Histones are the most abundant, highly charged polycationic globular proteins located in nuclei. DNA strands wrap histone octamers (two sets of H2A, H2B, H3, and H4) to form a structural unit called a nucleosome (Diagram 1A upper). Tens of modifications in histone isoforms have been found and new modifications continue to be identified. The possible combinations of modifications in individual histone molecules, that is, in histone code, exceed trillions. Considering that each histone octa-mer is wrapped by a 147 bp long nucleotide with a unique DNA sequence, combinations of histone codes and DNA sequences provide enormous information capacity. The histone code determines which gene should be expressed, while DNA sequences of a gene contain information on the functional structures of proteins and RNAs. ChIP-seq analyses using an antibody specific for H3K9me3 (trimethylated 9th lysine of histone), provide an address written in A, C, G, T bases where H3K9me3 locate on genome. However, the DNA-sequence address of a modified histone does not give information about its spatial distribution in a nucleus.

Microscopic imaging of heterochromatin-specific histone modifications such as H3K9me3 and H3K27me3 has shown the presence of distinct spatial distributions in senescent cells. Hutchinson-Gilford Progeria Syndrome is a rare, fetal genetic disease that induces premature senescence, and the syndrome is characterized by a defect in Lamin A protein (a scaffold protein of the nuclear envelope). Furthermore, instability of the nuclear envelope exhibits results in dramatic heterochromatin rearrangement. Oncogene-induced senescence (OIS) dramatically disturbs heterochromatin structure and the nuclear envelope. Oncogenes such as Ras, Raf, and Myc paradoxically induce cellular senescence if no additional mutations of tumor suppressor genes. We found that immunostaining of OIS cells using a nanoparticle conjugated antibody specific for H3K9me3 revealed distinct clustered patterns of senescence-associated heterochromatin foci (SAHF) (Diagram 1A lower). Furthermore, our observations that (i) Lamin A protein interacts with Heterochromatin-Protein 1 (HP1); (ii) HP1 specifically binds H3K9me3, and (iii) Oligomerization of HP1 proteins may explain how H3K9me3 locates along the nuclear envelope in normal growing cells. On the other hand, in senescent cells, H3K9me3 displayed a punctate intranuclear pattern. During OIS, H3K27me3 spatially rearranged to produce SAHF. These spatial arrangements of histone modifications in the nucleus appear to represent unique features of epigenetic status that might be useful for determining cellular status. However conventional histone imaging methods using organic fluorescent dyes are limited by photo-bleaching, poor resolution, and the obligatory use of secondary antibodies.

We first employed gold and silver plasmonic nanoparticles as probes to detect the spatial distributions of histone modifications during OIS. When two or more plasmonic nanoparticles are placed in close proximity, they exhibit plasmonic coupling, which induces spectral shifts in absorbance and scattering (Diagram 1B). These spectral shifts are sensitively dependent on interparticle distance, arrangement, and type of the nanoparticles (Diagram 1C). We utilized plasmonic nanoparticles conjugated with primary antibodies for H3K9me3 and H3K27me3 to achieve high-spatial and colourimetric imaging of histone markers in a single nucleus. The distance-dependent plasmonic coupling effect between the nanoprobes allowed distances between histone modifications to be estimated and interpreted. The proposed method based on spectral analyses of plasmonic probes provides a novel means of visualizing spatial changes of modified histones and other biomarkers at the single cell level and of predicting disease progression or the status of senescence.

This work was supported by a grant from the Research Fund of the University of Seoul (2019) to Hyunsung Park.