In vivo, histones are wrapped around by DNA in chromatin. Therefore, nucleosomes are more physiologically relevant substrates than histones and histone-derived peptides for in vitro studies. More importantly, some histone methyltransferases are significantly more active, as well as specific, when using nucleosomal substrates in HMT assays, such as DOT1L and NSD family enzymes. Nucleosomes are also widely used in histone methyltransferase screening assays to identify small molecular inhibitors for drug discovery. Histone H3.1 and Histone H3.3 are the two main Histone H3 variants found in plants and animals. They are known to be important for gene regulation. Histone H3.1 and H3.3 have been shown to demonstrate unique genomic localization patterns thought to be associated with their specific functions in regulation of gene activity. Specifically, Histone H3.3 primarily colocalizes with marks associated with gene activation (H3K4me3, H2BK120ub1, and RNA pol II occupancy). Deposition of the Histone H3.1 variant into the nucleosome correlates with the canonical DNA synthesis-dependent deposition pathway, whereas Histone H3.3 primarily serves as the replacement Histone H3 variant outside of S-phase, such as during gene transcription. Histones are linked to tumorigenesis primarily through alterations in their PTMs and the enzymes regulating these modifications, suggesting that they might disrupt the reading, writing, and/or erasing of these marks. Except for being near K9, which can be methylated or acetylated, R8 site itself can be methylated too. Mutations in histone H3 occur with high genetic penetrance within rare pediatric gliomas and sarcomas. Researchers found that mutations at H3 N-terminal residues at or near PTM sites including R2, R8, K18 and R26 might act as oncohistones.