The tumor suppressor HIC1 (hypermethylated in cancer 1) is O-GlcNAc glycosylated.
HIC1 (hypermethylated in cancer 1) is a transcriptional repressor containing five Krüppel-like C(2)H(2) zinc fingers and an N-terminal dimerization and autonomous repression domain called BTB/POZ. Here, we demonstrate that full-length HIC1 proteins are modified both in vivo and in vitro with O-linked N-acetylglucosamine (O-GlcNAc). This is a highly dynamic glycosylation found within the cytosolic and the nuclear compartments of eukaryotes. Analysis of [(3)H]Gal-labeled tryptic peptides indicates that HIC1 has three major sites for O-GlcNAc glycosylation. Using C-terminal deletion mutants, we have shown that O-GlcNAc modification of HIC1 proteins occurred preferentially in the DNA-binding domain. Nonglycosylated and glycosylated forms of full-length HIC1 proteins separated by wheat germ agglutinin affinity purification, displayed the same specific DNA-binding activity in electrophoretic mobility shift assays proving that the O-GlcNAc modification is not directly implicated in the specific DNA recognition of HIC1. Intriguingly, N-terminal truncated forms corresponding to BTB-POZ-deleted proteins exhibited a strikingly differential activity, as the glycosylated truncated forms are unable to bind DNA whereas the unglycosylated ones do. Electrophoretic mobility shift assays performed with separated pools of glycosylated and unglycosylated forms of a construct exhibiting only the DNA-binding domain and the C-terminal tail of HIC1 (residues 399-714) and supershift experiments with wheat germ agglutinin or RL-2, an antibody raised against O-GlcNAc residues, fully corroborated these results. Interestingly, these truncated proteins are O-GlcNAc modified in their C-terminal tail (residues 670-711) and not in the DNA-binding domain, as for the full-length proteins. Thus, the O-GlcNAc modification of HIC1 does not affect its specific DNA-binding activity and is highly sensitive to conformational effects, notably its dimerization through the BTB/POZ domain.
Lefebvre
Tony
T
UMR 8526 du CNRS, Institut de Biologie de Lille, Institut Pasteur de Lille, Lille Cédex BP447, France.
Pinte
Sébastien
S
Guérardel
Cateline
C
Deltour
Sophie
S
Martin-Soudant
Nathalie
N
Slomianny
Marie-Christine
MC
Michalski
Jean-Claude
JC
Leprince
Dominique
D
D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity; D006224:Cricetinae; D019175:DNA Methylation; D024202:Electrophoretic Mobility Shift Assay; D016147:Genes, Tumor Suppressor; D006001:Glycoconjugates / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D051741:Kruppel-Like Transcription Factors; D009369:Neoplasms / Q000378:metabolism* / Q000473:pathology; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D014157:Transcription Factors / Q000378:metabolism*; D014357:Trypsin / Q000494:pharmacology; D014909:Wheat Germ Agglutinins / Q000378:metabolism
D006001:Glycoconjugates; C096870:Hic1 protein, mouse; D051741:Kruppel-Like Transcription Factors; D010446:Peptide Fragments; D014157:Transcription Factors; D014909:Wheat Germ Agglutinins; D014357:Trypsin; D000117:Acetylglucosamine