Sequence-Specific Asymmetric Binding of Linker Histone to Nucleosome

Abstract

Linker histones (LHs) are critical for the formation of higher-order chromatin structure. Earlier studies showed that LHs exhibit a general preference for AT-rich DNA linkers. To account for this preference, we proposed that the asymmetric LH binding to nucleosome is stabilized by hydrophobic interaction between the ‘wing’ domain of LH and the thymine methyl groups in the major groove of linker DNA (Cui and Zhurkin, Nucl. Acids Res. 2009). To test this hypothesis, we prepared several constructs (based on the strongly positioned nucleosome ‘601’ designed by Lowary and Widom) with variable DNA linkers. The AT-rich constructs contained the TTT:AAA motif at the entry-exit point, whereas the GC-rich constructs had no A:T pairs at the nucleosome boundary.The band-shift experiments (6% PAGE) showed that the LH affinity to nucleosome is ∼1.7 times higher in the case of the AT-rich linker. Although this difference is not very significant, it was reliably reproduced. (The apparent Kd = 20 ± 3 nM [LH] for the AT-rich construct and 35 ± 5 nM for the GC-rich construct. Note that these Kd values reflect a high amount, 50 nM, of the wild type 601 construct used as a ‘competitor’ during gel electrophoresis.)In the second set of experiments we measured the LH-induced protection of linker DNA from the EcoRV cleavage. The EcoRV site, GATATC, was incorporated in the linker, 6-11 bp away from the entry-exit point of nucleosome (where LH is predicted to bind the DNA minor groove). In agreement with our model, the EcoRV cleavage rate of the AT-rich linker constitutes ∼60% of the corresponding rate for the GC-rich linker. The results are consistent with our hypothesis on the asymmetric binding of LH to the AT-rich linker DNA (which is tacitly ignored in the other models).