Regardless of the deposition pathway, aminoacid 31 in histone variant H3 is essential at gastrulation in Xenopus

Abstract

14 The closely related replicative H3 and non-replicative H3.3 variants show specific 15 requirement during development in vertebrates. Whether it involves distinct mode of 16 deposition or unique roles once incorporated into chromatin remains unclear. To disentangle 17 the two aspects, we took advantage of the Xenopus early development combined with 18 chromatin assays. Our previous work showed that in Xenopus, depletion of the non-19 replicative variant H3.3 impairs development at gastrulation, without compensation through 20 provision of the replicative variant H3.2. We systematically mutated H3.3 at each four 21 residues that differ from H3.2 and tested their ability to rescue developmental defects. 22 Surprisingly, all H3.3 mutated variants functionally complemented endogenous H3.3, 23 regardless of their incorporation pathways, except for one residue. This particular residue, the 24 serine at position 31 in H3.3, gets phosphorylated onto chromatin in a cell cycle dependent 25 manner. While the alanine substitution failed to rescue H3.3 depletion, a phosphomimic 26 residue sufficed. We conclude that the time of gastrulation reveals a critical importance of the 27 H3.3S31 residue independently of the variant incorporation pathway. We discuss how this 28 single evolutionary conserved residue conveys a unique property for this variant in 29 vertebrates during cell cycle and cell fate commitment. 30 31 32 1 65 the only non-centromeric histone H3 is mostly related to H3.3, and achieves both replicative 66 and non-replicative variant functions, illustrating the capacity of survival with a single variant 67 (Dion et al. 2007; Jamai et al. 2007; Rufiange et al. 2007). Intriguingly, however, humanized 68 2 S. cerevisiae with all histones replaced by human orthologs survived better with hH3.1 than 69 hH3.3 (Truong and Boeke 2017). The better adaptation to H3.1 could argue for non-essential 70 roles of H3.3 in S. cerevisae, a unicellular organism. However, in metazoans like Drosophila 71 melanogaster, the replicative variant can compensate for the loss of H3.3 during development 72 in somatic tissues, although the adults are sterile (Loppin et al. 2005; Bonnefoy et al. 2007; 73 Hodl and Basler 2009; Sakai et al. 2009; Orsi et al. 2013). Since the sterility could simply 74 reflect a shortage of maternal H3.3 to replace protamine from sperm chromatin after 75 fertilization, the most parsimonious hypothesis suggests that the nature of the variant itself 76 might not necessarily be essential. Similarly, H3.3 is dispensable in Caenorhabditis elegans, 77 since its removal does not result in lethality but rather to reduced fertility and viability in 78 response to stress (Delaney et al. 2018). In Arabidopsis thaliana, in contrast, replicative and 79 non-replicative H3 variants are clearly essential. The absence of H3.3 leads to embryonic 80 lethality and also partial sterility due to defective male gametogenesis (Wollmann et al. 131 132 Results 133 Conservation of histone H3 variants and their chaperones to assay H3.3 defects...