How the very first step in nucleosome assembly, deposition of histone H3-H4 as tetramers or dimers on DNA, is accomplished remains largely unclear. Here, we report that yeast chromatin assembly factor 1 (CAF1), a conserved histone chaperone complex that deposits H3-H4 during DNA replication, binds a single H3-H4 heterodimer in solution. We identify a new DNA-binding domain in the large Cac1 subunit of CAF1, which is required for high-affinity DNA binding by the CAF1 three-subunit complex, and which is distinct from the previously described C-terminal winged-helix domain. CAF1 binds preferentially to DNA molecules longer than 40 bp, and two CAF1-H3-H4 complexes concertedly associate with DNA molecules of this size, resulting in deposition of H3-H4 tetramers. While DNA binding is not essential for H3–H4 tetrasome deposition in vitro, it is required for efficient DNA synthesis-coupled nucleosome assembly. Mutant histones with impaired H3-H4 tetramerization interactions fail to release from CAF1, indicating that DNA deposition of H3-H4 tetramers by CAF1 requires a hierarchical cooperation between DNA binding, H3-H4 deposition and histone tetramerization.DOI: http://dx.doi.org/10.7554/eLife.23474.001

Vertebrates exhibit specific requirements for replicative H3 and non-replicative H3.3 variants during development. To disentangle whether this involves distinct modes of deposition or unique functions once incorporated into chromatin, we combined studies in Xenopus early development with chromatin assays. Here we investigate the extent to which H3.3 mutated at residues that differ from H3.2 rescue developmental defects caused by H3.3 depletion. Regardless of the deposition pathway, only variants at residue 31-a serine that can become phosphorylated-failed to rescue endogenous H3.3 depletion. Although an alanine substitution fails to rescue H3.3 depletion, a phospho-mimic aspartate residue at position 31 rescues H3.3 function. To explore mechanisms involving H3.3 S31 phosphorylation, we identified factors attracted or repulsed by the presence of aspartate at position 31, along with modifications on neighboring residues. We propose that serine 31-phosphorylated H3.3 acts as a signaling module that stimulates the acetylation of K27, providing a chromatin state permissive to the embryonic development program.

A hallmark of Wnt/β-Catenin signaling is the extreme diversity of its transcriptional response, which varies depending on the cell and developmental context. What controls this diversity is poorly understood. In all cases, the switch from transcriptional repression to activation depends on a nuclear increase in β-Catenin, which detaches the transcription factor T cell factor 7 like 1 (Tcf7l1) bound to Groucho (Gro) transcriptional co-repressors from its DNA-binding sites and transiently converts Tcf7/Lymphoid enhancer binding factor 1 (Lef1) into a transcriptional activator. One of the earliest and evolutionarily conserved functions of Wnt/β-Catenin signaling is the induction of the blastopore lip organizer. Here, we demonstrate that the evolutionarily conserved BarH-like homeobox-2 (Barhl2) protein stabilizes the Tcf7l1-Gro complex and maintains the repressed expression of Tcf target genes by a mechanism that depends on histone deacetylase 1 (Hdac-1) activity. In this way, Barhl2 switches off the Wnt/β-Catenin-dependent early transcriptional response, thereby limiting the formation of the organizer in time and/or space. This study reveals a novel nuclear inhibitory mechanism of Wnt/Tcf signaling that switches off organizer fate determination.

Chromatin organization in the nucleus provides a vast repertoire of information in addition to that encoded genetically. Understanding how this organization impacts genome stability and influences cell fate and tumorigenesis is an area of rapid progress. Considering the nucleosome, the fundamental unit of chromatin structure, the study of histone variants (the bricks) and their selective loading by histone chaperones (the architects) is particularly informative. Here, we report recent advances in understanding how relationships between histone variants and their chaperones contribute to tumorigenesis using cell lines and Xenopus development as model systems. In addition to their role in histone deposition, we also document interactions between histone chaperones and other chromatin factors that govern higher-order structure and control DNA metabolism. We highlight how a fine-tuned assembly line of bricks (H3.3 and CENP-A) and architects (HIRA, HJURP, and DAXX) is key in adaptation to developmental and pathological changes. An example of this conceptual advance is the exquisite sensitivity displayed by p53-null tumor cells to modulation of HJURP, the histone chaperone for CENP-A (CenH3 variant). We discuss how these findings open avenues for novel therapeutic paradigms in cancer care.

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...

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How the very first step in nucleosome assembly, deposition of histone H3-H4 as tetramers or dimers on DNA, is accomplished remains largely unclear. Here, we report that yeast chromatin assembly factor 1 (CAF1), a conserved histone chaperone complex that deposits H3-H4 during DNA replication, binds a single H3-H4 heterodimer in solution. We identify a new DNAbinding domain in the large Cac1 subunit of CAF1, which is required for high-affinity DNA binding by the CAF1 three-subunit complex, and which is distinct from the previously described C-terminal winged-helix domain. CAF1 binds preferentially to DNA molecules longer than 40 bp, and two CAF1-H3-H4 complexes concertedly associate with DNA molecules of this size, resulting in deposition of H3-H4 tetramers. While DNA binding is not essential for H3-H4 tetrasome deposition in vitro, it is required for efficient DNA synthesis-coupled nucleosome assembly. Mutant histones with impaired H3-H4 tetramerization interactions fail to release from CAF1, indicating that DNA deposition of H3-H4 tetramers by CAF1 requires a hierarchical cooperation between DNA binding, H3-H4 deposition and histone tetramerization.