Abstract
AbstractHomeobox genes encode proteins with a highly conserved DNA-binding motif and provoke morphological diversification of body segments by differentially controlling the expression of downstream targets. Here, we have identified hbx4, one of many homeobox genes in Dictyostelium discoideum and investigated its role during growth and development. In suspension, Hbx4-overexpressing cells, Hbx4^OE^, showed defects in cytokinesis and growth rate. During development, Hbx4^OE^ and hbx4-disrupting cells, hbx4&175; made differences in shape of mound and slug, cell-type proportioning from wild type KAx3 cells. These phenotypes were similar to those of mutant defective in cadA encoding Ca^2+^-dependent cell adhesion molecule so that we investigated the relationship between hbx4 and cadA. Overexpression of Hbx4 inhibited the expression of cadA and cAMP also failed to stimulate cadA in Hbx4^OE^. Furthermore, gel mobility shift assay showed the promoter of cadA contained Hbx4-binding site, indicating Hbx4 negatively regulates the expression of cadA. Proteome analysis revealed that overexpression of Hbx4 repressed the rdiA and abpB encoding rho guanine nucleotide dissociation inhibitor1, RhoGDI1 and actin bundling protein 34, ABP34, respectively. And the overexpression of cadA in Hbx4^OE^ cells rescued the defects and increased mRNA level of rdiA, abpB and one of Rho GTPase, rac1b. These results suggested that Hbx4 can modulate cytokinesis, cell sorting and cell-type proportioning by repressing cadA that regulates GTPase-dependent signaling pathway.
Highlights
Homeobox genes encode proteins with a highly conserved DNA-binding motif and provoke morphological diversification of body segments by differentially controlling the expression of downstream targets
To address the possibility that this phenomenon results from cytokinesis defect, we performed co-immunostaining with DAPI for DNA and TRITC-phalloidin for Factin as described in method
The rate of increase in cell number was significantly lower in Hbx4OE cells and slightly higher in hbx4 ̄ cells than that of KAx3 cells (Supplementary Fig. 4a) while the rates of increase in cell mass monitored as the turbidity of the culture were similar in KAx3, Hbx4OE and hbx4 ̄ cells (Supplementary Fig. 4b)
Summary
Homeobox genes encode proteins with a highly conserved DNA-binding motif and provoke morphological diversification of body segments by differentially controlling the expression of downstream targets. We identified the hbx[4] (DDB_G0272967), homeobox-containing gene 4, which encodes conserved homeodomain (Supplementary Fig. 1a) and prepared overexpression (Hbx4OE) and disruption (hbx4 ̄) mutants. Hbx4OE cells produced large multinucleate cells and hbx4 ̄ cells showed mono- or dinucleate similar to KAx3 (Fig. 2a).
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