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S.G. Kellaway et al.
dominant mechanism of action of RUNX1-EVI1 is not the displacement of RUNX1. This finding was confirmed by examining the proximity of RUNX1 and RUNX1-EVI1 ChIP peaks (Online Supplementary Figure S5A). The RUNX1 and RUNX1-EVI1 peak summits were distributed similar- ly prior to and following induction of RUNX1-EVI1, both overlapping the same sites and next to each other. More strikingly, induction caused a large movement of RUNX1 within the genome with the loss of over a third of pre- existing RUNX1 sites and considerably more gained RUNX1 sites. Most gained sites were not associated with RUNX1-EVI1 binding (Figure 5B) nor did they have any specific motif enrichment not present in the shared sites (Online Supplementary Figure S5B) but were instead found in promoters (Online Supplementary Figure S5C). This movement was a real re-distribution as the overall level of RUNX1 protein was unchanged (Figure 1B).
When RUNX1-EVI1 bound in concert with RUNX1, or displaced RUNX1, its binding sites were enriched for RUNX and GATA motifs (Figure 5C and D; Online Supplementary Figure S5D), suggesting that RUNX1-EVI1 can also bind via the RUNT homology domain. Unique RUNX1-EVI1 ChIP peaks, however, were enriched for ETS-like motifs (Figure 5C), which may be indicative of binding via the EVI1 portion of the protein27 in the absence of RUNX1, an example of which is shown in Figure 5D and the Online Supplementary Figure S5D.
RUNX1-EVI1 disrupts RUNX1 and EVI1 driven gene regulatory networks
We next integrated the RUNX1 and RUNX1-EVI1 ChIP- seq data with the DNaseI-seq and RNA-seq data to inves- tigate how the binding of RUNX1-EVI1 and the move- ment of RUNX1 influenced changes in gene expression. We observed that the DHS that were lost following induc- tion of RUNX1-EVI1 were associated with lost RUNX1 binding as well. However, RUNX1 moved to chromatin which was already accessible and DHS which were gained were associated with RUNX1-EVI1 binding (Figure 6A; Online Supplementary Figure S6A). When considering genes which were at least 2-fold deregulated, those which were downregulated - particularly in HP - were associated with reduced RUNX1 binding (Figure 6B), indicating that RUNX1-EVI1 induction interfered with gene activation by RUNX1. The proportion of changed genes associated with lost RUNX1 binding increased throughout differentiation, as the reliance on RUNX1 increased. Both up- and down- regulated genes at all stages were associated with new RUNX1 binding sites, again matching the trend in chro- matin accessibility. RUNX1-EVI1 bound de-regulated genes were predominantly upregulated in HP cells, but more were downregulated in HE cells. We therefore com- pared how the gene expression changes related to endoge- nous EVI1 and RUNX1 binding sites, by plotting which genes were associated with EVI1 binding from a public EVI1 ChIP dataset,27 and the genes associated with our RUNX1 ChIP in uninduced HP against gene expression changes following RUNX1-EVI1 (Figure 6C; Online Supplementary Figure S6B). Genes which changed expres- sion, were either upregulated or downregulated, were enriched for EVI1 binding and depleted for RUNX1 bind- ing, particularly in the HP. Therefore, neither RUNX1 nor EVI1 is solely associated with up- or downregulation of their target genes but instead we see a complex and stage- specific pattern of interference.
Finally, to examine which changes were a direct response to binding and which were a result of the cells’ changing identity, we employed gene set enrichment analysis comparing the genes upregulated in HP following induction of RUNX1-EVI1 to those downregulated fol- lowing small interfering RNA knockdown of RUNX1- EVI1 in a human cell line19 (Figure 6D) and observed a good correlation. These genes include hematopoietic genes (Online Supplementary Table S6) such as Gata2 (a RUNX1 target) and Meis1 (a target of both RUNX1 and RUNX1-EVI1). By contrast, the genes downregulated fol- lowing RUNX1-EVI1 induction in HP did not correlate well with those upregulated following RUNX1-EVI1 knockdown with the exception of a small subset of genes such as Mpo and Rab44 which are neither RUNX1 nor RUNX1-EVI1 targets.
These results suggest that RUNX1-EVI1 is likely to interfere with the repressive activities of both RUNX1 and EVI1 with the balance of lineage decisions depending on the differentiation stage at the time point of induction.
Discussion
RUNX1-EVI1 expression is only found as a secondary event in myeloid malignancies. Our study shows that its expression as sole oncogene in untransformed myeloid progenitor cells is incompatible with blood cell differenti- ation. We also found that RUNX1-EVI1 induction disrupts the RUNX1 driven endothelial-hematopoietic transition, in a similar fashion to RUNX1-ETO.28 Expression of RUNX1-EVI1 HP cells disrupted their colony forming capacity and led to extensive de-regulation of gene expres- sion. However, the underlying molecular cause was differ- ent. As with RUNX1-ETO, genes of the stem cell program were upregulated, but the arrest in differentiation after RUNX1-EVI1 induction was associated with the rapid activation of a multi-lineage gene expression program and a profound disturbance of hematopoietic lineage specifica- tion.
Induction of RUNX1-EVI1 in cells committed to the hematopoietic fate is associated with the activation of a pan-lineage hematopoietic gene expression program and a failure in fully downregulating factors associated with a vascular gene expression program. This behavior is remi- niscent of mutations in lineage commitment factors, such as PAX5. Knock-out of PAX5 leads to a block in B-cell dif- ferentiation which is associated with an inability to acti- vate the B-cell gene expression program, but also an inabil- ity to repress the myeloid program,29,30 generating progen- itors with a multi-lineage gene expression pattern and the inability to commit to a specific lineage. Alongside the dif- ferentiation associated phenotype, we found that RUNX1-EVI1 caused a partial cell cycle arrest and increase in apoptosis which is likely to be associated with increased expression of Cdkn1c, leading to the stage-spe- cific deregulation of cell cycle genes. Cdkn1c encodes the cell cycle inhibitor p57Kip2 which is important in mainte- nance of the adult hematopoietic stem cell compartment,31 and has been shown to be deregulated by both EVI1 and MDS-EVI1 thus causing to cell cycle mis-regulation.13,32
Our results indicate that the phenotype caused by RUNX1-EVI1 induction is a result of interference with both RUNX1 and EVI1 driven gene regulatory networks. Similar to PAX5, both RUNX1 and EVI1 interact with co-
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haematologica | 2021; 106(6)