Quantitative analysis of HP1alpha binding to nucleosomal arrays.
Elucidating how the metazoan genome is organised into distinct functional domains is fundamental to understanding all aspects of normal cellular growth and development. The "histone code" hypothesis predicts that post-translational modifications of specific histone residues regulate genomic function by selectively recruiting nuclear factors that modify chromatin structure. A paradigm supporting this hypothesis is the preferential binding of the silencing protein heterochromatin protein 1 (HP1) to histone H3 trimethylated at K9. However, a caveat to several in vitro studies is that they employed histone N-terminal tail peptides to determine dissociation constants, thus ignoring any potential role of DNA and/or the underlying chromatin structure in the recruitment of HP1. Using a well-defined in vitro chromatin assembly system (employing a 12-208 DNA template), we describe here, the use of a fluorescence spectroscopic method that enabled us to measure and quantify the relative binding affinities of HP1alpha to unmodified and variant nucleosomal arrays. Using this approach, we previously demonstrated that mouse HP1alpha (i) binds with high affinity to naked DNA, (ii) has an intrinsic affinity for highly folded chromatin, (iii) has a 2-fold higher affinity for nucleosomal arrays when H2A is replaced with H2A.Z, and (iv) binds to DNA or chromatin in a non-cooperative manner.
Fan
Jun Y
JY
The John Curtin School of Medical Research, The Australian National University, P.O. Box 334, Canberra, ACT 2601, Australia. jun.fan@anu.edu.au
Zhou
Jiansheng
J
Tremethick
David J
DJ
D000818:Animals; D042002:Chromatin Assembly and Disassembly / Q000502:physiology*; D000090266:Chromobox Protein Homolog 5; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008956:Models, Chemical; D017711:Nonlinear Dynamics; D009707:Nucleosomes / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods*; D017186:Titrimetry
D002868:Chromosomal Proteins, Non-Histone; D009707:Nucleosomes; D000090266:Chromobox Protein Homolog 5