It is involved in hindbrain

It is involved in hindbrain Wortmannin segmentation and patterning. Hoxa1 misregulation has been associated with mammary carcinogenesis. We used a stringent high throughput yeast two hybrid approach to systematically test pairwise combinations, using Hoxa1 both as a bait and as a prey against the human ORFeome v3. 1 resource, which contains 12,212 ORFs representing 10,214 genes. Of the 59 Hoxa1 interactions identified, 45 could be validated by in vivo affinity binding assays in co transfected animal cells. A striking subset of the validated interactors are not proteins involved in gene regulation. Rather, these inter actors are adaptor proteins or modulators of the Bone Morphogenetic Proteins Tumor Growth Factor B, Tumor Necrosis Factor, Receptor Tyrosine Kinases and integrins signal transduction pathways.

Other interactors participate in cell adhesion or endosomal trafficking. We detected 41 interactions in live cells by Bimolecular Fluorescence Complementation. Depending on the different proteins identified, interactions either take place in the cytoplasm, in the nucleus, in association with vesicles or show a variable pattern from cell to cell, underscoring a dynamic inter play with Hoxa1. Numerous identified Hoxa1 partners reported to interact with each other within known pathways share similar intracellular patterns of Hoxa1 interaction by BiFC. We conclude that Hoxa1 can con tact several subunits of multi molecular functional plat forms involved in cell signaling, cell adhesion, or cell shape regulation.

Results A proteome wide yeast two hybrid screening for Hoxa1 interactors The yeast two hybrid is a powerful approach for large scale screenings to identify binary protein protein interactions. DB Hoxa1 was tested pairwise against 12,212 open reading frame derived pro teins from the human ORFeome version 3. 1 fused to the Gal4 activation domain. In this configur ation, we detected 40 distinct interactions. We also screened in the other configuration, Hoxa1 as a prey against the full hORFeome in fusion with the Gal4 DB. In the second configuration we detected 28 interactions, of which 8 were also detected in the DB Hoxa1 AD ORFs configuration. A total of 59 candidate Hoxa1 interactors were identified. We found the Hoxa1 homodimerization interaction and 8 out of the 9 Hoxa1 interactions, previously described in the literature.

Co purification from animal cells validate forty five Hoxa1 interactors To validate the 59 interactions identified by the Y2H screen by an orthogonal assay we turned to affinity co purification of a FLAG Hoxa1 fusion protein co expressed with glutathione S transferase tagged candidate interactors in transfected COS7 or HEK293T cells. In absence of GST partners, there was no or very weak GSK-3 back ground binding of FLAG Hoxa1 onto the glutathione agarose beads. As positive controls we measured Hoxa1 dimer formation and the reproducible interaction between Hoxa1 and Pbx1a.

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