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Definition of the transcriptional activation domains of three human HOX proteins depends on the DNA-binding context - PubMed

Definition of the transcriptional activation domains of three human HOX proteins depends on the DNA-binding context

M A Viganò et al. Mol Cell Biol. 1998 Nov.

Abstract

Hox proteins control developmental patterns and cell differentiation in vertebrates by acting as positive or negative regulators of still unidentified downstream target genes. The homeodomain and other small accessory sequences encode the DNA-protein and protein-protein interaction functions which ultimately dictate target recognition and functional specificity in vivo. The effector domains responsible for either positive or negative interactions with the cell transcriptional machinery are unknown for most Hox proteins, largely due to a lack of physiological targets on which to carry out functional analysis. We report the identification of the transcriptional activation domains of three human Hox proteins, HOXB1, HOXB3, and HOXD9, which interact in vivo with the autoregulatory and cross-regulatory enhancers of the murine Hoxb-1 and human HOXD9 genes. Activation domains have been defined both in a homologous context, i.e., within a HOX protein binding as a monomer or as a HOX-PBX heterodimer to the specific target, and in a heterologous context, after translocation to the yeast Gal4 DNA-binding domain. Transfection analysis indicates that activation domains can be identified in different regions of the three HOX proteins depending on the context in which they interact with the DNA target. These results suggest that Hox proteins may be multifunctional transcriptional regulators, interacting with different cofactors and/or components of the transcriptional machinery depending on the structure of their target regulatory elements.

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Figures

FIG. 1
FIG. 1

(A) Schematic representation of the HOXD9 full-length protein and deletion mutants expressed by the pSGHOXD9 series of expression plasmids and of the pTHCR luciferase reporter plasmid. Patterned boxes indicate the HD. (B) Cotransfection assay in HeLa cells. Cells were transfected with 4 μg of reporter plasmid (HCR) and cotransfected with 0.5 to 5 μg of the different expression plasmids. The amount of transfected DNA was kept constant (10 μg) by addition of pSG5 plasmid. Bars represent the luciferase activity of transfected cell extracts (mean ± standard error of the mean [SEM] of at least four independent experiments, each carried out in duplicate), expressed as fold activation over the basal activity of the promoter-only reporter construct. Values were normalized by cotransfection of 0.1 μg of a pCMV–β-gal plasmid as an internal standard. (C) Immunoblot analysis of HeLa cells transfected with 5 μg of the indicated HOXD9 expression plasmids. Nuclear extracts (10 μg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis blotted onto nitrocellulose filters, and probed with a monoclonal antibody recognizing the HOXD9 homeodomain. M, molecular mass markers (in kilodaltons).

FIG. 2
FIG. 2

(A) Schematic representation of the Gal4 fusion proteins containing the HOXD9 N-terminal domain (positions 1 to 265) or its deletion mutants (positions 75 to 265, 142 to 265, and 222 to 265) and of the pTUAS luciferase reporter plasmid. Solid boxes represent the Gal4 1–147 DBD. (B) Transcriptional activity of HOXD9-Gal4 chimeras in HeLa cells transfected with 1 μg of reporter plasmid (UAS) and cotransfected with 1 to 5 μg of the different expression plasmids. Luciferase activity is expressed as fold activation over the basal activity of the promoter-only reporter construct (see the legend of Fig. 1 for details).

FIG. 3
FIG. 3

Best-fit alignment of the N-terminal regions of group 9 human (all capitals), mouse (m), and Xenopus (x) Hox proteins. Numbers indicate amino acid positions within the HOXD9 protein. Amino acids at the borders of the deletions generated in the HOXD9 N terminus (Fig. 1A) are indicated in boldface type.

FIG. 4
FIG. 4

(A) Schematic representation of the HOXB1 full-length protein and deletion mutants, of the PBX-1a protein, and of the pAdMLARE reporter plasmid. The solid boxes represent the HOXB1 HD and PBX HD, and the hatched boxes represent the conserved PBC-A and PBC-B domains of PBX1. (B) Transcriptional activity of the HOXB1-PBX1a complexes in P19 cells transfected with 4 μg of reporter plasmid (ARE) and cotransfected with 2 μg of the full-length HOXB1 (columns 2 and 6) or the deletion mutants B1Δ1–38 (columns 3 and 7), B1Δ1–90 (columns 4 and 8), or B1Δ1–155 (columns 5 and 9), and 4 μg of PBX1a (columns 1 and 6 to 9). Luciferase activity is expressed as fold activation over the basal activity of the promoter-only reporter construct (see the legend of Fig. 1 for details).

FIG. 5
FIG. 5

Best-fit alignment of the N-terminal regions of group 1 human (all capitals), mouse (m), rat (r), chicken (c) zebra fish (z), and Xenopus (x) Hox proteins. Numbers indicate amino acid positions within the HOXB1 protein. Amino acids at the borders of the deletions generated in the HOXB1 N terminus (Fig. 4A) are indicated in boldface type.

FIG. 6
FIG. 6

(A) Schematic representation of the Gal4 fusion proteins containing the HOXB1 N-terminal domain (positions 1 to 164) or its deletion mutants (positions 38 to 164 and 90 to 164), and of the pTUAS luciferase reporter plasmid. Solid boxes indicate the Gal4 1 147 DBD. (B) Transcriptional activity of the HOXB1-Gal4 chimeras in COS7 cells transfected with 2 μg of reporter plasmid (UAS) and cotransfected with 2 to 6 μg of B1(1–164)-Gal4, B1(38–164)-Gal4, B1(90–164)-Gal4, and Gal4-DBD. Luciferase activity is expressed as fold activation over the basal activity of the promoter-only reporter construct (see the legend of Fig. 1 for details).

FIG. 7
FIG. 7

(A) Schematic representation of the HOXB3 full-length protein and deletion mutants and of the pTCBS luciferase reporter plasmid. Patterned boxes indicate the HOXB3 HD. (B) Transcriptional activity of the HOXB3 mutants in COS7 cells transfected with 2 μg of the pTCBS reporter plasmid (CBS) and cotransfected with 1 to 6 μg of the indicated HOXB3 mutant. Luciferase activity is expressed as fold activation over the basal activity of the promoter-only reporter construct (see the legend of Fig. 1 for details). (C) EMSA analysis of the binding of in vitro-synthesized HOXB3 full-length protein and deletion mutants (6 μl of reticulocyte lysate [lanes 2 to 7]) to a labeled double-stranded oligonucleotide containing a HOX consensus binding site. Lane 1, free probe. ns, nonspecific binding.

FIG. 8
FIG. 8

(A) Schematic representation of the fusion proteins between the HOXB3 N terminus or C terminus and the Gal4 1–147 DBD and of the pTUAS reporter plasmid. (B) Transcriptional activity of the HOXB3-Gal4 chimeras in COS7 cells transfected with 2 μg of reporter plasmid (UAS) and cotransfected with 2 to 6 μg of B3(1–182)-Gal4 and B3(273–431)-Gal4, 4 to 6 μg of Gal4-B3(1–182) and Gal4-B3(273–431), and 6 μg of Gal4-DBD expression plasmids. Luciferase activity is expressed as fold activation over the basal activity of the promoter-only reporter construct (see the legend to Fig. 1 for details).

FIG. 9
FIG. 9

(A) Schematic representation of the HOXB1 and HOXB3 proteins, the HOXB3/B1 chimeric protein, and the HOXB3/B1 deletion mutants. Numbers indicate amino acid positions. Shaded and open boxes indicate N- and C-terminal regions from the HOXB1 and HOXB3 proteins, respectively, in the HOXB3/B1 chimeras. Solid and patterned boxes indicate regions from the HOXB1 and HOXB3 HD, respectively. pAdMLARE is represented in Fig. 4A. (B) Transcriptional activity of HOXB3/B1 mutants in P19 cells transfected with 4 μg of reporter plasmid (ARE) and cotransfected with 2 μg of HOXB3/B1 (columns 1 and 7), B3/B1Δ72–150 (columns 2 and 8), B3/B1Δ1–123 (columns 3 and 9), B3/B1Δ238–325 (columns 4 and 10), B3/B1Δ238–396 (columns 5 and 11), B3/B1Δ1–123/Δ238–396 (columns 6 and 12), and 4 μg of PBX1a (columns 7 to 13). Luciferase activity is expressed as fold activation over the basal activity of the promoter-only reporter construct (see the legend of Fig. 1 for details). (C) EMSA analysis of the binding of in vitro-synthesized (4 μl of reticulocyte lysate for each protein) PBX (lane 1), HOXB3-PBX (lane 2) HOXB1-PBX (lane 3), HOXB3/B1-PBX (lane 4), B3/B1Δ72–150-PBX (lane 5), B3/B1Δ1–123-PBX (lane 6), B3/B1Δ238–325-PBX (lane 7), and B3/B1Δ238–396-PBX (lane 8) complexes to a labeled double-stranded oligonucleotide containing the b1-ARE R3 repeat.

FIG. 9
FIG. 9

(A) Schematic representation of the HOXB1 and HOXB3 proteins, the HOXB3/B1 chimeric protein, and the HOXB3/B1 deletion mutants. Numbers indicate amino acid positions. Shaded and open boxes indicate N- and C-terminal regions from the HOXB1 and HOXB3 proteins, respectively, in the HOXB3/B1 chimeras. Solid and patterned boxes indicate regions from the HOXB1 and HOXB3 HD, respectively. pAdMLARE is represented in Fig. 4A. (B) Transcriptional activity of HOXB3/B1 mutants in P19 cells transfected with 4 μg of reporter plasmid (ARE) and cotransfected with 2 μg of HOXB3/B1 (columns 1 and 7), B3/B1Δ72–150 (columns 2 and 8), B3/B1Δ1–123 (columns 3 and 9), B3/B1Δ238–325 (columns 4 and 10), B3/B1Δ238–396 (columns 5 and 11), B3/B1Δ1–123/Δ238–396 (columns 6 and 12), and 4 μg of PBX1a (columns 7 to 13). Luciferase activity is expressed as fold activation over the basal activity of the promoter-only reporter construct (see the legend of Fig. 1 for details). (C) EMSA analysis of the binding of in vitro-synthesized (4 μl of reticulocyte lysate for each protein) PBX (lane 1), HOXB3-PBX (lane 2) HOXB1-PBX (lane 3), HOXB3/B1-PBX (lane 4), B3/B1Δ72–150-PBX (lane 5), B3/B1Δ1–123-PBX (lane 6), B3/B1Δ238–325-PBX (lane 7), and B3/B1Δ238–396-PBX (lane 8) complexes to a labeled double-stranded oligonucleotide containing the b1-ARE R3 repeat.

FIG. 10
FIG. 10

Best-fit alignment of the N-terminal (A) and C-terminal (B) regions of group 3 human (all capitals) and mouse (m) Hox proteins. Numbers indicate amino acid positions within the HOXB3 protein. Amino acids at the borders of the deletions generated in the HOXB3 N-terminus (Fig. 7A) are indicated in boldface type.

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