Expansion microscopy allows high resolution single cell analysis of epigenetic readers

Interactions between epigenetic readers and histone modifications play a pivotal role in gene expression regulation and aberrations can enact etiopathogenic roles in both developmental and acquired disorders like cancer. Typically, epigenetic interactions are studied by mass spectrometry or chromatin immunoprecipitation sequencing. However, in these methods, spatial information is completely lost. Here, we devise an expansion microscopy based method, termed Expansion Microscopy for Epigenetics or ExEpi, to preserve spatial information and improve resolution. We calculated relative co-localization ratios for two epigenetic readers, lens epithelium derived growth factor (LEDGF) and bromodomain containing protein 4 (BRD4), with marks for heterochromatin (H3K9me3 and H3K27me3) and euchromatin (H3K36me2, H3K36me3 and H3K9/14ac). ExEpi confirmed their preferred epigenetic interactions, showing co-localization for LEDGF with H3K36me3/me2 and for BRD4 with H3K9/14ac. Moreover addition of JQ1, a known BET-inhibitor, abolished BRD4 interaction with H3K9/14ac with an IC₅₀ of 137 nM, indicating ExEpi could serve as a platform for epigenetic drug discovery. Since ExEpi retains spatial information, the nuclear localization of marks and readers was determined, which is one of the main advantages of ExEpi. The heterochromatin mark, H3K9me3, is located in the nuclear rim whereas LEDGF co-localization with H3K36me3 and BRD4 co-localization with H3K9/14ac occur further inside the nucleus.     

The centromeric heterochromatin of Costus spiralis: poorly methylated and transiently acetylated during meiosis

The centromeric region of Costus spiralis is characteristically composed of a small heterochromatic DAPI(+) band flanked by a discrete decondensed region. High concentrations of serine 10 of histone H3 (H3S10ph) around the DAPI(+) band in pachytene chromosomes and the location of this heterochromatin at the chromosome region directed towards the poles during metaphase-anaphase I confirm its integration into the centromeric region. Antibodies against both typical components of euchromatin histones (histone H4 acetylated at lysine 5 (H4K5ac) and histone H3 dimethylated at lysine 4 (H3K4me2)) and heterochromatin (dimethylated lysine 9 of H3 (H3K9me2) and anti-5-methylcytosine (5-mC)) were used to characterize the centromeric chromatin of this species during meiosis. In pachytene chromosomes, the decondensed terminal euchromatin of the chromosome arms were seen to be richer in H4K5ac and H3K4me2 histones, while the more condensed proximal region was relatively stronger labeled with anti-H3K9me2 and anti-5-methylcytosine (5-mC). The centromeric region itself, including the DAPI(+) band, was poor in all of these chromatin modifications, but it was highly enriched in H4K5ac at pachytene. Before and after this stage, the centromeric region was poorly labeled with anti-H4K5ac. Hypomethylation and hyperacetylation of any kind of heterochromatin has rarely been reported, and it may be related to the dominant role of the centromere domain over the heterochromatin repeats.