Differential dissociation of chromatin digests: a novel approach revealing a hierarchy of DNA-protein interactions within chromatin domains.
We describe here a novel approach to the dissection of chromatin structure by extracting DNA fragments from digested nuclei irreversibly immobilized (via proteins) on Celite columns. Three successive gradients (NaCl, LiCl-urea, temperature) are used to release three families of DNA fragments: namely, the 'DNA adherence' classes DNA-0, DNA-I and DNA-II, respectively. This 'protein image' DNA chromatography separates DNA fragments in accordance with the tightness of their bonds with proteins in situ. There are at least two DNA-skeleton attachment sites differing from each other by their resistance to the dissociating agents used as well as their susceptibility to DNAase I and S1 nuclease treatments, DNA cross-linking and single-stranded breaks. Several lines of evidence show a specific, topological rather than chemical, DNA-protein linkage at the tight attachment site. A hierarchy of chromatin loops demarcated by these attachment sites was determined. The technique described is generally applicable and can be used both to probe DNA-protein interactions and to map specific DNA sequences within the chromatin domain.
Laboratory of Tumor Biochemistry, All-Union Cancer Research Center, Moscow, USSR.
A V
AV
Lichtenstein
M M
MM
Zaboikin
N I
NI
Sjakste
R P
RP
Alechina
D002458:Cell Fractionation / Q000379:methods; D002843:Chromatin / Q000302:isolation & purification* / Q000378:metabolism; D002845:Chromatography / Q000379:methods; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D009690:Nucleic Acid Conformation; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*
D002843:Chromatin; D011506:Proteins; D004247:DNA