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. 2012 Oct 22;12:29.
doi: 10.1186/1471-213X-12-29.

Protein Interactions of the Transcription Factor Hoxa1

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Free PMC article

Protein Interactions of the Transcription Factor Hoxa1

Barbara Lambert et al. BMC Dev Biol. .
Free PMC article

Abstract

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.

Figures

Figure 1
Figure 1
Validation of 45 out of the 59 interactions revealed for Hoxa1 by affinity co-purification on glutathione-agarose beads. Candidate interactors were fused with a GST-tag and co-expressed in transfected cells with a FLAG-Hoxa1 fusion protein. Western blots were run to detect FLAG-Hoxa1 from cell extracts before (Input) or after (Co-P) purification. The Hoxa1-Hoxa1 or PBX1A-Hoxa1 interactions were used as positive controls (see lanes with arrowheads). Negative control corresponds to transfected cells with the only FLAG-Hoxa1 fusion protein (C-, lanes with blue arrows). Some interactors which could not be confirmed by co-purification are also shown (red asterisks).
Figure 2
Figure 2
Bimolecular Fluorescence Complementation assay reveals the Hoxa1-PBX1A interaction in culture cells. COS7 cells were transfected with combinations of VN173, VN173-PBX1A, VC155 and VC155-Hoxa1 expression vectors. Upon interaction between PBX1A and Hoxa1, the VN173 and VC155 moieties of the Venus fluorescent protein brought together provide a fluorescent signal (A). Fluorescence complementation does not appear when the VN173 or VC155 fragments are expressed instead of the corresponding fusion proteins (B-C). As a control, expression of Venus fragments is detected by immunocytochemistry (anti-GFP). The BiFC signal shows colocalization with the anti-GFP immunofluorescence (D-F).
Figure 3
Figure 3
Bimolecular Fluorescence Complementation assay reveals the Hoxa1-mediated interactions in culture cells. MCF10A cells were transfected with VN173-hORF and VC155-Hoxa1 fusion proteins. Upon interaction between the partner proteins, the VN173 and VC155 moieties of the Venus fluorescent protein brought together provide a fluorescent signal. The interactions between Hoxa1 and its interactors can be classified according to their intracellular pattern: (A) nuclear, (B) cytoplasmic or associated to vesicles, (C) nuclear and cytoplasmic and/or vesicular.
Figure 4
Figure 4
Hoxa1 and TRAF1 intracellular distributions overlap. MCF10A cells were transfected with GST-TRAF1 and Hoxa1 expression vectors. The immunolocalization of GST-TRAF1 and Hoxa1 (anti-GST, anti-TRAF1 and anti-Hoxa1 immunocytofluorescence) reveals that the partner proteins display partially overlapping intracellular distribution.

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