Variants of core histones and their roles in cell fate decisions, development and cancer

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1 Variants of core histones and their roles in cell fate decisions, development and cancer Marcus Buschbeck 1,2 and Sandra B. Hake 3,4 Abstract Histone variants endow chromatin with unique properties and show a specific genomic distribution that is regulated by specific deposition and removal machineries. These variants in particular, H2A.Z, macroh2a and H3.3 have important roles in early embryonic development, and they regulate the lineage commitment of stem cells, as well as the converse process of somatic cell reprogramming to pluripotency. Recent progress has also shed light on how mutations, transcriptional deregulation and changes in the deposition machineries of histone variants affect the process of tumorigenesis. These alterations promote or even drive cancer development through mechanisms that involve changes in epigenetic plasticity, genomic stability and senescence, and by activating and sustaining cancer-promoting gene expression programmes. 1 Josep Carreras Leukaemia Research Institute, Campus Institut Català d Oncologia Germans Trias i Pujol, Campus Can Ruti, Badalona, Spain. 2 Program for Predictive and Personalized Medicine of Cancer, Germans Trias i Pujol Research Institute, Badalona, Spain. 3 Center for Integrated Protein Science Munich and Department of Molecular Biology, BioMedical Center, Ludwig-Maximilians University Munich, Planegg-Martinsried, Germany. 4 Present address: Institute for Genetics, Justus Liebig University Giessen, Giessen, Germany. mbuschbeck@ carrerasresearch.org; sandra.hake@ gen.bio.uni-giessen.de doi: /nrm Published online 1 Feb 2017 One of the most abundant and most conserved protein families in eukaryotic cells is the histone family. Histones package genetic information into the nuclear space and contribute to the regulation of all DNA template- based reactions. Core histones bind DNA as part of the nucleosome, the building block of chromatin, and linker histones bind to DNA in the internucleosomal space. Aside from the so-called canonical histones, which comprise the majority of any given histone species in any cell, evolution drove the emergence of histone variants, which endow chromatin with special properties in a locus-specific manner. With respect to the core histones, eight variants of H2A (H2A.X, H2A.Z.1, H2A.Z.2.1, H2A.Z.2.2, H2A Barr body deficient (H2A.Bbd; also known as H2A.B), macroh2a1.1, macroh2a1.2 and macroh2a2) and six variants of H3 (H3.3, histone H3 like centromeric protein A (CENP-A), H3.1T, H3.5, H3.X (also known as H3.Y.2) and H3.Y (also known as H3.Y.1)) have been identified in human somatic cells. In addition, two testis-specific variants of H2B (histone H2B type WT (H2BFWT; also known as H2B.W) and testis- specific histone H2B (TSH2B; also known as histone H2B type 1A)) have been found. However, no variants of H4 have yet been discovered in higher eukaryotes 1. Surprisingly, in lower eukaryotes such as trypanosomes 2 and some urochordates 3, H4 variants have been found, which suggests either that functional specialization of this histone family is evolutionarily possible or that these variants do exist in higher eukaryotes but their detection has so far REVIEWS been unsuccessful. Notably, the different variants are distinct and unique in terms of their gene and protein sequences, as well as the timing of their transcription, how their RNA is processed and when they are deposited on DNA during the cell cycle (FIG. 1). The canonical histones are deposited on DNA during replication, and in this Review we hereafter refer to these as replication-coupled histones. Each of the replication- coupled histones is encoded by multiple genes and, with a few exceptions, most of these genes are organized into clusters throughout the genome 4,5 (FIG. 1a). This probably ensures that the expression of replication-coupled histones, which takes place during S phase, generates large and equal amounts of all four nucleosome core histone proteins, thereby providing a constant source of nucleosomes at newly replicated DNA strands and allowing efficient chromatin packaging. By contrast, histone variants are typically encoded by a single gene (as in the case of H2A.X 6 ) or two genes (in the cases of H3.3, H2A.Z and macroh2a). These genes are located outside the replication-coupled histone clusters that are present on other chromosomes, providing the possibility of variant-specific transcription and deposition throughout the cell cycle. Whereas most histone variants are expressed in somatic tissues, some are exclusively or predominantly found in the male germline 7. In summary, each histone variant has a unique temporal expression and, as we discuss below, this unique expression pattern accounts for specific cellular functions of the variants. NATURE REVIEWS MOLECULAR CELL BIOLOGY ADVANCE ONLINE PUBLICATION MacmilanPublishersLimited,partofSpringerNature.Alrightsreserved.

2 a H2A H2B H3 H2A H2B H3 H4 H2A H2B H3 H4 b H2A 58.3% H2A.Z % H2A.Z % H2A.Z % H2A.X Replication-coupled histones 62.0% macroh2a % macroh2a % macroh2a2 38.1% H2A.Bbd H2A H2B H2B 84.9% TSH2B 33.3% H2BFWT H % H % H3.3 H3 H4 6 H2B H2A H3 H4 45.1% CENP-A 97.0% H3.1t 77.8% H3.Y 93.3% H % H3.X Figure 1 Comparing replication-coupled and variant human core histones. a Whereas individual genes encode histone variants, at least in humans, replication-coupled histones are encoded by multiple gene copies (each gene is represented by a coloured square in the top right panel) that are present in clusters (lower right panel) on different human chromosomes. Note the presence of several different clusters of histone genes in one chromosomal region. No distinction between genes and pseudogenes has been made in this figure. Chromosome 6 is shown here, as it harbours the largest cluster of histone genes. b A depiction of human variants of histone H2A (yellow), H2B (orange) and H3 (blue), with variants shown in pale yellow, pale orange and pale blue, respectively. Rectangles represent core regions, and lines represent flexible histone tails. No variants of H4 (green) have yet been discovered in humans. Testis-specific histone variants are highlighted by light purple boxes, and alternative splice isoforms by light green boxes. Percentages indicate total amino acid sequence conservation (% sequence identity) of the variants relative to their replication-coupled counterparts (for H3, two replication-coupled isoforms are present (H3.1 and H3.2); in the figure, sequence conservation was calculated for H3.1). CENP-A, histone H3 like centromeric protein A; H2BFWT, histone H2B type WT; TSH2B, testis-specific histone H2B. Part a is adapted with permission from REF. 4, Springer. Urochordates Also called tunicates. Small marine invertebrates that exhibit a simplified chordate body plan and are within the chordate phylum, which includes the closest relatives of vertebrates. Pseudogenes Genes and gene copies that have lost their protein-coding function. Replication-coupled and variant histone- encoding RNAs differ in their structure and processing. Replicationcoupled histone genes lack introns and contain a specific 40 bp sequence downstream of the translation stop codon that forms a consensus stem loop structure that is responsible for the processing of the 3ʹ ends 8. By contrast, several histone variant genes, albeit not all, contain introns that need to be spliced during RNA processing, which provides the chance to generate alternative splice isoforms and, in turn, to further increase histone diversity and also in the case of core histones nucleosome diversity 9. Examples of core histone variant proteins generated by alternative splicing are macroh2a1.1 and macroh2a1.2 (REF. 10), as well as H2A.Z.2.1 and the primate- specific H2A.Z.2.2 (REF. 11). In addition, similarly to most cellular mrnas, most histone variant mrnas have a poly(a) tail 12. At the protein level, all replication- coupled and variant histones differ in their primary sequences, and sometimes in their secondary and tertiary conformations. In some cases for example, for the replication-coupled H3.2 and the histone variant H3.3 they differ in only a few amino acids, whereas other variants such as those in the H2A family can have largely distinct sequence stretches or even unique domains (FIG. 1b). This Review focuses on mammalian core histone variants. We briefly discuss how these histones are deposited on DNA, and how they affect chromatin and its functions. However, we devote the majority of this article to outlining the normal functions of histone vari ants in development and their pathophysiological roles in cancer. The evolution of histone variants, their roles in environmental responses and their biochemical regulation have been comprehensively reviewed elsewhere Deposition and chromatin occupancy Replication-coupled and variant core histone proteins differ not only in their temporal expression pattern but also in their spatial localization, both in the 3D nuclear space and in their distribution along the linear genome. Replication-coupled histone proteins are distributed rather equally in chromatin, whereas histone variantcontaining nucleosomes show specific and unique distrib utions. Generally, nucleosomes are generated when ~147 bp of DNA are wrapped around an octamer of histones that is formed of an (H3 H4) 2 tetramer and two dimers of H2A H2B, which are packaged on either side 18. To ensure spatially and temporally controlled histone deposition and eviction, which are necessary to modulate chromatin organization and consequently 2 ADVANCE ONLINE PUBLICATION

3 Histone chaperones Histone-binding proteins that facilitate histone-dependent processes, including histone deposition on and removal from chromatin. Chromatin remodellers Frequently multimeric complexes that use ATP to catalyse changes in chromatin structure, including the exchange of histones. DNA template-based processes biological functions, speci fic chaperone and ATP-dependent remodelling complexes have evolved 19 (FIG. 2a; TABLE 1). Histone chaperones participate in multiple steps of nucleosome formation 20 and bind histones directly after their synthesis to control their stability or degradation 21. Some chaperones assist in the cytoplasm nucleus trafficking of histones directly after their synthesis, in part through regulating their interaction with importins 22 ; others affect histone post-translational modifications (PTMs) by facilitating the binding of histones to the responsible enzyme 8 ; and others facilitate histone interactions to promote nucleosome formation 23,24. By contrast, chromatin remodellers use ATP to organize chromatin structure by sliding or ejecting assembled nucleosomes and, importantly, can allow the exchange of labile H2A H2B dimers with dimers of histone variants 25. One of the first histone variant-specific chaperone complexes to be discovered was theh3.3 specific histone cell cycle regulation-defective homologue A (HIRA). As part of a separate complex, chromatin assembly factor 1 (CAF1; also known as CNOT7) is involved in H3.1 and H3.2 deposition during replication, which reveals the existence of distinct deposition machineries and pathways responsible for DNA synthesisindependent and DNA synthesis-dependent chromatin assembly 24. Later, another H3.3 specific depo sition complex namely, the death domain-associated protein 6 (DAXX) ATRX complex was identified 26. Both the HIRA complex and the DAXX ATRX complex have non-redundant roles in the deposition of H3.3 in different chromatin environments and have different functional outcomes 26. Whereas HIRA-deposited H3.3 at sites of naked DNA, such as regions of gene activation, might a Ribosome b Homotypic nucleosome Heterotypic nucleosome Replicationcoupled H2A H2A variant Stabilization of free histones H2A variants H3 variant Histone chaperones H2B Direct effects of histone variant deposition Nucleosome structure and stability change Change in nucleosome occupancy Histone exchange Nuclear import Histone chaperone and chromatinremodelling complex Nucleosome assembly and deposition Indirect effects of histone variant deposition PTM reader Variantspecific reader Trimethylation PTM changes Due to amino acid sequence Due to chromatin environment Open versus closed chromatin Changes to the histone code DNA and its read-out Figure 2 The roles of histone chaperones and remodellers, and the impact of histone variants on chromatin. a Specific histone chaperone and chromatin-remodelling complexes recognize histone variants when they are complexed with their replication-coupled partner histone (after synthesis, the H2A variant shown here (pale yellow) pairs with H2B (orange), whereas H3 pairs with H4 (not shown)). Chaperones stabilize histones and prevent their degradation. They also have a role in the nuclear import of histones and facilitate histone exchange, as well as nucleosome assembly and deposition. ATP-dependent chromatin-remodelling enzymes slide nucleosomes on DNA or allow the exchange of labile H2A H2B dimers with dimers that contain an H2A variant. b The incorporation of histone variants (pale yellow and pale blue) into chromatin can result in homotypic or heterotypic nucleosomes (top) and can have direct (middle) or indirect (bottom) effects on chromatin structure and function. Histone variants can directly influence nucleosome structure and stability, thus resulting in changes in the local chromatin environment (for example, they can affect the propensity for chromatin remodelling). The composition of the nucleosome can also have indirect effects on chromatin organization and function. The presence of histone variants can induce the recruitment of specific readers of histone post-translational modifications (PTMs) and/or the recruitment of variant-specific binding complexes, which can then induce local chromatin changes. NATURE REVIEWS MOLECULAR CELL BIOLOGY ADVANCE ONLINE PUBLICATION 3

4 Table 1 Histone variants and their regulation by histone chaperones and chromatin remodellers Histone or variant H2A* H2B* complex Tissues (location within the genome) Chaperones and remodellers Function Refs All (genome-wide) FACT (chaperone) Deposition and exchange of histones 167 NAP1 (chaperone) Nuclear import and deposition 168,169 Nucleolin (chaperone) Deposition and exchange of histones 170 H2A.X All (genome-wide) FACT (chaperone) Deposition and exchange of histones 167 H2A.Z.1 H2A.Z.2.1 All (regulatory regions (promoters and enhancers) and heterochromatin) All (regulatory regions (promoters and enhancers) and heterochromatin) p400 (remodeller) H2A.Z deposition 11 SRCAP (remodeller) H2A.Z deposition 11 ANP32E (chaperone) INO80 (remodeller) H2A.Z chromatin removal at sites of DNA damage H2A.Z removal, which promotes homologous recombination p400 (remodeller) H2A.Z deposition 11 SRCAP (remodeller) H2A.Z deposition 11 H2A.Z.2.2 All but enriched in the brain p400 (remodeller) H2A.Z deposition 11 MacroH2A1.1 MacroH2A1.2 MacroH2A2 All (facultative and constitutive heterochromatin) All (facultative and constitutive heterochromatin) All (facultative and constitutive heterochromatin) SRCAP (remodeller) H2A.Z deposition 11 Not known Not known ATRX (remodeller) Negative regulation, possibly indirect 33 Not known Not known H2A.Bbd Testis and brain (euchromatin) NAP1 (chaperone) Assembly and disassembly of H2A.Bbd nucleosome core particles H2BFWT Testis (not known) SWI SNF (remodeller) Remodelling and mobilization of nucleosomes TH2B Testis (not known) Not known Not known H3.1* H4* or H3.2* H4* complex All (genome-wide) FACT (chaperone) Deposition and exchange of histones 167 H3.3 All (regulatory elements of active genes and repetitive, heterochromatic sequences) NAP1 (chaperone) Nuclear import and deposition 168,169 ASF1 (chaperone) Histone import and transfer to CAF1 174 NASP1 (chaperone) Protection of histones from degradation 21 CAF1 (chaperone) Histone deposition and tetramer formation 175 FACT (chaperone) Deposition and exchange of histones 167 NAP1 (chaperone) Nuclear import and deposition 168,169 ASF1 (chaperone) Histone import and transfer to HIRA 174 NASP1 (chaperone) Protection of histones from degradation 21 DAXX ATRX (chaperone and remodeller, respectively) Deposition at heterochromatin 26,28,176 HIRA (chaperone) Deposition at gene promoters 175,177 DEK (chaperone) Supply of H3.3 to DAXX ATRX in PML bodies 178 CENP-A All (centromere) HJURP (chaperone) Deposition at centromeres 179,180 H3.Y H3.X Testis, brain and tumours (euchromatin) Testis, brain and tumours (not known) Not known Regulation of cell cycle genes 181 Not known Not known 181 H3.1t Testis (not known) Not known Not known 182 H3.5 Testis (euchromatin) Not known Not known 183 ANP32E, acidic leucine-rich nuclear phosphoprotein 32 family member E; CAF1, chromatin assembly factor 1; CENP-A, histone H3 like centromeric protein A; DAXX, death domain-associated protein 6; FACT, facilitates chromatin transcription; H2BFWT, histone H2B type WT; HIRA, histone cell cycle regulationdefective homologue A; HJURP, Holliday junction recognition protein; NAP1, nucleosome assembly protein 1; NASP1, nuclear autoantigenic sperm protein; PML, promyelocytic leukaemia; SWI SNF, switch sucrose non-fermentable; SRCAP, SWI2 SNF2 related CBP activator protein; TH2B, testis histone 2B. * Canonical replication-coupled histones. 4 ADVANCE ONLINE PUBLICATION

5 Retrotransposons Ubiquitous genetic elements that are able to amplify themselves. Centromere A chromosome region that mediates attachment to the mitotic spindle during mitosis. protect chromatin integrity 27, H3.3 incorporation via DAXX ATRX contributes to the proper establishment of heterochromatin 26, Furthermore, ATRX is important for the repression of a subset of retrotransposons in mouse embryonic stem (ES) cells and mouse embryonic fibroblasts (MEFs), but controversy exists about whether this is mediated by H3.3 deposition 31 or by ATRX facilitating the repressive trimethylation of H3 at K9 (K9me3), which could be largely H3.3 independent 32. Part of the controversy might be resolved by considering the cell state and cell type in which the analysis has been performed. Retrotransposons are bound by H3.3 in ES cells but not in MEFs, which suggests that H3.3 might not be required for the maintenance of the repressive heterochromatic state in the latter cell type 31. H3.3 and ATRX also contribute to telomere maintenance in ES cells and in differentiated cells 29,30. Chromatin remodellers can also affect the distribution of histone variants other than H3.3. For example, ATRX negatively regulates the amount of macroh2a present at genes close to the subtelomeric region 33. Similarly to the deposition of H3.3, two independent chaperone and remodelling complexes are needed for the deposition of all three human H2A.Z isoforms 11. These two H2A.Z specific multicomponent complexes namely, p400 and SWI2 SNF2 related CBP activator protein (SRCAP) evolutionarily arose from a single SWR1 chromatin-remodelling complex that is found in lower eukaryotes 34, and they are clearly distinct from the facilitates chromatin transcription (FACT), nucleosome assembly protein 1 (NAP1) and nucleolin complexes that are responsible for the deposition of the replicationdependent histone H2A (TABLE 1). It is so far not exactly clear whether these complexes are functionally redundant or act independently, although data suggest that they can form distinct subcomplexes with other proteins, and have different roles in H2A.Z deposition or even ejection Functions in chromatin How do histone variants function when incorporated into chromatin? This is currently a subject of intense research. Different, interconnected modes of action can be envisioned (FIG. 2b). A direct influence on nucleosome structure is possible. For instance, nucleosome core particles that contain the histone variant H2A.Bbd, which lacks an acidic patch in the carboxy terminus, organize only bp of DNA (instead of bp) and have reduced salt stability compared with those that contain H2A 38,39. As a result, H2A.Bbd incorporation leads to the formation of less compact chromatin and, in the in vitro setting, this facilitates transcription 40. The primatespecific alternative splice variant H2A.Z.2.2 severely destabilizes nucleosomes in vitro and in vivo owing to its unique and shortened C terminus, which is responsible for a more dynamic structure of the protein and loosens its binding to H3 within the nucleosome 11. Whether histone variants favour distinct stoichiometric compositions of nucleosomes, sometimes referred to as quaternary structures, is still under debate. In particu lar, there is controversy about whether nucleosomes that contain the H3 variant CENP-A are tetrameric hemisomes or conservative octamers (as reviewed in REF. 41). It is possible that both forms of CENP-A-containing nucleosomes exist in vivo depending on the cell cycle stage and the phosphoryl ation status of its chaperone Holliday junction recognition protein (HJURP) 42 (TABLE 1). As some variants cause pronounced structural changes in nucleosomes, the presence of multiple variants in the same nucleosome is particularly intriguing. Aside from homotypic nucleosomes that contain two copies of the same histone, heterotypic nucleosomes which contain a replication-coupled histone and a variant histone, or two different variants also exist (FIG. 2b). This allows for greater variability in nucleosome formation, stability and structure, as explained in the proposed nucleosome code hypothesis 43. For example, nucleosomes that contain H2A.Z and H3.3 are less stable than replication-coupled nucleosomes, and they are present at the nucleosome-depleted regions of active promoters, enhancers and insulators Such labile H3.3 H2A.Zcontaining nucleosomes are proposed to serve as place holders that prevent the formation of stable nucleosomes around regulatory regions. As such, they might be easily displaced by transcription factors and regulatory complexes that are not able to bind DNA in the presence of a nucleosome composed of replication- coupled histones, thus facilitating gene regulation 44. In addition, over expression of CENP-A can lead to its mislocalization from the centromere to chromosome arms and can also lead to the formation of CENP-A H3.3 heterotypic nucleosomes that form unexpectedly stable structures relative to homotypic CENP-A nucleosomes 47. Such CENP-A H3.3 nucleosomes are found at regulatory sites of transcription factor binding where they seem to replace labile H3.3 H2A.Z-containing nucleo somes in a DAXX-dependent manner 48. Interestingly, this stable CENP-A H3.3 nucleosome participates in the occlusion of binding sites for the transcription repressor CTCF and might therefore contribute to the regulation of transcription 48. In summary, the variant composition of nucleosomes might directly influence gene expression by altering nucleosome stability and, in turn, by facilitating or interfering with the recruitment of transcription regulators. Aside from directly influencing nucleosome structure, the deposition of histone variants can have indirect effects on chromatin organization and biological processes (FIG. 2b). Variants can be modified by diverse PTMs (listed in REFS 49 51). Owing to differences in amino sequence and also preferential deposition in specific genome regions (such as H3.3 deposition in active promoter regions), variant and replication-coupled histones differ in the level and type of some of these PTMs. The recruitment of variant-specific or PTM-specific reader molecules further modifies chromatin at sites of variant deposition. Indeed, microcephalin (MCPH1) acts as a histone variant-specific PTM reader; it recognizes phosphorylation at C terminal serine and tyrosine residues in H2A.X following DNA damage 52, and serves as an early sensor of damage and as a mediator between chromatin and the repair machinery. Although modifications of NATURE REVIEWS MOLECULAR CELL BIOLOGY ADVANCE ONLINE PUBLICATION 5

6 Inner cell mass Pluripotent cells of the blastocyst. Kinetochore A multiprotein complex assembled on the centromere that mediates the interaction with the microtubules of the spindle. histone variants themselves can recruit specific proteins, a histone variant can also provide binding specificity in combination with PTMs on a replication-coupled histone that is present in the same nucleosome. Bromodomaincontaining protein 2 (BRD2), for example, recognizes H4 hyperacetylated nucleosomes that contain the histone variant H2A.Z.1 and facilitates transcriptional activation 53. Similarly, the candidate tumour-suppressor protein zinc finger MYND domain-containing protein 11 (ZMYND11), which regulates the activity of RNA polymerase II, has recently been identified as a reader of H3.3K36me3, thereby linking histone variant-mediated enhancement of transcriptional elongation to tumour suppression 54. Some chromatin-interacting proteins need to be post-translationally modified themselves to be able to bind to unmodified histone variants. One example is the binding of automodified poly(adp-ribose) polymerase 1 (PARP1; also known as ARTD1) to nucleosomes that contain macroh2a1.1 (REF. 55); binding promotes the acetylation of surrounding chromatin by recruiting the acetyltransferase CREB-binding protein (CBP) 56. In summary, histone variants are deposited into chromatin by specialized chaperone and remodelling complexes in a spatially and temporally restricted manner. They can then influence chromatin either directly by changing nucleosome structure and stability, or indirectly by the acquisition of different histone modifications and the recruitment of distinct histone variant-interacting proteins and chromatin-modifying enzymes, which form the so called variant network. Physiological roles Unsurprisingly, owing to their involvement in chromatin organization and function, histone variants are involved in cell fate transitions. Their expression is dynamically regulated during embryonic development, and their special ized functions in lineage commitment or reversion have been dissected in ex vivo cultures of ES cells and during somatic cell reprogramming. Histone variants in early embryonic development. During early mouse embryonic development, the expression of histone variants is highly dynamic (FIG. 3a). H3.3 and H2A.X are abundant during the first cell divisions 57,58. MacroH2A becomes detectable again between the 8 cell and 16 cell stage 57. H2A.Z is largely absent from pluripotent cells and is first expressed at the outer layer of the inner cell mass as differentiation proceeds 59. It remains to be analysed whether CENP-A levels vary during early embryogenesis and whether there are differences in the expression of the different gene products and isoforms of macroh2a and H2A.Z. In this context, macroh2a1.1 is almost absent in mouse ES cells 60, and macroh2a2 is the predominantly expressed form of macroh2a during zebrafish development 61. Such develop mental changes in the expression levels of histone variants need to be considered in the context of their contribution to the cellular histone pool (FIG. 3b). H2A.Z.1, H3.3 and CENP-A are essential for mouse embryonic development, but macroh2a isoforms and H2A.X are not (TABLE 2). The Drosophila melanogaster homologue of CENP-A, CenH3 (also known as Cid), is essential and sufficient to form a functional centromere 62, and targeted deletion of the gene encoding mouse CENP-A causes mitotic defects and embryonic lethality at day 6.5 (REF. 63). Similarly, depletion of CENP-A in human cells is toxic, and rescue experiments demonstrate the importance of its targeting domain for mammalian centromere integrity 64,65. In addition, the knockout of both genes encoding H3.3 (H3f3a and H3f3b) is embryonic lethal in mice at day 6.5, possibly as a consequence of cellular apoptosis induced by mitotic defects 66. Mice with reduced H3.3 expression as a consequence of single gene knockout and mice carrying hypomorphic alleles can be normal 66 but can display phenotypes such as mild growth retardation, neonatal lethality, and male and female steril ity 67,68. Although the two H3.3 encoding genes are partially redundant, the knockout phenotypes are generally more severe for H3f3b, which suggests that this gene has a larger or broader contribution to H3.3 protein function. H3f3b is particularly important for the first cell division after fertiliza tion, as demonstrated by the failure of zygote cleavage when H3f3b is inactivated in the oocyte 69. The first cell division of the zygote requires the establishment of heterochromatin, which is a pre requisite for the formation of a functional kinetochore. In the zygote, H3.3 is a maternal factor that first becomes asymmetrically enriched in the paternal pronucleus 58, the heterochromatin of which is particularly sensitive to the depletion of H3.3 (REF. 70). Mistargeting of CENP-A to chromosome arms has also been proposed to contribute to the mitotic defects in H3.3 deficient cells 68. H2A.Z is first detected at sites of heterochromatin, which suggests that it similar to H3.3 at an earlier stage might be required for heterochromatin formation and the mainten ance of a functional centromere 59. In support of this, knockdown of H2AZ leads to chromosomal segregation defects and reduced binding of heterochromatin protein 1α (HP1α) in cell lines 71. H2A.Z deficient mouse embryos die at the pre-implantation stage of the blastocyst 72, which coincides with the time point of the first detectable expression of H2A.Z 59. In contrast to mice that lack H3.3, H2A.Z or CENP-A, H2A.X-deficient mice and macroh2a deficient mice are viable. H2A.X deficient mice, however, carry various defects: they are smaller, are subfertile and have a reduced number of lymphocytes, which leads to immune deficiencies 73,74. In line with a major role for H2A.X in the DNA damage response, H2A.X-deficient mice are hypersensitive to γ-radiation 74. Mice that systemically lack macroh2a1 develop without displaying an overt phenotype, but they have alterations in fat and sugar metabolism, although there is controversy over whether the phenotype represents an improvement or impairment of metabolic fitness Mice that lack both macroh2a1 and macroh2a2 show normal early development, but have reduced prenatal and postnatal growth 78. In conclusion, histone variants have essential and nonessential functions in embryonic development and viability. It is striking that all essential histone variants have been implicated in the regulation of kinetochore 6 ADVANCE ONLINE PUBLICATION

7 a Zygote 2-cell 4-cell 8-cell 16-cell Early blastocyst ICM Late blastocyst Epiblast Differentiated cells H3.3 H2A.X H2A.Z MacroH2A b Histone pool H3 H2A H3.3 ~35% ~8% c H2A.Z ~1% MacroH2A ~1% H2A.X ~6% ES cell ipsc Selfrenewal Differentiation H2A.Z H3.3 TH2A, TH2B MacroH2A Differentiated cell Reprogramming Figure 3 The dynamic expression and function of histone variants during embryonic development and cell fate transitions, and the contribution of variants to the histone pool. a The expression levels of several histone variants change during the early stages of embryonic development. H3.3 and H2A.X are already expressed in the zygote, whereas macroh2a and H2A.Z are expressed later at the 8 cell stage and in the blastocyst, respectively 57,58. The sizes of the blue, green, yellow and orange circles represent the expression level of each histone variant during the early developmental process (that is, larger circles indicate higher expression than do smaller circles), but the indicated expression levels cannot be compared between the variants. b The average contribution of some histone variants to the total pool of the respective histone species has been determined in a few mammalian tissues and immortal cell lines 51,130,165, and the estimated average is depicted. The absolute contribution to the histone pool, however, can vary widely for specific cell types; for H3.3 this ranges from % of the H3 pool 51, and for H2A.X this ranges from % of the H2A pool in most cells but reaches 25.0% in a brain tumour-derived cell line 130. c Some histone variants have been shown to affect the differentiation of embryonic stem (ES) cells or the inverse process, the reprogramming of differentiated cells into induced pluripotent stem cells (ipscs). Testis histone 2A (TH2A) and TH2B are germline-specific variants that have been characterized in rodents 96. See TABLE 2 for additional details and references. ICM, inner cell mass. Part a is adapted from REF. 166, Macmillan Publishers Ltd. formation and chromosome segregation, and this might be linked to a role for histone variants in the establishment of heterochromatin. Mitotic defects can be a trigger for early developmental failure and are likely to obscure additional functions of histone variants that would have become apparent at later developmental stages. Histone variants in ES cells and lineage commitment. The transient developmental state of cells of the inner cell mass of blastocyst, in which pluripotency is coupled with fast proliferation, can be perpetuated in cultures of ES cells. ES cells are thus an important tool for investi gating early events in lineage commitment and the balance between cell self-renewal and differenti ation. MacroH2A, H2A.Z and H3.3 have been linked to regulating this balance in mouse ES cells 60,79,80 (FIG. 3c). However, it is important to note that as multiple feedback loops couple the inverse expression of genes regulating pluripotency and lineage commitment 81, it is difficult to distinguish whether delays in lineage commitment are caused by the reduced activation of pro-differentiation genes or the defective repression of pluripotency genes. NATURE REVIEWS MOLECULAR CELL BIOLOGY ADVANCE ONLINE PUBLICATION 7

8 Totipotent cells Cells that can give rise to all embryonic and extra-embryonic tissues. In mouse ES cells, macroh2a1.2 is the predominantly expressed isoform; its levels are low but increase during differentiation 60. Human and mouse macroh2a1.2 are found on developmental genes that are marked by H3K27me3. This histone mark is laid down by Polycomb repressive complex 2 (PRC2) and leads to the silencing of pluripotency genes. In line with this, knockdown of H2AFY (which encodes macroh2a1) generally reduces the induction of differentiation programmes 60,82. By preventing the establishment of stable nucleosomes and thereby inducing dynamic nucleosome depletion, H2A.Z facilitates the binding of chromatin-regulatory complexes (which both activate and repress gene expression) to H2A.Z targeted sites 83. This includes the binding of the pluripotency factor OCT4 (also known as POU5F1) to activate the expression of self-renewal genes and the binding of PRCs to lineage-specifying genes that must be silenced 79,83. It will be interesting to evaluate the extent to which this function of H2A.Z affects the activity of various regulatory complexes and transcription factors, particularly comparing those able to bind DNA in the context of a nucleosome and those that require its removal. It is conceivable that the latter might be particularly sensitive to H2A.Z mediated nucleosome destabilization. Considering that a main function of H2A.Z is to facilitate the access of regulatory complexes to the genome, it is plausible that the requirement for H2A.Z is more pronounced during cell fate transitions than during the maintenance of a cellular state. Thus, unsurprisingly, the loss of H2A.Z affects differentiation more than self-renewal in mouse ES cells 79,83. Similarly to H2A.Z, H3.3 also facilitates the recruitment of repressive complexes such as PRC2 in ES cells 80. In contrast to H3.3, H2A.Z and macroh2a, H2A.X has not been linked to cell fate transitions but contributes to maintaining the highly proliferative state of ES cells through its function in the DNA damage response pathway (discussed below) 84,85. It is worth noting that a key feature of the chromatin of pluripotent cells, and even more so that of totipotent cells, is high histone mobility and exchange, which contribute to the plastic nature of these stem cells 86. It will be interest ing to study whether the function of histone variants differs throughout development, and if so, whether this is governed by changes in histone mobility. Histone variants in somatic cell reprogramming. Somatic cell reprogramming refers to the reversion of the differentiated state to pluripotency. Methods to Table 2 The functions of histone variants in physiology and cancer Histone Function Knockout mouse phenotype Refs General ES cells Reprogramming Cancer H2A.X DNA damage response Sustains fast proliferation H2A.Z.1 H2A.Z.2 MacroH2A1 MacroH2A2 H2A.Bbd TH2A and TH2B Facilitates the binding of regulatory complexes and increases nucleosome dynamics Facilitates the binding of regulatory complexes Gene repression and signal-induced gene activation Gene repression and signal-induced gene activation Associates with sites of active transcription and replication and increases nucleosome dynamics Paternal genome activation in zygote H3.3 Gene activation, genome integrity and the establishment of heterochromatin CENP-A Kinetochore attachment, chromosome segregation Required for differentiation and facilitates self-renewal Not determined Tumour suppressor that promotes genome integrity Viable but show male infertility 73,74, 84,85, 130 Not determined Oncogenic Lethal 72,83, 107 Not determined Not determined Oncogenic Not determined 108 Facilitates differentiation and the induction of differentiation genes Not determined owing to low expression Acts as barrier Acts as barrier Tumour suppressor with isoform-specific differences Tumour suppressor Viable but show metabolic defects Viable; reduced growth of knockout mice lacking both macroh2a1 and macroh2a2* 60,76, 77,92, 100,101, ,78, 95,100 Not determined Not determined Not determined Not determined 185,186 Not determined Facilitator Not determined Not determined 96 Facilitates differentiation Facilitator Pro-oncogenic Knockouts of both H3f3a and H3f3b show early embryonic lethality; H3f3b knockouts show growth deficiency during development and do not survive birth; H3f3a knockouts are viable but show male infertility Not determined Not determined Potentially oncogenic 26,31, 66,69, 80,88, 89 Lethal 48,63 CENP-A, histone H3 like centromeric protein A; ES, embryonic stem; H3f3a, H3 histone family member 3A; H3f3b, H3 histone family member 3B; TH2A, testis histone 2A; TH2B, testis histone 2B. *Knockout of macroh2a2 in zebrafish causes malformations ADVANCE ONLINE PUBLICATION

9 Table 3 Point mutations in histones and their chaperones in human cancer Mutated protein H3.3 and replicationcoupled H3.1 ATRX Mutation Cancer tissue Function Refs K27M G34R, G34V and G34L Paediatric brain tumours and juvenile bone tumours Paediatric brain tumours and juvenile bone tumours Inhibits the K27 methyltransferase activity of Polycomb repressive complex 2 Unknown function, but tight association with ATRX and DAXX mutations 118,119, 122, K36M Chondroblastoma Inhibits K36 methyltransferases 121,128 Various along coding sequence Cancers of the peripheral and central nervous system; less frequent in various other types of cancer DAXX Various Various; most frequent in the nervous system DAXX, death domain-associated protein 6. Mostly inactivating 118,188 Considered to be inactivating; less frequent than ATRX mutations 118,188 reprogramme somatic cells include somatic cell nucleus transfer (SCNT) into an enucleated oocyte, cell fusion and reprogramming into induced pluripotent stem cells (ipscs) by Yamanaka transcription factors (namely, OCT4, SOX2, Krueppel-like factor 4 (KLF4) and MYC) 87. The efficiency of experi mental reprogramming is generally low and decreases as cells further differentiate, which suggests that the stable differentiated epigenome of somatic cells presents a barrier to reprogramming. Several studies have addressed the influence of H3.3, macroh2a and testis-specific histone variants on the efficiency of reprogramming (FIG. 3c, TABLE 2). The induction of pluripotency by SCNT involves extensive chromatin remodelling in the donor nucleus, and this is facilitated by HIRA and H3.3 in the donor cell 88,89. Interestingly, H3.3 also contributes to maintaining the memory of an active chromatin state of the muscle cell-specific gene MyoD (which encodes myoblast determin ation protein 1) in the donor nucleus, even though there is no active transcription after SCNT 90. It remains to be seen whether the ability of H3.3 to facilitate the induction of pluripotency also applies to reprogramming induced by cell fusion and by transcription factor- induced reprogramming. MacroH2A1, by contrast, is rapidly removed during SCNT 91, and its depletion facilitates reprogramming by SCNT 92. Similarly, macroh2a comprises a barrier to transcription factor-induced reprogramming, and its removal from chromatin enhances the rate of successful reprogramming. This seems to be particu larly relevant for human cells and the macroh2a2 variant Enforced expression of the rodent germlinespecific variants testis histone 2A (TH2A) and TH2B (also known as also H2B.1) facilitates the formation of ipscs, whereas their suppression inhibits this process, mirroring their function in paternal genome activation in the fertilized oocyte 96. Mechanistically, TH2A and TH2B facilitate the formation of an open chromatin structure as shown by an increase in DNase I accessibility. Taken together, the influence of histone variants on reprogramming reflects their expression during early development. Variants expressed in the zygote such as H3.3, TH2A and TH2B act as facilitators of reprogramming, whereas somatically expressed macroh2a constitutes part of the epigenetic barrier that makes reprogramming an inefficient process. Histone variants in cancer In cancer, histone variants and their regulators are frequently deregulated at the level of transcription (as summarized in REF. 97) and in some case by mutations (TABLES 2,3). In this section, we discuss how the deregulation of histone variants promotes cancer by several mechanisms (also see FIG. 4). Histone variants increase epigenetic plasticity. Histone variants can help to switch epigenetic states by stabilizing or destabilizing chromatin structure at the level of the nucleosome and fibre 9. Key characteristics of cancer include unrestricted proliferation, apoptosis resistance, and the abilities to metastasize and to evade the immune system 98, and all of these properties can probably be acquired through mechanisms that involve increased epigenetic plasticity. As discussed above, studies on somatic cell reprogramming support a role for macroh2a in the stabilization of differentiated epigenomes. Collectively, current data support a tumour-suppressive role for macroh2a1.1 and macroh2a2, whereas the function of macroh2a1.2 depends on the specific cancer context (as discussed in REF. 99). MacroH2A expression is downregulated as disease progresses; its re expression reduces the metastatic potential of melanoma 100, whereas sirna-mediated depletion of macroh2a1 was shown to increase the aggressiveness of teratoma and breast cancer cells 60,101. Levels of the splice isoform macroh2a1.1 inversely correlate with proliferation. Accordingly, this variant is downregulated in many types of cancer, and its low levels are associated with poor prognosis 102,103. Similarly to the function of macroh2a1.2 in ES cells and epidermal stem cells 60, downregulation of macroh2a1 in bladder cancer cells enhances their stem-like properties 104. In contrast to macroh2a, HIRA-mediated deposition of H3.3 facilitates reprogramming, at least reprogramming by SCNT 88. The frequent inactivation of DAXX and ATRX by mutation or genomic loss in cancers provokes the intriguing hypothesis that in these conditions a shift to HIRA-mediated functions of H3.3 could contribute to cancer, potentially by increasing cellular plasticity. Further supporting the role of an oncogenic function, H3.3 is frequently overexpressed in various types of cancer 97. However, H3.3 has tumour-suppressive function NATURE REVIEWS MOLECULAR CELL BIOLOGY ADVANCE ONLINE PUBLICATION 9

10 Initiation of the DDR H2A.X MacroH2A Tumour suppressor Nucleosome stability Differentiation HR Epigenetic plasticity Nucleosome turnover Expression of oncogenes NHEJ? Telomere DSBs PcG Centromere chc HJURP Euchromatin CENP-A MacroH2A Inactivating mutations Shift to HIRA-mediated deposition ATRX DAXX Mislocalization; risk of neocentromere formation Epigenetic plasticity Nucleosome turnover NHEJ H3.3 Mutations inhibit histone PTM machineries H2A.Z p400 and SRCAP complexes HIRA Figure 4 Deregulation of the histone variant network in cancer. Specific chaperones (such as death domainassociated protein 6 (DAXX)) and chromatin remodellers (such as ATRX) and the complexes that contain p400 and SWI2 SNF2 related CBP activator protein (SRCAP)) deliver histone variants to specific chromatin environments (indicated by arrows). The machinery involved in macroh2a deposition is currently unknown (question mark). H2A.Z and histone cell cycle regulation-defective homologue 4 (HIRA)-deposited H3.3 promote epigenetic plasticity and nucleosome dynamics, and are frequently overexpressed in cancer (upward-pointing arrows). On the contrary, macroh2a has tumour-suppressive capabilities, stabilizes nucleosomes, promotes differentiation and was found to be downregulated in cancer (downward-pointing arrows). When overexpressed in cancer, histone H3 like centromeric protein A (CENP-A) is misplaced to chromosome arms by the H3.3 deposition machinery (which comprises ATRX and DAXX), where it participates in the formation of more stable nucleosomes, thereby potentially affecting chromatin structure and accessibility to chromatin-interacting proteins (including transcription regulators). Mislocalized CENP-A could also initiate the formation of neocentromeres that could drive chromosome breakage. The CENP-A-depositing complex Holliday junction recognition protein (HJURP) was also shown to be upregulated in cancer, whereas components of p400 containing complex and SRCAP-containing complex, which are involved in H2A.Z deposition, were found to be upregulated in some cancers and downregulated in others. The histone variant network can also be affected by mutations (red stars). For example, H3.3 can harbour mutations that affect the distribution of post-translational histone modifications (PTMs) and, consequently, the epigenetic landscape of the cell. In addition, ATRX and DAXX can be inactivated by mutations, causing a shift towards HIRA-mediated H3.3 deposition. ATRX also has a role in inhibiting the deposition of macroh2a1 on telomeres. In ATRX-deficient cells, macroh2a1 accumulates at subtelomeric regions. Histone variants also affect genome integrity through the engagement in the DNA damage response (DDR). H2A.X is important for initiating the DDR and thereby safeguards genomic stability. H2A.Z is also important for the proper execution of DNA repair, but its sustained presence promotes error-prone repair through non-homologous end joining (NHEJ). NHEJ is also promoted by H3.3. By contrast, macroh2a1.2 favours repair through homologous recombination (HR), which is error-free. In cancers, the overexpression of H2A.Z and H3.3, and the frequent downregulation of macroh2a, could potentially promote NHEJ and genomic instability. chc, constitutive heterochromatin; DSBs, double-strand breaks; PcG, Polycomb-group protein-bound facultative heterochromatin. See TABLE 2 for additional details and references. Epithelial mesenchymal transition (EMT). The switch of cells from an epithelial to a mesenchymal morphology. in glioblastoma, whereby it antagonizes the self-renewal capacity of stem-like cells to promote differentiation and is downregulated in malignant cells 105. The role of H3.3 in cancer development is, thus, likely to be complex. Aside from H3.3, H2A.Z is also overexpressed in different types of cancer 97. As discussed above, studies in mouse ES cells have shown that H3.3 and H2A.Z facili tate the access of regulatory complexes to chromatin, and data suggest that they might contribute to cancer development by increasing epigenetic plasticity. Indeed, H2A.Z overexpression is an indicator of poor prognosis in melanoma, hepatocellular carcinoma and breast cancer In hepatocellular carcinoma, H2A.Z.1 increases epithelial mesenchymal transition (EMT) and tumour growth 107. Both H2A.Z.1 and H2A.Z.2 are overexpressed in advanced-stage melanoma and, in particular, H2A.Z.2 contributes to proliferation by facilitating the transcription of oncogenes 108. Furthermore, the two chromatin remodellers that mediate H2A.Z deposition namely, p400 and SRCAP are overexpressed in some cancers 97, and p400 promotes oncogenic WNT signalling and counteracts the tumour-suppressive function of the histone acetyltransferase KAT5 (also known as TIP60) ADVANCE ONLINE PUBLICATION

11 G2 M checkpoint A control mechanism that allows cell cycle progression only in the absence of DNA damage. Homologous recombination The exchange of DNA sequences between different chromosome copies. Non-homologous end joining (NHEJ). A DNA repair pathway that ligates DNA break ends independently of their sequence. Whereas H2A.Z and H3.3 have oncogenic properties, macroh2a seems to be tumour suppressive; this is reflected by the opposing influence of H2A.Z and H3.3 compared with macroh2a on chromatin stabil ity and dynamics. MacroH2A itself has intrinsic histone H1 like functions 110,111, whereas H3.3 (but not H2A.Z) impairs the binding of the linker histone H1 to DNA 112,113. H1 linker histones bind to the DNA between nucleosomes, stabilize DNA nucleosome inter actions and contribute to fibre compaction (as reviewed in REF. 114). In line with this, H2A.Z-containing and H3.3 containing nucleosomes are particularly labile 9, and the presence of these variants at promoters correlates with particularly high nucleosome turnover 115, whereas macroh2a1 has been found to be even less mobile than is the replicationcoupled histone H4 (REF. 116), and macroh2a containing nucleosomes are relatively more stable 117. Taken together, the observed overexpression of dynamic histone variants such H2A.Z and H3.3 and the reduction of stabilizing histone variants such as macroh2a in various cancers suggest that these changes could contribute to tumorigenesis by increasing epigenetic plasticity. Mutations of H3 variants affect histone methylation and drive childhood cancers. Mutations at K27, K36 and G34 in the amino-terminal tail of H3.3, but also in that of the replication-coupled H3.1, are frequent in paediatric brain tumours and juvenile bone tumours (TABLE 3). H3.3 mutations are more frequent than are H3.1 mutations, which suggests that there is an increased positive selection pressure on mutations of the two H3.3 encoding genes, the expression of which contributes ~35% of the total H3 protein pool (FIG. 3b; thus, it is most likely that H3.3 mutations have a greater functional impact than do mutations of any of the multiple genes that encode the replication-coupled histone H3.1). The cancer type-specific appearance of mutations in these two different H3.3 coding genes might reflect different expression levels of these genes in the cell of origin. In adults, mutations in the genes that encode H3.3 and H3.1 are extremely rare, which suggests that there is a limited developmental time window in which these selective pressures operate: mutated clones could arise early during embryonic development, and expand during infancy and puberty. H3 mutations exert their oncogenic potential by reprogramming the epigenetic landscape. For example, a K27M substitution in H3.3 targets a residue that is post-translationally modified by PRC2 and was found to inhibit its catalytic activity 122. In general, PRCs maintain the repression of genes that drive alternative fates during differentiation and development, and their deregulation frequently occurs and contributes to tumour patho genesis 123. PRC2 catalyses H3K27me3 (REF. 124). High-grade gliomas, which feature the K27M mutation of H3.3, show reduced global K27 methylation and DNA hypomethylation, and these changes are major drivers of altered gene expression 125. The global reduction of H3K27 methylation is accompanied by a locally restricted increase in the enrichment of H3K27 methylation at hundreds of genes 126. In a study that used human ES cells as a system in which to model the K27M mutation of H3.3 and its impact on cell fate, K27M mutated H3.3 was found to synergize with the loss of the key tumour-suppressor gene TP53 (which encodes p53) and the activation of plateletderived growth factor receptor-α (PDGFRα) signalling, which reset neural progenitor cells derived from the ES cells back to a developmentally more primitive stem cell state 127. Similarly to K27M, the experimental introduction of analogous mutations of other post-translationally modified K residues in the N-terminal tails of H3.3 was shown to also affect the methylation status of histones, in particular K36me3 (REF. 122). A K36M substitution in H3.3 occurs in chondroblastoma 121 and was shown to inhibit at least two H3K36 histone methyltransferases (namely, multiple myeloma SET domain-containing protein (MMSET; also known as NSD2) and SETD2) and result in a global reduction of K36 methylation of H3 histones 128. As the K36M substitution prevents K36 methylation of H3.3, this mutation will also inevitably interfere with the binding of H3.3 specific K36me3 readers, such as ZMYND11 (REFS 54,129). In short, mutations in the N terminal tails of H3 variants, in particular H3.3, are prevalent in paediatric and juvenile cancers, but not in adult cancers, which suggests that these mutations drive tumorigenesis through a developmentally timed mechanism. Histone variants in DNA damage repair and genomic stability. DNA damage and repair are double-edged swords in cancer. Therapeutics aim to kill cells by inducing high levels of DNA damage, but low levels of DNA damage coupled with error-prone repair drive cancer progression by promoting genomic or genetic instability. Phosphorylation of the histone variant H2A.X at its unique serine at position 139 (the product of which is referred to as γh2a.x) is an early event in the detection of DNA double-strand breaks (DSBs) 130. γh2a.x spreads around the DNA damage site to amplify the signal to the repair machinery (as reviewed in REF. 131). At low levels of genotoxic stress, γh2a.x helps to activate the G2 M checkpoint and cell cycle arrest 132, and H2A.X-deficient mice are hypersensitive to radiation 74 and develop T cell lymphomas on a p53 null background 73. Mammalian cells repair DSBs via homologous recombination and non-homologous end joining (NHEJ; reviewed in REF. 133), the latter of which is more likely to generate genomic translocations and gene fusions. H2A.Z controls a crucial step in both NHEJ and homologous recombination, as its incorporation at a DNA damage site generates an open chromatin structure that is a suitable template for the function of the DNA repair machinery 134. Removal of H2A.Z by the histone chaperone acidic leucine-rich nuclear phosphoprotein 32 family member E (ANP32E) initiates homologous recombination 37, whereas the sustained presence of H2A.Z favours NHEJ 135. In addition to H2A.Z, H3.3 contributes together with PARP1 and the chromatin remodeller chromodomain helicase DNA binding NATURE REVIEWS MOLECULAR CELL BIOLOGY ADVANCE ONLINE PUBLICATION 11