| Autor: |
Lefebvre, Tony, Pinte, Sébastien, Guérardel, Cateline, Deltour, Sophie, Martin-Soudant, Nathalie, Slomianny, Marie-Christine, Michalski, Jean-Claude, Leprince, Dominique |
| Předmět: |
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| Zdroj: |
European Journal of Biochemistry; Oct2004, Vol. 271 Issue 19, p3843-3854, 12p |
| Abstrakt: |
HIC1(hypermethylated in cancer 1) is a transcriptional repressor containing five Krüppel-like C2H2 zinc fingers and an N-terminal dimerization and autonomous repression domain called BTB/POZ. Here, we demonstrate that full-length HIC1 proteins are modified bothin vivoandin vitrowith O-linkedN-acetylglucosamine (O-GlcNAc). This is a highly dynamic glycosylation found within the cytosolic and the nuclear compartments of eukaryotes. Analysis of[3H]Gal-labeled tryptic peptides indicates that HIC1 has three major sites forO-GlcNAc glycosylation. Using C-terminal deletion mutants, we have shown thatO-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 theO-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 againstO-GlcNAc residues, fully corroborated these results. Interestingly, these truncated proteins areO-GlcNAc modified in their C-terminal tail (residues 670–711) and not in the DNA-binding domain, as for the full-length proteins. Thus, theO-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. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
| Externí odkaz: |
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