Methylation-independent binding to histone H3 and cell cycle-dependent incorporation of HP1beta into heterochromatin.
We have examined HP1beta-chromatin interactions in different molecular contexts in vitro and in vivo. Employing purified components we show that HP1beta exhibits selective, stoichiometric, and salt-resistant binding to recombinant histone H3, associating primarily with the helical "histone fold" domain. Furthermore, using "bulk" nucleosomes released by MNase digestion, S-phase extracts, and fragments of peripheral heterochromatin, we demonstrate that HP1beta associates more tightly with destabilized or disrupted nucleosomes (H3/H4 subcomplexes) than with intact particles. Western blotting and mass spectrometry data indicate that HP1beta-selected H3/H4 particles and subparticles possess a complex pattern of posttranslational modifications but are not particularly enriched in me3K9-H3. Consistent with these results, mapping of HP1beta and me3K9-H3 sites in vivo reveals overlapping, yet spatially distinct patterns, while transient transfection assays with synchronized cells show that stable incorporation of HP1beta-gfp into heterochromatin requires passage through the S-phase. The data amassed challenge the dogma that me3K9H3 is necessary and sufficient for HP1 binding and unveil a new mode of HP1-chromatin interactions.
Stem Cell and Chromatin Group, Laboratory of Biology, The University of Ioannina, School of Medicine and Ioannina Biomedical Research Institute/Foundation for Research and Technology, 45 110 Ioannina, Greece.
George K
GK
Dialynas
Dimitra
D
Makatsori
Niki
N
Kourmouli
Panayiotis A
PA
Theodoropoulos
Kevin
K
McLean
Stefan
S
Terjung
Prim B
PB
Singh
Spyros D
SD
Georgatos
D000595:Amino Acid Sequence; D000818:Animals; D002453:Cell Cycle; D002467:Cell Nucleus / Q000378:metabolism; D000090266:Chromobox Protein Homolog 5; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry*; D004285:Dogs; D006367:HeLa Cells; D006570:Heterochromatin / Q000737:chemistry*; D006657:Histones / Q000737:chemistry*; D006801:Humans; D008745:Methylation; D008969:Molecular Sequence Data; D011485:Protein Binding; D051381:Rats
C000715593:CBX1 protein, human; D002868:Chromosomal Proteins, Non-Histone; D006570:Heterochromatin; D006657:Histones; D000090266:Chromobox Protein Homolog 5