Functional domain structure of human heterochromatin protein HP1(Hsalpha): involvement of internal DNA-binding and C-terminal self-association domains in the formation of discrete dots in interphase nuclei.
Human heterochromatin protein HP1(Hsalpha) possesses two evolutionarily conserved regions in the N- and C-terminal halves, so-called chromo and chromo-shadow domains, and DNA-binding domain in the internal non-conserved region. Here, to examine its in vivo properties, we expressed HP1(Hsalpha) as a fusion product with green fluorescent protein in human cells. HP1(Hsalpha) was observed to form discrete dots in interphase nuclei and to localize in the centromeric region of metaphase chromosomes by fluorescence microscopy. Interestingly, this dot-forming activity was also found in the N-terminal half retaining the chromo and DNA-binding domains and in the C-terminal chromo-shadow domain. However, the chromo domain alone stained nuclei homogeneously. To correlate this dot-forming activity with self-associating activity in vitro, the chromo and chromo-shadow domain peptides were independently expressed in Escherichia coli, affinity purified, and chemically cross-linked with glutaraldehyde. In a SDS-polyacrylamide gel, the former mainly produced a dimer, while the latter produced a ladder of bands up to a tetramer. When passed through a gel filtration column in a native state, these peptides were exclusively separated as a dimer and a tetramer, respectively. These results suggested that the internal DNA-binding and C-terminal chromo-shadow domains are both involved in heterochromatin formation in vivo.
Yamada
T
T
Laboratory of Applied Molecular Biology, Department of Applied Biochemistry, Osaka Prefecture University, Sakai, Osaka, 599-8531, Japan.
Fukuda
R
R
Himeno
M
M
Sugimoto
K
K
D001665:Binding Sites; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D000090266:Chromobox Protein Homolog 5; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003432:Cross-Linking Reagents; D004247:DNA / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D005976:Glutaral; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007399:Interphase; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D002868:Chromosomal Proteins, Non-Histone; D003432:Cross-Linking Reagents; D008164:Luminescent Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D000090266:Chromobox Protein Homolog 5; D049452:Green Fluorescent Proteins; D004247:DNA; D005976:Glutaral