Rice specific Histone H4 Variant forecasts a stress dependent epigenetic program by virtue of distinct nucleosome properties [MNase-Seq]

Probing the rice genome for novel histone coding genes revealed a genera specific, histone H4 variant with several variations in N-terminal tail sequence motifs, expressing in a tissue-specific manner. Genetic perturbation of the H4 variant in rice led to defects in growth and fertility. Probing the chromatin binding sites of the H4 variant with a custom antibody revealed its binding at specific genes and its knockout led to mis-expression of stress-associated genes. Stress-challenged plants showed loss of the H4 variant and re-distribution of other H4 linked histone marks. In vitro reconstituted rice nucleosomes with H4 variant histones displayed atypical properties in terms of stability, intra-nucleosome binding and structure, when compared to the canonical nucleosomes. In order to understand the extent of DNA wound by the canonical H4 and H4 variant nucleosomes, MNase protection assay followed by deep sequencing was performed. In vitro reconstituted nucleosomes (NCPs) with Widom601 nucleosome positioning sequence and arbitrary sequences on either side is subjected to limited nuclease activity using MNase. Post MNase digestion the DNA fragments were purified and deep sequenced. Alignment of these fragments reveal the extent and location of histone-octamer protected zones.