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

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, Imp␤, Kap␤2, Imp4, Imp5, Imp7, Imp9, and Imp␣, show the H3 tail binding more tightly than the H4 tail. The H3 tail binds Kap␤2 and Imp5 with K D 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 Imp␤, Kap␤2, and Imp4. However, Imp5, Imp7, Imp9, and Imp␣ 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 Lys 14 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.New nucleosomes are assembled in the nucleus during S phase as new core histones H2A, H2B, H3, and H4 are synthesized, assembled into H2A/H2B and H3/H4 dimers in the cytoplasm, and then imported into the nucleus for deposition onto replicating chromatin (1-8). Little is known about cytoplasmic assembly/processing of H2A and H2B, but assembly/processing of H3 and H4 are better understood. In the cytoplasm, H3 and H4 are passed from one histone chaperone to another for folding, assembly into H3/H4 dimers, and acetylation.

Karyopherin-β2 Recognition of a PY-NLS Variant that Lacks the Proline-Tyrosine Motif

“…The very strong Epitope 1 also contains two acetylation sites as 20–30% of cytoplasmic H3 is acetylated at Lys14 (Kapβ2-binding hotspot) and/or Lys18 (Jasencakova et al, 2010). Lys14 acetylation abolished Kapβ2 binding, suggesting that the modification may attenuate nuclear import of the small pool of cytoplasmic H3/H4 acetylated at H3 Lys14 (Soniat et al, 2016). …”

“…Kapβ2 binds H4 tail residues 5–20 but the affinity is ~11-fold weaker than the H3 tail (H4, K D 871 nM vs. H3, K D 77 nM) (Soniat et al, 2016). rpL23A uses a 43-residue β-like import receptor binding (BIB) sequence to bind Kapβ2 and other Importins in cell lysates, but biochemical studies with recombinant proteins are not available (Jakel and Gorlich, 1998).…”

“…Despite importance of the H3 tail, the histone fold domain of H3 may also contribute to Importin binding. A recent study shows that both histone tails and histone fold domains are important for Importin binding (Soniat et al, 2016). A complete picture of how H3 is transported awaits structures of Importins bound to full-length H3/H4 dimers.…”

“…The H3 tail binds Impβ, Kapβ2, Imp4, Imp5, Imp7, Imp9 and Impα with affinities ranging from 50–500 nM (Soniat et al, 2016). H3 residues 11–27 are also important for binding the other Importins (Soniat et al, 2016).…”

A promiscuous split intein with expanded protein engineering applications

“…Initial studies revealed poor incorporation of the requisite truncated histone-intein fusion construct (Cfa C WT -H3 ) into native chromatin of HEK293T cells, possibly as a result of removal of recognition sequences required for nuclear localization and/or an inability to be recognized by histone chaperones (SI Appendix, Fig. S13) (28). This problem was solved by fusing the missing 28 residues of H3 to the N terminus of Cfa C , in effect embedding the intein fragment within the histone (Fig.…”