Recognition Elements in the Histone H3 and H4 Tails for Seven Different Importins

Michael Soniat,Tolga Cağatay,Yuh Min Chook

doi:10.1074/jbc.m116.730218

Michael Soniat, Tolga Cağatay + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m116.730218

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Abstract

N-terminal tails of histones H3 and H4 are known to bind several different Importins to import the histones into the cell nucleus. However, it is not known what binding elements in the histone tails are recognized by the individual Importins. Biochemical studies of H3 and H4 tails binding to seven Importins, Impβ, Kapβ2, Imp4, Imp5, Imp7, Imp9, and Impα, show the H3 tail binding more tightly than the H4 tail. The H3 tail binds Kapβ2 and Imp5 with KD values of 77 and 57 nm, respectively, and binds the other five Importins more weakly. Mutagenic analysis shows H3 tail residues 11–27 to be the sole binding segment for Impβ, Kapβ2, and Imp4. However, Imp5, Imp7, Imp9, and Impα bind two separate elements in the H3 tail: the segment at residues 11–27 and an isoleucine-lysine nuclear localization signal (IK-NLS) motif at residues 35–40. The H4 tail also uses either one or two basic segments to bind the same set of Importins with a similar trend of relative affinities as the H3 tail, albeit at least 10-fold weaker. Of the many lysine residues in the H3 and H4 tails, only acetylation of the H3 Lys14 substantially decreased binding to several Importins. Lastly, we show that, in addition to the N-terminal tails, the histone fold domains of H3 and H4 and/or the histone chaperone Asf1b are important for Importin-histone recognition.

Highlights

SEPTEMBER 30, 2016 VOLUME 291 NUMBER 40 ins for transport into the nucleus (9 –18)
Activities of the eight ␤-Importins (Imp␤, Kap␤2, Imp4, Imp5, Imp7, and Imp9) were verified by RanGTP binding, and activity of Imp␣ was verified by classical NLS binding (Fig. 1, C and E)
The results inform on both the NLS organization in the histone tails and general sequence elements that bind different Importins

Summary

Results

Seven Different Human Importins Bind the H3 and H4 Tails—H3 and H4 tails were shown to mediate nuclear import of the histones by binding to several different Importins (12, 16 –18, 29). Band densities for bound Importins and histone tails in pulldown binding assays were measured, and their ratios were compared in a histogram to estimate relative strengths of H3 tail and H4 tail binding to Imp␤, Kap␤2, Imp, Imp, Imp, Imp, and Imp␣ (Fig. 1B). Comparison of bound Importin bands suggests that Importin-H3 tail interactions can be roughly divided into two groups: 1) Kap␤2 and Imp, known from ITC to bind tightly (KD values Ͻ100 nM), and 2) Imp␤, Imp, Imp, Imp, and Imp␣, which all seem to bind more weakly. ITC data show that mutation of all basic residues within H3 residues 11–27 (mutant MBP-H3 tail(K14A/K17A/K18A/ K23A/R26A/K27A) decreases Imp affinity ϳ15-fold (KD of 862 nM for the mutant versus 57 nM for wild-type H3 tail; Table 1) Pulldown of this mutant (GST-H3 tail(K14A/ K17A/K18A/K23A/R26A/K27A)) still shows Imp binding, suggesting additional binding element(s) beyond H3 residues 11–27 (Fig. 2B).

KD determined by ITC

Discussion

Experimental Procedures