results suggest that RUNX1-EVI1 induction causes a cell cycle and differentia- tion arrest that is associated with a disturbance of the bal- ance between the hematopoietic and vascular/smooth muscle fate.
Disturbed lineage specification is caused by chromatin changes associated with altered RUNX1 binding
In order to understand how RUNX1-EVI1 induction reprograms the chromatin landscape, we performed and integrated ChIP-seq analysis for both RUNX1 and RUNX1-EVI1 in HP, with data from DNaseI-seq experi- ments performed on sorted cKit+ HP, and HE (cKit+, Tie2+, CD41-/+). Induction of RUNX1-EVI1 led to changes to chromatin accessibility in the HE and HP cells and an increased proportion of distal DNaseI hypersensi- tive sites (DHS) (Online Supplementary Figure S4A). Few DHS changed at promoter sites (Online Supplementary Figure S4B). We therefore focussed on the analysis of distal DHS and ranked them by the fold change in tag count at each site. 1,296 DHS were gained and 858 lost in the HE when RUNX1-EVI1 was expressed (Figure 4A). The gained sites showed a specific enrichment of RUNX motifs, whilst the sites lost contained SOX, TEAD and AP-1 motifs. In HP cells, RUNX1-EVI1 induction had a completely different effect as here we observed a loss rather than a gain of RUNX motif enrichment (Figure 4B). Taken together, this result suggested a shift in chromatin patterns in HE from those of the vascular/endothelial lin- eages11,22 towards a HP-like pattern. We confirmed this result by plotting the HP DNaseI-seq peaks alongside those of the HE (Figure 4C) and by performing a correla- tion analysis (Figure 4D). These analyses demonstrated that the HE chromatin pattern was more similar to that of HP cells following induction of RUNX1-EVI1, despite the cells still displaying surface markers and an overall gene expression signature of the HE (Figures 1C and 3A). Furthermore, in HP we also saw a shift from the ETS motif to a PU.1 specific motif, which was consistent with the upregulation of Spi1 (encoding PU.1) expression, indicat- ing that the chromatin accessibility pattern was being rewired towards myelopoiesis.
In order to test how these results related to the interplay of RUNX1-EVI1 with RUNX1, we compared ChIP-seq for RUNX1 with and without induction of RUNX1-EVI1, to the binding of RUNX1-EVI1 itself in cKit+ HP. The anti- body we used against human EVI1 did not recognize the endogenous murine EVI1 and thus exclusively measured binding of the exogenous protein. We analyzed only high- confidence ChIP-seq peaks, which had been filtered for the presence of a DHS at the same site, to minimize noise associated with the technical difficulty of these ChIP experiments.
Around half of RUNX1-EVI1 binding sites overlapped with those of RUNX1 (Figure 5A), including those RUNX1 sites that were either maintained or gained following RUNX1-EVI1 induction. This result suggests that the pre-
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