Large scale projects are underway to map the epigenomes of various eukaryotes, including humans. The objective is usually to define the distribution across the genome of modified histones, various non-histone proteins or methylated cytosines, and then link these modifications to genomic functions [1
]. Genome-wide analyses have been made possible by coupling the long-established technique of chromatin immunoprecipitation (ChIP) with either high density DNA microarrays (ChIP-chip) or next-generation DNA sequencing (ChIP-seq) [4
]. These powerful technologies require material from large numbers of cells and the data generated inevitably represent a mean value derived from cells with differing patterns of expression from a significant subset of genes. Differences can arise through intrinsic transcriptional noise or because cells are in different phases of the cell cycle. Such cell to cell heterogeneity inevitably limits the precision with which histone modifications can be linked to chromatin function. In principle, this issue can be addressed by using immunomicroscopy to examine the distribution of histone modifications at the single cell level. Metaphase chromosome spreads provide a source of material in which individual chromosomes can be identified and in which the entire human epigenome can be scanned in a single cell. This approach has several additional advantages: there is little or no transcription at metaphase, removing a major source of variability between cells, consistency from cell to cell can be monitored, fluorescent probes are extremely sensitive (offering detection at the single gene level if required) and the procedure is quick (once experimental conditions are established) and relatively cheap. It should also be noted that immunostaining, if properly controlled, can detect modified histones and other proteins across the entire genome, including repeat-rich regions that are inaccessible to sequencing-based approaches [4
]. While microscopy cannot match the ultimate resolving power of ChIP-seq, it has the potential to provide a valuable complementary approach to epigenomic mapping.
Immunolabeling of metaphase chromosomes is a well established technique and has revealed dramatic regional differences in the distribution of specific histone modifications, particularly the distinctive pattern of modifications present on centric (constitutive) heterochromatin in plants and animals [5
] and the facultative heterochromatin of the inactive X chromosome in female mammals [8
]. Immunolabeling of meiotic (pachytene) chromosomes in maize has shown regional variation in levels of various methylated histone isoforms, with distinctive differences between heterochromatin and euchromatin [10
Surprisingly, there has been only limited use of metaphase chromosome immunostaining to map histone modifications across individual chromosomes [11
], and no systematic attempt to explore the genome-wide distribution of multiple histone modifications.
Here we describe a systematic analysis of the distribution of selected histone modifications across metaphase chromosomes from normal human cells. Antibodies to histone modifications previously linked to active transcription (H3K9ac, H3K27ac and H3K4me3, described collectively as active modifications) all highlight the same 10- to 50-Mb genomic regions, giving a characteristic and consistent banding pattern. Bands closely correspond to regions rich in genes and CpG islands (CGIs). In contrast, H3K27me3, a mark associated with gene silencing, shows a preference for telomeric regions and defines bands that only occasionally overlap with gene rich regions. At 10-Mb resolution, active modifications have similar, though not identical, distributions across interphase [13
] and metaphase chromosomes, while H3K27me3 is clearly different. The results suggest that there is extensive remodeling of the epigenome as cells enter mitosis, but that a high-level memory of some components of the interphase epigenome is retained into metaphase.