Characterization of Nkx3.2 DNA binding specificity and its requirement for somitic chondrogenesis.
We have previously shown that Nkx3.2, a member of the NK class of homeoproteins, functions as a transcriptional repressor to promote somitic chondrogenesis. However, it has not been addressed whether Nkx3.2 can bind to DNA in a sequence-specific manner and whether DNA binding by Nkx3.2 is required for its biological activity. In this work, we employed a DNA binding site selection assay, which identified TAAGTG as a high affinity Nkx3.2 binding sequence. Sequence-specific binding of Nkx3.2 to the TAAGTG motif in vitro was confirmed by electrophoretic mobility shift assays, and mutagenesis of this sequence revealed that HRAGTG (where H represents A, C, or T, and R represents A or G) comprises the consensus DNA binding site for Nkx3.2. Consistent with these findings, the expression of a reporter gene containing reiterated Nkx3.2 binding sites was repressed in vivo by Nkx3.2 co-expression. In addition, we have generated a DNA nonbinding point mutant of Nkx3.2 (Nkx3.2-N200Q), which contains an asparagine to glutamine missense mutation in the homeodomain. Interestingly, despite being defective in DNA binding, Nkx3.2-N200Q still retains its intrinsic transcriptional repressor function. Finally, we demonstrate that unlike wild-type Nkx3.2, Nkx3.2-N200Q is unable to activate the chondrocyte differentiation program in somitic mesoderm, indicating that DNA binding by Nkx3.2 is critical for this factor to induce somitic chondrogenesis.
Kim
Dae-Won
DW
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Kempf
Herve
H
Chen
Raymond E
RE
Lassar
Andrew B
AB
D016475:3T3 Cells; D020816:Amino Acid Motifs; D000818:Animals; D001216:Asparagine / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D002642:Chick Embryo; D019902:Chondrocytes / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D017930:Genes, Reporter; D005973:Glutamine / Q000737:chemistry; D005982:Glutathione Transferase / Q000378:metabolism; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D018398:Homeodomain Proteins; C107965:Nkx3-2 protein, mouse; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D005973:Glutamine; D001216:Asparagine; D004247:DNA; D005982:Glutathione Transferase