Structure and function correlation in histone H2A peptide-mediated gene transfer

Abstract

Histone H2A has been found to be efficient in DNA delivery into a number of cell lines. We have reasoned that this DNA-delivery activity is mediated by two mechanisms: (i) electrostatically driven DNA binding and condensation by histone and (ii) nuclear import of these histone H2A⅐DNA polyplexes via nuclear localization signals in the protein. We have identified a 37-aa N-terminal peptide of histone H2A that is active in in vitro gene transfer. This peptide can function as a nuclear localization signal and can bind DNA. Amino acid substitutions that replace positively charged residues and͞or DNA-binding residues of this peptide obliterate transfection activity. The introduction of a proline in the first turn of an ␣-helix of this 37-mer obliterates transfection activity, suggesting that the integrity of the ␣-helical structure of the N-terminal region of histone H2A is related to its transfection activity.transfection ͉ nuclear localization signal ͉ DNA binding G ene transfer into eukaryotic cells has the potential to treat a large number of incurable diseases. The goal of nonviral gene therapy is to mimic the successful viral mechanisms for overcoming cellular barriers that block efficient expression of the target gene while minimizing the toxicities associated with gene delivery (1). The capabilities of a synthetic nonviral vector could include specific binding to the cell surface, entry, endosomal escape, translocation to the nucleus, and in some cases stable integration into the target cell genome. The rate-limiting step of current nonviral gene delivery techniques is the transfer of encapsulated plasmids from the endosomes to the nucleus (2). The potential advantages of protein͞peptide gene transfer include ease of use, production, purity, homogeneity, ability to target nucleic acids to specific cell types, the potential for cost-effective large-scale manufacture, modular attachment of targeting ligands, and the lack of limitation on the size or type of the nucleic acid that can be delivered (1). The critical first step for efficient gene delivery is the formation of the polyplex, or complex between DNA and protein͞peptide (1-3).Eukaryotic DNA within the nucleus is packaged by wrapping around a histone octamer consisting of two molecules each of histone H2A, H2B, H3, and H4, and this packaging has dramatic effects on DNA metabolism and gene regulation (reviewed by Van Holde in ref. 4). We determined that histone octamers mediate transfection into a number of cell lines and that this activity is localized to H2A. It was not observed with other histones or cationic peptides (5, 6). Previous studies have demonstrated that histones H1 (7-12), H2A (5, 6, 13, 14), and H3 and H4 (15) are effective mediators of transfection. The postulated mechanisms by which histone H1 increases gene transfection are through DNA condensation, DNase protection, and the mediation of nuclear import. In these studies, histone H1 was considered to be the subclass of histones that mediates efficient gene transfer in the presence of chlo...

Improvement of exogenous DNA nuclear importation by nuclear localization signal‐bearing vectors: a promising way for non‐viral gene therapy?

“…Other cationic peptides with a less canonical NLS sequence (with regard to that of SV40 antigen T), such as protamine (Wienhues et al, 1987), histone H2 (Balicki et al, 2002) and melittin-derived peptides , have been shown to display DNA compaction and enhanced in vitro transfection efficiency properties.…”

Transgene expression efficiency from plasmid DNA delivered as a complex with histone H3

“…Histones have the NLS sequence, and importance of the NLS sequence was reported for histone H2A (Wagstaff et al, 2007;Balicki et al, 2002), suggesting nuclear entry through the nuclear pores. However, this interpretation should be reviewed carefully, since the DNA−H3 complexes were ~1,000 nm in size (Table 1).…”

“…The nuclear localizing signal (NLS) sequence of histone H2A is important for the transfection efficiency, and the additional attachment of the NLS peptide enhances both the nuclear entry of the H2B−DNA complex and transgene expression (Wagstaff et al, 2007;Balicki et al, 2002). Thus, the histone−DNA complexes are expected to penetrate the cell membrane by a non-endocytotic pathway(s) and enter the nucleus through nuclear pores.…”

Advances in Gene Delivery Systems

“…A significant number of polymers, such as polyethylenimine, [93][94][95] polyamidoamine, [96][97][98] polyallylamine, [99] chitosan, [100] dendrimers, [101] cationic proteins [102] and peptides, [103] have been studied for in vivo and in vitro gene delivery. These cationic polymers condense DNA into small particles and prevent DNA from degradation.…”