Selection of homeotic proteins for binding to a human DNA replication origin

• Published in: Journal of molecular biology Vol. 299; no. 3; pp. 667 - 680
• Main Authors: de Stanchina, E, Gabellini, D, Norio, P, Giacca, M, Peverali, F A, Riva, S, Falaschi, A, Biamonti, G
• Format: Journal Article
• Language: English
• Published: Netherlands 09.06.2000
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Cell Division; Cell Line; DNA - genetics; DNA - metabolism; DNA Footprinting; Gene Expression Regulation; Genes, Homeobox - genetics; Homeodomain Proteins - chemistry; Homeodomain Proteins - genetics; Homeodomain Proteins - metabolism; HOXA13 protein; HOXC10 protein; HOXC13 protein; Humans; lamin B2; Lamin Type B; Lamins; Mice; Molecular Sequence Data; Nuclear Proteins - genetics; Open Reading Frames - genetics; Organ Specificity; Protein Binding; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Replication Origin - genetics; replication origins; RNA, Messenger - analysis; RNA, Messenger - genetics; Substrate Specificity; Transfection; Two-Hybrid System Techniques
• ISSN: 0022-2836
• DOI: 10.1006/jmbi.2000.3782

We have previously shown that a cell cycle-dependent nucleoprotein complex assembles in vivo on a 74 bp sequence within the human DNA replication origin associated to the Lamin B2 gene. Here, we report the identification, using a one-hybrid screen in yeast, of three proteins interacting with the 74 bp sequence. All of them, namely HOXA13, HOXC10 and HOXC13, are orthologues of the Abdominal-B gene of Drosophila melanogaster and are members of the homeogene family of developmental regulators. We describe the complete open reading frame sequence of HOXC10 and HOXC13 along with the structure of the HoxC13 gene. The specificity of binding of these two proteins to the Lamin B2 origin is confirmed by both band-shift and in vitro footprinting assays. In addition, the ability of HOXC10 and HOXC13 to increase the activity of a promoter containing the 74 bp sequence, as assayed by CAT-assay experiments, demonstrates a direct interaction of these homeoproteins with the origin sequence in mammalian cells. We also show that HOXC10 expression is cell-type-dependent and positively correlates with cell proliferation.