HOXA1 binds RBCK1/HOIL-1 and TRAF2 and modulates the TNF/NF-κB pathway in a transcription-independent manner.

HOX proteins define a family of key transcription factors regulating animal embryogenesis. HOX genes have also been linked to oncogenesis and HOXA1 has been described to be active in several cancers, including breast cancer. Through a proteome-wide interaction screening, we previously identified the TNFR-associated proteins RBCK1/HOIL-1 and TRAF2 as HOXA1 interactors suggesting that HOXA1 is functionally linked to the TNF/NF-κB signaling pathway. Here, we reveal a strong positive correlation between expression of HOXA1 and of members of the TNF/NF-κB pathway in breast tumor datasets. Functionally, we demonstrate that HOXA1 can activate NF-κB and operates upstream of the NF-κB inhibitor IκB. Consistently, we next demonstrate that the HOXA1-mediated activation of NF-κB is non-transcriptional and that RBCK1 and TRAF2 influences on NF-κB are epistatic to HOXA1. We also identify an 11 Histidine repeat and the homeodomain of HOXA1 to be required both for RBCK1 and TRAF2 interaction and NF-κB stimulation. Finally, we highlight that activation of NF-κB is crucial for HOXA1 oncogenic activity.

Protein interactions of the transcription factor Hoxa1.

BACKGROUND: Hox proteins are transcription factors involved in crucial processes during animal development. Their mode of action remains scantily documented. While other families of transcription factors, like Smad or Stat, are known cell signaling transducers, such a function has never been squarely addressed for Hox proteins. RESULTS: To investigate the mode of action of mammalian Hoxa1, we characterized its interactome by a systematic yeast two-hybrid screening against ~12,200 ORF-derived polypeptides. Fifty nine interactors were identified of which 45 could be confirmed by affinity co-purification in animal cell lines. Many Hoxa1 interactors are proteins involved in cell-signaling transduction, cell adhesion and vesicular trafficking. Forty-one interactions were detectable in live cells by Bimolecular Fluorescence Complementation which revealed distinctive intracellular patterns for these interactions consistent with the selective recruitment of Hoxa1 by subgroups of partner proteins at vesicular, cytoplasmic or nuclear compartments. CONCLUSIONS: The characterization of the Hoxa1 interactome presented here suggests unexplored roles for Hox proteins in cell-to-cell communication and cell physiology.

Pentapeptide insertion mutagenesis of the Hoxa1 protein: mapping of transcription activation and DNA-binding regulatory domains.

The mode of action of Hoxa1, like that of most Hox proteins, remains poorly characterized. In an effort to identify functional determinants contributing to the activity of Hoxa1 as a transcription factor, we generated 18 pentapeptide insertion mutants of the Hoxa1 protein and we assayed them in transfected cells for their activity on target enhancers from the EphA2 and Hoxb1 genes known to respond to Hoxa1 in the developing hindbrain. Only four mutants displayed a complete loss-of-function. Three of them contained an insertion in the homeodomain of Hoxa1, whereas the fourth loss-of-function mutant harbored an insertion in the very N-terminal end of the protein. Transcription activation assays in yeast further revealed that the integrity of both the N-terminal end and homeodomain is required for Hoxa1-mediated transcriptional activation. Furthermore, an insertion in the serine-threonine-proline rich C-terminal extremity of Hoxa1 induced an increase in activity in mammalian cells as well as in the yeast assay. The C-terminal extremity thus modulates the transcriptional activation capacity of the protein. Finally, electrophoretic mobility shift assays revealed that the N-terminal extremity of the protein also exerts a modulatory influence on DNA binding by Hoxa1-Pbx1a heterodimers.