Mechanisms underlying the nuclear transport of histones and histone-related proteins

Abstract

The CCAAT-specific transcription factor NF-Y is a heterotrimeric complex composed of NF-YA, NF-YB, and NF-YC. Each subunit contains an evolutionarily conserved core region, comprising a histone fold motif (HFM) in the case of NF-YB and NF-YC. Here, we report that in higher eukaryotes the subunits of NF-Y are imported into the nucleus by distinct pathways. Importin β mediates nuclear import of NF-YA, whereas heterodimerization of NF-YB and NF-YC is a prerequisite for nuclear accumulation conferred by importin 13. The monomeric HFM containing subunits NF-YB and NF-YC lack a functional nuclear localization signal (NLS) but the minimal sequence regions necessary for nuclear localization basically correspond to their HFMs. This indicates that in the NF-YB/NF-YC complex a distinct binding platform derived from the HFM of both subunits mediates the interactions with importin 13. This assumption was supported by the fact that binding of importin 13 to NF-YB/NF-YC was competed by NF-YA which also only binds to the dimerized HFM. NF-YA contains a nonclassical NLS (ncNLS) at the C-terminus of the protein, and mutational analysis indicates that a certain number of positively charged amino acid residues in the NLS sequence is required for nuclear accumulation. Importin β binding is restricted to the monomeric, uncomplexed NF-YA subunit.Redundant receptor-mediated pathways exist for the nuclear import of core histones. Previously, in vivo transfection experiments demonstrated that both the amino-terminal tails and the central globular domains of all core histones can serve as regions involved in nuclear targeting. However, here we report that import receptors only interact directly with the amino-terminal tails of core histones (in vitro binding and nuclear import assays). The result that globular histone domains (containing a HFM) are not recognized by importins is in line with the data obtained from the HFM containing subunits NF-YB and NF-YC and strongly suggests that the HFM represents a protein-protein interaction domain but not a NLS per se. Hence, the nuclear transport competence of globular domains of core histones in vivo depends most likely on the formation of histone heterodimers (oligomers). The results of in vitro nuclear import assays showed that the globular domain of H2B can be piggyback transported into the nucleus via the ncNLS in the amino-terminal tail of the corresponding partner histone H2A. These results further indicate that histone import signals are still accessible to importin binding when H2A and H2B are dimerized.