When overexpressed, MEIS1 is able to transform tumour suppressor-deficient mouse cells in the absence of other oncogenes 4 and to accelerate the occurrence of HoxA9-dependent Acute Myeloid Leukemia (AML) 18– 20. MEIS1 has a crucial role in embryonic hematopoiesis 16, 17, and is involved in leukemogenesis in children and adults. All known functions of PREP1 depend on the association with PBX. In particular, overexpression of PREP1 inhibits the growth of MEIS1-dependent tumour cells, competing in vivo for the MEIS1 DNA-binding sites 4, 5. Because of these properties PREP1 is considered a tumour suppressor. Human tumours are low in PREP1 11, 15, while PREP1 overexpression slows-down the growth of tumour cells in vitro and in vivo 4. Prep1 i/i mice develop spontaneous tumours also at the heterozygous state and Prep1 haplo-insufficient mice accelerate the development of oncogenes-dependent tumours 9, 11, 14. Indeed, down-regulation of PREP1 induces DNA damage and consents oncogenes like Ras or Meis1 to evade senescence and transform 12. Prep1 hypomorphic ( Prep1 i/i) mouse fibroblasts accumulate DNA damage, chromosomal abnormalities and display increased basal and genotoxicity-induced apoptosis 12. The tumour-suppressor role of PREP1 is associated with the maintenance of genomic stability 12, control of DNA replication timing 13, and protection of the nuclear envelope structure (Purushotaman D.

PREP1 is essential in embryonic development 9, 10 and in the adult functions as a tumour-suppressor 11. Unveiling the PBX-interaction surface of PREP or MEIS would allow not only to better understand their molecular function but would also represent the basis for searching inactivating therapeutic molecules. PREP1 or MEIS1 dimerize with PBX1 and direct its DNA binding to specific and only partially overlapping sequences 4– 8. PREP1 (Pbx-regulating protein 1, aka PKNOX1), and MEIS1 (Myeloid ecotropic insertion site 1) are transcription factors belonging to the three amino acids loop extension (TALE) homeodomain family 1, and play essential roles during embryonic development and cancer by associating to pre-B-cell leukemia (PBX) cofactors 2, 3. Moreover, the extensive mutagenic analysis of PREP1 identifies the role of individual hydrophobic HR1 and HR2 residues, both in vitro and in vivo. Although partial, this analysis provides for the first time some ideas on the tertiary structure of the complexes not available before. This difference is also reflected by the different binding affinity of the two proteins to PBX1. The HR1 domain of MEIS1, however, seem to play a less stringent role in PBX1 interaction with respect to that of PREP1. The HR2 domain has a fundamental role in binding the PBC-B domain of PBX1 in both PREP1 and MEIS1. The data confirm the existence of two distinct interaction sites within the PBC domain of PBX1 and unravel differences among the highly similar binding sites of MEIS1 and PREP1. Here, we have combined cross-linking mass-spectrometry and systematic mutagenesis to detail the binding geometry of the PBX1-PREP1 (and PBX1-MEIS1) complexes, under native in vivo conditions. This interaction stabilizes the two proteins and allows their translocation into the nucleus and thus their transcriptional activity. Both onco-suppressor PREP1 and the oncogene MEIS1 bind to PBX1.

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