The pioneer transcription factor PBX as a downstream target of MNX1 [ACT]

Recently we have identified the upregulation of the homebox domain protein MNX1 associated with the paediatric t(7;12) AML as the initiating event for Leukaemia in mice. We demonstrated that MNX1 triggers abnormal histone modifications, specifically H3K4me3 and H3K27me3, by interacting with SAM-dependent methyltransferases. In the current study our aim was investigate downstream targets of MNX1 and the relationship with histone modification. Employing TMT Mass spectrometry and RNA-Seq integrative analysis identified the pioneer transcription factor PBX1 as a downstream target of MNX1. We observe that MNX1 binds to the promoter region of PBX1, leading to increased expression of PBX1. This binding of MNX1 to the PBX1 promoter was associated with enhanced H3K4me3 and decreased H3K27me3 in the same promoter region. Interestingly, this binding of MNX1, but not the histone modification on the PBX1 promoter was transient and diminishes as MNX1-FL cells progress to leukaemia in mice. To gain further insight, we conducted a comparative analysis of H3K4me3 using ACT-Seq and RNA-Seq data between MNX preleukemia FL cells and leukaemia MNX1 BM blasts. Our results indicate that this phenomenon is not limited to PBX1 but is rather widespread throughout the genome, identifying several downstream targets for MNX1 mediated H3K4me3 modification. Investigating the PBX1 and 4 motifs identified from Chip-Seq, revealed a significant enrichment in MNX1 H3K4me3 and ATAC-seq peaks. This suggest that PBX family might play an important role in the function of MNX1, which was further confirmed by showing MNX1 induced PBX1 expression to be downregulated using the sinefungin inhibitor, which we have previously reported to inhibit leukemia induction by MNX1.