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 H2A.DNA 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 and/or DNA-binding residues of this peptide obliterate transfection activity. The introduction of a proline in the first turn of an alpha-helix of this 37-mer obliterates transfection activity, suggesting that the integrity of the alpha-helical structure of the N-terminal region of histone H2A is related to its transfection activity.

Figure 1

Schematic of the peptides tested for transfection used in this study. Schematic representation of key peptides synthesized to determine the active region of the histone H2A molecule in the mediation of DNA transfection. Intermediate levels of transfection (+) are defined here as peak transfection levels yielding β-galactosidase activity between 5 and 15,000 pg; high levels of transfection (++) yield higher, and background or low levels of transfection (−) yield lower β-galactosidase activity than those defined for intermediate levels of transfection activity. These peptides were tested in a matrix in a 96-well plate. The dashed line in the representation of peptide 17 illustrates that it is a fusion of the N and C termini of histone H2A, respectively. ■, designates the first and last amino acids of histone H2A. *, see the Table 1 legend.

Figure 2

DNA binding to the N-terminal region of histone H2A. The N-terminal 35 residues of histone H2A (black ribbon) bind DNA (gray backbone, white bases, cpk model) in the context of the histone octamer (PDB code 1AOI, www.rcsb.org; ref. 18). The α1–turn–α2 motif (residues 17–35) binds three adjacent phosphates highlighted in dark gray along one DNA strand. The N-terminal extension lies within the adjacent DNA minor groove. This figure was generated with MOLSCRIPT (19) and RASTER3D (20).

Figure 3

Confocal microscopy of transfected COS-7 cells. Confocal microscopy of COS-7 cells 24 h after transfection with polyplexes of pCMVβ DNA and histone H2A; 10% of this DNA was FITC-labeled and 10% of this histone H2A was rhodamine-labeled. (A) Differential interference contrast (DIC) image of COS-7 cells. (B) The same cells as in A depicting the localization of FITC-labeled DNA transfected with polyplexes of fluorescein-labeled pCMVβ DNA. (C) The same cells as in A depicting the localization of rhodamine-labeled histone H2A. (D) The same cells as in A depicting the colocalization of FITC-labeled DNA and rhodamine-labeled DNA. The same cells as in A depicting the colocalization of FITC-labeled DNA and rhodamine-labeled DNA in both the cytoplasm and nucleus, suggesting that the DNA and histone H2A in these polyplexes not only forms a complex but is capable also of nuclear entry where the target gene can be transcribed.

Figure 4

Indirect immunofluorescence of β-galactosidase fusions in COS-7 cells. Results of COS-7 cells transfected with pCMVβ (control, A), pCMVβ-9 (B), and pCMVβ-2 (C) are shown. β-Galactosidase is detected in the cytoplasm of cells in A and C and in both the cytoplasm and nuclei of cells in B. These results demonstrate that the sequence of peptide 9 contains a complete and functional nuclear localization signal, whereas that of peptide 2 does not.