Dynamic interaction of HMGA1a proteins with chromatin.
High-mobility-group proteins A1 (HMGA1; previously named HMGI/Y) function as architectural chromatin-binding proteins and are involved in the transcriptional regulation of several genes. We have used cells expressing proteins fused to green fluorescent protein (GFP) and fluorescence recovery after photobleaching (FRAP) to analyze the distribution and dynamics of HMGA1a in vivo. HMGA1-GFP proteins localize preferentially to heterochromatin and remain bound to chromosomes during mitosis. FRAP experiments showed that they are highly mobile components of euchromatin, heterochromatin and of mitotic chromosomes, although with different resident times. For a more-detailed investigation on the interaction of HMGA1a with chromatin, the contribution of the AT-hook DNA-binding motifs was analyzed using point-mutated HMGA1a-GFP proteins. Furthermore, by inhibiting kinase or histone deacetylase activities, and with the help of fusion proteins lacking specific phosphorylation sites, we analyzed the effect of reversible modifications of HMGA1a on chromatin binding. Collectively our data show that the kinetic properties of HMGA1a proteins are governed by the number of functional AT-hooks and are regulated by specific phosphorylation patterns. The higher residence time in heterochromatin and chromosomes, compared with euchromatic regions, correlates with an increased phosphorylation level of HMGA1a. The regulated dynamic properties of HMGA1a fusion proteins indicate that HMGA1 proteins are mechanistically involved in local and global changes in chromatin structure.
Harrer
Monika
M
Department of Cell and Developmental Biology, University of Würzburg, Würzburg 97080, Germany.
Lührs
Hardi
H
Bustin
Michael
M
Scheer
Ulrich
U
Hock
Robert
R
D001483:Base Sequence; D015153:Blotting, Western; D002453:Cell Cycle; D002843:Chromatin / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D025741:HMGA1a Protein / Q000378:metabolism*; D006801:Humans; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D002843:Chromatin; D011993:Recombinant Fusion Proteins; D025741:HMGA1a Protein; D049452:Green Fluorescent Proteins