Viviana Peña

Jazmin J Hernandez-Kantun

Walter H Adey

L E Line

Gall

Assessment of coralline species diversity in the European coasts supported by sequencing of type material: the case study of Lithophyllum nitorum (Corallinales, Rhodophyta)

Keywords: Biodiversity, Corallinales, DNA barcoding, distribution, Lithophyllum, maerl beds, Mediterranean, North East Atlantic, systematics, type collections

A constant effort in sequencing an extensive number of specimens originating from comprehensive sampling had return an unprecedented amount of information fostering our understanding of diversity, evolution and distribution of coralline algae; however, many sequences lack reliable assignation of a taxonomic name, specially at the species level. Recently, the sequencing of type material allowed to bridge this gap by providing a direct link between the DNA sequence and the type bearing name. For instance, in the genus Lithophyllum, the identity of three species, generally abundant along the European Atlantic and the Mediterranean, was demonstrated by including sequences of the type material. Nevertheless, for less conspicious species, such as Lithophyllum nitorum, data are still needed to assess distribution, anatomy, phylogenetic affinities and taxonomic status. Using DNA sequences recovered from the type material of L. nitorum, further recent collections were resolved as conspecific and used to improve the description and refine the distribution of this species. Lithophyllum nitorum consisted of subtidal, thin crusts overgrowing fragments of dead maerl, pebbles and drifted fragments of fishing lines. The presence of uniporate conceptacles markedly protruding can be considered an external morphological feature useful to identify L. nitorum from other subtidal Lithophyllum species with similar habitat. Lithophyllum nitorum is reported for the Mediterranean Sea and confirmed in the North East Atlantic, a similar distribution as for L. incrustans and L. hibernicum.

INTRODUCTION

Lithophyllum Philippi is considered one of the most speciose non-geniculate coralline algal genera with 130 specific/infraspecific names currently accepted (Guiry & Guiry, 2017). This genus is mainly distributed in tropical and temperate waters, where it contributes to the formation of characteristic calcareous bioconstructions such as the Caribbean algal ridges (Adey, 1978), the coralligenous and trottoir communities in the Mediterranean Sea [START_REF] Ballesteros | Mediterranean coralligenous assemblages: a synthesis of present knowledge[END_REF][START_REF] Pezzolesi | -Genetic and morphological variation in an ecosystem engineer, Lithophyllum byssoides (Corallinales, Rhodophyta)[END_REF], and singular maerl beds in the Norht East Atlantic [START_REF] Peña | Lithophyllum spp. form unusual maerl beds in the North East Atlantic: the case study of L. fasciculatum (Lamarck) Foslie, 1898, in Brittany[END_REF][START_REF] Heesch | Sequencing type material resolves the identity and distribution of the generitype Lithophyllum incrustans, and related European species L. hibernicum and L. bathyporum. (Corallinales, Rhodophyta)[END_REF]. In this latter region, recent literature pointed out 12 species of Lithophyllum occurring under different growth-forms, from the intertidal to subtidal : the generitype Lithophyllum incrustans Philippi, together with L. bathyporum Athanasiadis & Ballantine, L. byssoides (Lamarck) Foslie, L. crouaniorum Foslie, L. cystosirae (Hauck) Heydrich, L. decussatum (Ellis & Solander) Philippi, L. dentatum (Kützing) Foslie, L. hibernicum Foslie, L. nitorum W.H.Adey & P.J.Adey, L. orbiculatum (Foslie) Foslie, L. papillosum (Zanardini ex Hauck) Foslie and L. vickersiae Me.Lemoine [START_REF] Chamberlain | Rhodophyta Part 2B Corallinales[END_REF]Hernandez-Kantún et al., 2015, reviewed in Lugilde et al., 2016). So far, only three taxa -L. incrustans, L. hibernicum and L. bathyporum-have been subject of molecular studies using type material and recent collections from a wide geographic area [START_REF] Peña | b -Phymatolithon lusitanicum sp. nov. (Hapalidiales, Rhodophyta): the third most abundant maerlforming species in the Atlantic Iberian Peninsula[END_REF]. It allowed a reassessment of the phylogenetic affinities of these species, and the consideration of earlier collections identified as L. duckerae (Cornwall), L. fasciculatum and L. dentatum (Ireland and Britany) as L. hibernicum and L. incrustans, respectively. These results also demonstrated that L. hibernicum and L. incrustans have a high morphological plasticity, for which features mainly related with epithallial cells and sporangial conceptacles were useful for their identification [START_REF] Peña | b -Phymatolithon lusitanicum sp. nov. (Hapalidiales, Rhodophyta): the third most abundant maerlforming species in the Atlantic Iberian Peninsula[END_REF][START_REF] Vázquez | -Tipo de división en esporocistes de Lithophyllum hibernicum (Corallinales, Rhodophyta) y su implicación en el ciclo vital, en el Atlántico europeo[END_REF]. A fourth species -L. byssoides-has been also reassessed based on Atlantic and Mediterranean collections; however, molecular information from the lectotype (Nullipora byssoides Lamarck) is still pending to be obtained for a better clarification [START_REF] Pezzolesi | -Genetic and morphological variation in an ecosystem engineer, Lithophyllum byssoides (Corallinales, Rhodophyta)[END_REF].

With regards to the habitat, while the crustose Lithophyllum hibernicum (as L. incrustans in the literature before 2015) and L. orbiculatum are common in the intertidal, and L. crouaniorum in the subtidal [START_REF] Adey | Studies on the biosystematics and ecology of epilithic crustose Corallinaceae of the British Isles[END_REF][START_REF] Chamberlain | -A redescription of Lithophyllum crouanii (Rhodophyta, Corallinales) in the British Isles with and assessment of its relationship to L. orbiculatum[END_REF][START_REF] Chamberlain | -A redescription of Lithophyllum orbiculatum (Rhodophyta, Corallinales) in the Britis Isles and areassessment of generic delimitation i the Lithophylloideae[END_REF][START_REF] Chamberlain | Rhodophyta Part 2B Corallinales[END_REF], L. nitorum is considered a rare species, occurring on rock and shells only in the sublittoral zone down to 46 m [START_REF] Adey | Studies on the biosystematics and ecology of epilithic crustose Corallinaceae of the British Isles[END_REF][START_REF] Chamberlain | Rhodophyta Part 2B Corallinales[END_REF]. Lithophyllum nitorum was described by [START_REF] Adey | Studies on the biosystematics and ecology of epilithic crustose Corallinaceae of the British Isles[END_REF], and it was mainly characterized by small, adherent crusts relatively thin, with orange-pink colour, a smooth surface without orbital ridges, highly to somewhat glossy, a single layer of epithallial cells (rarely two), short subepithallial cells, cortical cells of contiguous filaments usually laterally aligned, conceptacles markedly protruding, usually bisporangial with a pore canal tapering to the surface and lined by small papillae, and sporangial chambers not globular with a small columella [START_REF] Adey | Studies on the biosystematics and ecology of epilithic crustose Corallinaceae of the British Isles[END_REF][START_REF] Chamberlain | Rhodophyta Part 2B Corallinales[END_REF]. In addition, [START_REF] Chamberlain | Rhodophyta Part 2B Corallinales[END_REF] pointed out the absence of secondary growth and growth over Lithothamnion sonderi Hauck as key characters to identify plants of L. nitorum in the British Isles. The type locality of L. nitorum is Port Erin Harbour (Isle of Man, 3-9 m depth, as epilithic in [START_REF] Adey | Studies on the biosystematics and ecology of epilithic crustose Corallinaceae of the British Isles[END_REF], but it was also reported southwards to northern Spain [START_REF] Chamberlain | Rhodophyta Part 2B Corallinales[END_REF]; in fact, [START_REF] Adey | Studies on the biosystematics and ecology of epilithic crustose Corallinaceae of the British Isles[END_REF] With the aim of re-assessing coralline species diversity and distributions along the European coasts, the sequencing of type specimens coupled with extensive sampling across a wide geographic area became the most efficient practice to refine the concept of a species [START_REF] Puillandre | Barcoding type specimens helps to identify synonyms and an unnamed new species in Eumunida Smith, 1883 (Decapoda: Eumunididae)[END_REF]. In the present study, we obtained DNA sequences (psbA and COI-5P) from the holotype of Lithophyllum nitorum, and we inferred species delimitation among recent collections made along the European coasts to assess the distribution of this species.

MATERIAL AND METHODS

Collections studied

We examined the holotype of Lithophyllum nitorum, collected in Port Erin Harbour, Isle of Man, at 3-9 m depth [START_REF] Adey | Studies on the biosystematics and ecology of epilithic crustose Corallinaceae of the British Isles[END_REF], and preserved in the herbarium of the Smithsonian Institution (US 70-10B, 10-30, Fig. 1). In addition, we studied eleven specimens identified as Lithophyllum sp. collected in recent subtidal surveys in Brittany (Trevignon, Fig. 2), Galicia (Ons and Cíes archipelagos, Islas Atlanticas National Park, Figs 3-7), and Mediterranean Spain (Granada, Figs 8-9) (Table 1). The Atlantic collections corresponded to thin crusts overgrowing dead maerl and pebbles associated to maerl beds between 11-15 m depth in 2011 (Figs 2-7), while the Mediterranean specimens consisted on crusts on a pebble and a drifted fragment of a fishing line at 15 m depth in 2012 (Figs 89). Recent collections were air-dried or oven-dried (50º C), preserved in plastic bags with silica gel before molecular and morphological studies, and deposited in the herbaria of Universidade de Santiago de Compostela (SANT) and Museum National d´Histoire Naturelle (PC) (acronyms follow [START_REF] Thiers | Index Herbariorum: A global directory of public herbaria and associated staff[END_REF].

DNA sequencing and phylogenetic analyses

Eleven specimens from recent subtidal collections were cleaned under a stereomicroscope and selected surfaces were ground for DNA extraction. Genomic DNA was extracted using a NucleoSpin® 96 Tissue kit (Macherey-Nagel, GmbH and Co. KG, Germany), and DNeasy® Blood & Tissue Kit (Qiagen, Valencia, CA) following manufacture's recommendations. DNA extractions and amplifications of the holotype of Lithophyllum nitorum housed in US were performed separately from recent collections and with negative controls run throughout at Smithsonian Laboratories of Analytical Biology, Washington DC, US; extraction of total genomic DNA of the holotype followed the modified protocol by [START_REF] Broom | Utility of psbA and nSSU for phylogenetic recontruction in the Corallinales based on New Zealand taxa[END_REF] of the Qiagen DNeasy Blood and Tissue Kit® (Qiagen, Crawley, UK). The mitochondrial COI-5P fragment was PCR-amplified using primer pairs GazF1/GazR1 [START_REF] Saunders | Applying DNA barcoding to red macroalgae: a preliminary appraisal holds promise for future applications[END_REF]. The chloroplast psbA gene was amplified using primer pairs psbA-F1/ psbA-R2 or psbA-F1/psbA600R [START_REF] Yoon | -A single origin of the peridinin-and fucoxanthin-containing plastids in dinoflagellates through tertiary endosymbiosis[END_REF], this latter used for the holotype. PCR reactions, thermal profiles, cleaning for sequencing and sequencing of the resulting products of the holotype followed [START_REF] Hernández-Kantún | -DNA sequencing, anatomy, and calcification patterns support a monophyletic, sub-arctic, carbonate reef-forming Clathromorphum (Hapalidiaceae, Corallinales, Rhodophyta)[END_REF]. Thermal profiles for PCR amplification of COI-5P and psbA fragments of recent collections followed [START_REF] Saunders | Methods for DNA barcoding photosynthetic protists emphasizing the macroalgae and diatoms[END_REF] and [START_REF] Bittner | Phylogénie des Corallinales (Rhodophyta) et analyse de leur diversité génétique dans le Pacifique Sud[END_REF], respectively; PCR reactions followed Peña et al. (2015a). Excess primers and nucleotides from PCR products (labeled CPVP, Table 1) were removed with shrimp alkaline phosphatase and exonuclease I enzymes, and sequenced using the Sanger method at Macrogen facilities (http://www.macrogen.com). PCR products labelled VPF and LLG (Table 1) were purified and sequenced by Genoscope (Bibliothèque du Vivant program, Centre National de Séquençage, France). Sequences were assembled and aligned with the assistance of CodonCode Aligner® (CodonCode Corporation, USA) and adjusted by eye using SeaView version 4 [START_REF] Gouy | -SeaView Version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building[END_REF]. Sequences were submitted to the Barcode of Life Data Systems (project 'NGCOR', BOLD, http://www.boldsystems.org; Ratnasingham & [START_REF] Hebert | -BOLD: The Barcode of Life Data System (www.barcodinglife.org)[END_REF] and GenBank (accession numbers listed in Table 1). Our analyses also comprised publicly available GenBank sequences of Atlantic and Mediterranean Lithophyllum spp., including those conspecific with type collections of the generitype L. incrustans, L. bathyporum and L. hibernicum (Hernández-Kantun et al., 2015) as well as the most relevant matches found with L. nitorum (> 90% similarity). These databases also encompassed sequences assigned to the genus Lithophyllum, and collected as associated crusts in Galician and Brittany maerl beds [START_REF] Pardo | Insights into species diversity of associated crustose coralline algae (Corallinophycidae, Rhodophyta) with Atlantic European maerl beds using DNA barcoding[END_REF]. According to recent phylogenetic studies [START_REF] Van Der | -DNA sequencing resolves species of Spongites (Corallinales, Rhodophyta) in the Northeast Pacific and South Africa, including S. agulhensis sp[END_REF][START_REF] Rösler | Phylogenetic relationships of Corallinaceae (Corallinales, Rhodophyta): taxonomic implications for reef-building corallines[END_REF], publicly available sequences for Spongites spp. corresponding to the "Southern Hemisphere group" were selected as outgroups for the psbA alignment; while sequences of Porolithon onkodes were selected as outgroups for the COI-5P alignment (Table 1).

The alignments comprised 25 COI-5P and 16 psbA sequences. Phylogenetic relationships were inferred using Maximum Likelihood (ML) and Bayesian inference (BI) using Mega 6 and MrBayes 3.2.1, respectively [START_REF] Ronquist | MRBAYES 3: Bayesian phylogenetic inference under mixed models[END_REF][START_REF] Tamura | MEGA6: Molecular Evolutionary Genetics Analysis version 6.0[END_REF]. Models of sequence evolution were estimated using the Akaike Information Criterion (AIC) and the Bayesian Information Criterion (BIC) obtained in jModeltest 2.1.3 [START_REF] Darriba | -jModelTest 2: more models, new heuristics and parallel computing[END_REF]. Maximum Likelihood and Bayesian analyses for the COI-5P and psbA alignments were performed under a generalized time-reversible with gamma+invariant sites heterogeneity model (GTR+G+I). The Bayesian analyses were performed under the same model with four Markov Chain Monte Carlo for 10 million generations, and tree sampling every 1000 generations.

Morphological studies of DNA-sequenced specimens

According to the previous phylogenetic analyses, representative specimens resolved conspecific to Lithophyllum nitorum were selected for anatomical examination with a scanning electron microscope (SEM, model JEOL JSM 6400, Universidade da Coruña, Spain). The examined specimens were deposited in the herbarium of Universidade de Santiago de Compostela (SANT). Cell length is the distance between primary pitconnections, and cell diameter is the measurement taken perpendicularly to this across the middle of the cell lumen. Conceptacle measurements were taken according to [START_REF] Adey | Studies on the biosystematics and ecology of epilithic crustose Corallinaceae of the British Isles[END_REF] and [START_REF] Chamberlain | Rhodophyta Part 2B Corallinales[END_REF].

RESULTS

The holotype of Lithophyllum nitorum was successfully sequenced only for the plastid psbA gene (fragment of 450 bp), but it did not result in any match in GenBank database obtaining the highest similarity (94%) with Lithophyllum impressum from NE Pacific and Uncultured Corallinales -LBC0599-from Vanuatu (Table 1, Fig. 10; searched on 9 November 2017). Our phylogenetic analyses of the psbA alignment (22 sequences ranging from 450 to 822 bp, with 222 variable sites) resolved this species, albeit with low support values, as the sister taxa of L. byssoides. Moreover, this phylogeny recovered a recent collection obtained from a Galician maerl bed (voucher SANT-Algae 31426, Ons archipelago) with high similarity with the holotype of L. nitorum (0.07 % divergence, 3 bp difference, Fig. 10). Based on this recent collection, the phylogenetic tree obtained from the ML analysis of the COI-5P alignment (25 sequences resulting 17 haplotypes, ranging from 445 to 664 bp in length, consisting of 216 variable sites) resolved another ten additonal specimens (grouped into four haplotypes) from Atlantic and Mediterranean waters as conspecific with it (Fig. 11). Thus, our results confirm the occurrence of L. nitorum along the Atlantic European coasts of Brittany and the Northwest of the Iberian Peninsula (Galicia), and they also expand its distribution range southwards to the Mediterranean Spain (Granada).

The recent collections resolved conspecific with the holotype of Lithophyllum nitorum (Fig. 1) and consisted of small crusts overgrowing fragments of dead maerl (ca. <3 cm), pebbles, cobbles, and a drifted fragment of fishing line (Figs 23456789). In contrast with the colour features provided in the literautre for L. nitorum, the colour of our specimens was dark to pale pink, and the surface was somewhat glossy only in some specimens (i.e. Fig. 3). Uniporate conceptacles were conspicuously protruding (Figs 12,13) and surrounded by a white rim (i.e. Figs 5,12). The SEM examination of several specimens showed very thin crusts, <500 µm thick, very adherent to the substrate with a monomerous construction and non-coaxial medulla (Fig. 14). Epithallial cells were mostly flattened, 1-2 µm tall x 7-8 µm diameter, disposed in 1-2 layers. Subepithallial cells were 5-8 µm tall x 3-10 µm diameter, as short as or shorter than cells subtending them (Figs 16,17).

Cortical cells of contiguous filaments were laterally aligned, 6-14 µm tall x 5-8 µm diameter, containing inclusions usually interpreted as floridean starch .

Secondary pit-connections were observed; cell fusions were absent (Fig. 15). Uniporate conceptacles were protruding (Fig. 18); old conceptacles were buried, not infilled, with elliptical chambers of 54-100 µm in height x 180-290 µm in diameter (Fig. 22).

According to the shape of the chamber, some buried and empty conceptacles in one plant collected in Ons archipelago (Galicia, voucher SANT-Algae 31426) corresponded to spermatangial conceptacles with chambers 21-22 µm in height x 84-87 µm in diameter (Fig. 19). While another recent collection from the same Galician locality (voucher SANT-Algae 31421) showed an uniporate tetrasporangial conceptacle protruding (Fig. 20), with pore canal of 50 µm in length, tapering towards the surface (from 60 µm to 20 µm diameter) (Fig. 21); the chamber was 150 µm in height x 250 µm in diameter , with a small calcified columella and tetrasporangia elongate, 117 µm in height x 24 µm in diameter, zonately divided (Fig. 20).

DISCUSSION

Based on a phylogenetic analysis of psbA sequences including the sequence obtained for the holotype of Lithophyllum nitorum, we were able to identify conspecific specimens in our recent collections from Atlantic and Mediterranean coasts (Fig. 11). However, none of the publicly available sequences from recent studies in Atlantic and Mediterranean Lithophyllum taxa (i.e. [START_REF] Heesch | Sequencing type material resolves the identity and distribution of the generitype Lithophyllum incrustans, and related European species L. hibernicum and L. bathyporum. (Corallinales, Rhodophyta)[END_REF][START_REF] Pezzolesi | -Genetic and morphological variation in an ecosystem engineer, Lithophyllum byssoides (Corallinales, Rhodophyta)[END_REF][START_REF] Pardo | Insights into species diversity of associated crustose coralline algae (Corallinophycidae, Rhodophyta) with Atlantic European maerl beds using DNA barcoding[END_REF] matched sequences from L. nitorum. According to the literature [START_REF] Adey | Studies on the biosystematics and ecology of epilithic crustose Corallinaceae of the British Isles[END_REF][START_REF] Chamberlain | Rhodophyta Part 2B Corallinales[END_REF], Lithophyllum nitorum is considered a rare, subtidal species distributed from the British Isles to northern Spain, and recorded on rocks and shells. Our present study based on DNA sequence data confirmed the presence of Lithophyllum nitorum in Brittany, the Northwest of the Iberian Peninsula (Galicia) and Mediterranean Spain. Furthermore, this study revealed the presence of this species on other substrates such as dead maerl, small pebbles or even drifted fragments of fishing lines. It is noteworthy to remark that all specimens collected are of small size and do not develop into large, conspicuous crusts; therefore, L. nitorum is likely to be easily overlooked. In agreement with the literature reports [START_REF] Adey | Studies on the biosystematics and ecology of epilithic crustose Corallinaceae of the British Isles[END_REF][START_REF] Chamberlain | Rhodophyta Part 2B Corallinales[END_REF] and our morphological observations, the presence of uniporate conceptacles markedly protruding can be considered an external morphological feature useful to identify L. nitorum from other Lithophyllum species that also occur in the subtidal, such as L. crouaniorum, L. bathyporum, L. hibernicum or L. incrustans [START_REF] Chamberlain | Rhodophyta Part 2B Corallinales[END_REF][START_REF] Heesch | Sequencing type material resolves the identity and distribution of the generitype Lithophyllum incrustans, and related European species L. hibernicum and L. bathyporum. (Corallinales, Rhodophyta)[END_REF]. On the other hand, L. nitorum is described in the literature as orange-pink crusts, highly to somewhat glossy [START_REF] Adey | Studies on the biosystematics and ecology of epilithic crustose Corallinaceae of the British Isles[END_REF][START_REF] Chamberlain | Rhodophyta Part 2B Corallinales[END_REF] while in our collections the colour varied from dark to pale pink and only some specimens were somewhat glossy. Most of our specimens of L. nitorum have empty uniporate conceptacles, but one Galician plant is considered a male gametophyte based on the shape of spermatangial conceptacles. [START_REF] Chamberlain | Rhodophyta Part 2B Corallinales[END_REF] pointed out that gametangial plants are unknown in the British Isles; Adey & Adey (1973, Table XII) recorded spermatangial conceptacles, however collection details are not indicated. According to the measurements provided in [START_REF] Adey | Studies on the biosystematics and ecology of epilithic crustose Corallinaceae of the British Isles[END_REF], the Galician specimen had smaller chambers (21-22 µm in height x 84-87 µm in diameter vs.

22 µm in height x 90-118 µm in diameter). In our study, only one Galician specimen examined showed tetrasporangial conceptacles that are in agreement with the literature (chamber 150 µm in height x 250 µm in diameter vs 100-135 x 255-385 µm µm and 94-156 µm x 245-385 µm in Adey &[START_REF] Adey | Studies on the biosystematics and ecology of epilithic crustose Corallinaceae of the British Isles[END_REF]Chamberlain &Irvine, 1994, respectively). For the British Isles, [START_REF] Chamberlain | Rhodophyta Part 2B Corallinales[END_REF] reported only bisporangial conceptacles, while [START_REF] Adey | Studies on the biosystematics and ecology of epilithic crustose Corallinaceae of the British Isles[END_REF] indicated that tetrasporangia were rare in the distribution range hitherto known for L. nitorum along the Atlantic European coasts. Likewise, we observed buried conceptacles not infilled which has not been reported earlier [START_REF] Adey | Studies on the biosystematics and ecology of epilithic crustose Corallinaceae of the British Isles[END_REF][START_REF] Chamberlain | Rhodophyta Part 2B Corallinales[END_REF]. The distribution range hitherto reported for L. nitorum is restricted from the British Isles to northern Spain [START_REF] Adey | Studies on the biosystematics and ecology of epilithic crustose Corallinaceae of the British Isles[END_REF][START_REF] Chamberlain | Rhodophyta Part 2B Corallinales[END_REF]; our results confirm records for Brittany and Galicia (Northwest of Spain), and they also expand the distribution of the species southwards to the Spanish coast of the Mediterranean Sea (Granada). Our Mediterranean record based on molecular evidences are congruent with previous observations of extant plants and Neogene fossil records in southern Spain and Balearic Islands [START_REF] Braga | Taxonomy of fossil coralline algal species: Neogene Lithophylloideae (Rhodophyta, Corallinaceae) from southern Spain[END_REF]. All recent collections assigned to L. nitorum were subtidal, made between 11-15 m, in agreement with the wide depth range provided in the literature (3-30 m in the British Isles, down to 46 m in northern Spain, [START_REF] Adey | Studies on the biosystematics and ecology of epilithic crustose Corallinaceae of the British Isles[END_REF][START_REF] Chamberlain | Rhodophyta Part 2B Corallinales[END_REF]. The Atlantic collections from Brittany and Galicia were found on dead maerl or small pebbles associated with maerl beds. A recent DNA barcoding study carried out on maerl beds from these two Atlantic regions [START_REF] Pardo | Insights into species diversity of associated crustose coralline algae (Corallinophycidae, Rhodophyta) with Atlantic European maerl beds using DNA barcoding[END_REF] revealed higher species diversity in associated crustose coralline algae than in maerl-forming species (9 vs 3, as total taxa obtained for each growth-form); two out of the nine crustose taxa corresponded to Lithophyllum hibernicum (Brittany) and an unidentified Lithophyllum sp.3 (Galicia) overgrowing pebbles, both with uniporate conceptacles not protruding (Fig. 3 in [START_REF] Pardo | Insights into species diversity of associated crustose coralline algae (Corallinophycidae, Rhodophyta) with Atlantic European maerl beds using DNA barcoding[END_REF]. The record of L. nitorum as an additional encrusting species associated with maerl beds in Brittany and Galicia increases the associated diversity reported for this coastal habitat in the North East Atlantic (Peña et al., 2014a).

The present work confirms the effectiveness of sequencing type specimens of coralline algae to link the type bearing name to the many sequences obtained from recent specimens (i.e. Peña et al. 2014bPeña et al. , 2015b;;[START_REF] Hernández-Kantún | -DNA sequencing, anatomy, and calcification patterns support a monophyletic, sub-arctic, carbonate reef-forming Clathromorphum (Hapalidiaceae, Corallinales, Rhodophyta)[END_REF][START_REF] Peña | b -Phymatolithon lusitanicum sp. nov. (Hapalidiales, Rhodophyta): the third most abundant maerlforming species in the Atlantic Iberian Peninsula[END_REF] in the aim to establish a DNA Library of Life (Le [START_REF] Gall | -Toward the DNA Library of Life[END_REF]. The sequence of the holotype of Lithophyllum nitorum allowed us to identify recent collections that expanded its distribution range southwards to the Mediterranean. Lithophyllum nitorum is considered a rare species in the British Isles, while our results indicate that it occurs on different substrates in the subtidal, even associated to maerl beds in Brittany and Galicia.

According to the literature [START_REF] Adey | Studies on the biosystematics and ecology of epilithic crustose Corallinaceae of the British Isles[END_REF], L. nitorum occurs sporadically to the north while increased southwards in Cornwall and the Cantabrian Sea. Together with our confirmed records in the Northwest of Spain (Galicia) and in the Mediterranean, it might represent a temperate Lithophyllum species with warm-water affinities; a similar Atlantic-Mediterranean distribution is also reported for L. incrustans and L. hibernicum [START_REF] Heesch | Sequencing type material resolves the identity and distribution of the generitype Lithophyllum incrustans, and related European species L. hibernicum and L. bathyporum. (Corallinales, Rhodophyta)[END_REF]. Despite the considerable number of publicly available sequences of Lithophyllum taxa (more than 275 COI-5P and psbA GenBank sequences), none of them matched with L. nitorum; the presence of this uncommon, subtidal species with somewhat restricted distribution further supports Lithophyllum as a genetically diverse genus in the European coasts (i.e. [START_REF] Pezzolesi | -Genetic and morphological variation in an ecosystem engineer, Lithophyllum byssoides (Corallinales, Rhodophyta)[END_REF]. In this regard, if the diversification of the subfamily Lithophylloideae was estimated during the early and middle Miocene (~ 23-12 million years ago, [START_REF] Rösler | -Timing of the evolutionary history of Corallinaceae (Corallinales, Rhodophyta)[END_REF], the Miocene fossil records of L. nitorum in Mediterranean Spain [START_REF] Braga | Taxonomy of fossil coralline algal species: Neogene Lithophylloideae (Rhodophyta, Corallinaceae) from southern Spain[END_REF] would indicate an early divergence of this species. Finally, our results based on type material confirmed Lithophyllum nitorum as a distinct species from other European Lithophyllum taxa recently re-assessed (Hernández-Kantun et al., 2015). Numerous additional Lithophyllum species from the North East Atlantic and worldwide need to have their type specimens sequenced to correctly apply names.

SANT-Algae 31420

  recorded L. nitorum at most of the Bay of Biscay stations sampled, reaching 24% abundance at 37-46 m off Castro Urdiales (Cantabrian Sea) compared to 14% between 8-30 m in the British Isles, particularly abundant off the Lizard (Cornwall) while it occurrs sporadically northwards (ca. 0.2-0.7 % mean abundance, fig. 53 in Adey & Adey, 1973). In the Mediterranean Spain, Braga & Aguirre (1995) observed plants attributed to L. nitorum in Almería and in Cabrera (Balearic Islands), and reported it in southern Spain as minor component of fossil coralline associations in the Neogene (Miocene and Pliocene, ca. 23 million years ago).

Figs 1- 9 .

 9 Figs 1-9. Lithophyllum nitorum. 1. Holotype specimen (voucher US 70-10B). 2, 3, 5-7.

Fig. 10 .

 10 Fig. 10. Phylogenetic tree inferred from ML and BI analyses of psbA sequences of

Table 1 .

 1 Sample information for species included in molecular analyses. In bold, holotype specimen of Lithophyllum nitorum. Additional COI-5P and psbA sequences downloaded from GenBank are detailed.

	Species	Voucher	GenBank	GenBank	Collection details	Herbarium
			accession	accession
			number (COI-	number
			5P)	(psbA)
	Lithophyllum nitorum	US 70-10B		MG648773	Port Erin Harbour, Isle of Man, UK (Adey	US 70-10B
					& Adey, 1973)
	L. nitorum	CPVP913	MG648771		Subtidal (15 m), maerl bed, Le Dragon,	SANT-Algae 31427
					Trevignon, France, 13/05/2011. Coll. W.
					Thomas, Y. Fontana.
	L. nitorum	CPVP760	MG648763		Subtidal (13 m), maerl bed, Ons, Ría de
					Pontevedra, Galicia, Spain, 07/04/2011. Coll.
					I. Bárbara, R. Barreiro, V. Peña.

Acknowledgments

Financial support came from the following institutions and projects: Action Transversale du Muséum National d'Histoire Naturelle ("Taxonomie moléculaire: DNA Barcode et gestion durable des collections" and "Biodiversité actuelle et fossile. Crises, stress, restaurations et panchronisme: le message systématique" and"Emergences"), Spain´s Ministerio de Economía y Competitividad (CTM2010-18787, partially founded by FEDER) and Xunta de Galicia (10MMA103003PR); ASSEMBLE (grant agreement nº. 227799) supported sampling in Brittany. Acquisition of molecular data was carried out at the CNRS-UMS 2700 in Service de Systématique Moléculaire (MNHN, Paris). VPF acknowledges support by Universidade da Coruña (Contrato programa-Campus Industrial de Ferrol). JJHK acknowledges support from the Smithsonian Institution through a postdoctoral fellowship. We sincerely thank Ignacio Bárbara, Rodolfo Barreiro and Cristina Pardo for fieldwork and/or lab collaboration. We acknowledge to Parque

Nacional Marítimo Terrestre das Illas Atlánticas de Galicia for permission to carry out our surveys. We are also grateful to both reviewers for their helpful comments on the manuscript.


Kirti Prakash
email: kirtiprakash1.61@gmail.com

David Fournier
email: dfournie@uni-mainz.de

Evidence for the implication of the histone code in building the genome structure

Keywords: chromatin domains, chromosomes, histone code, chromatin folding, genome structure

Histones are punctuated with small chemical modifications that alter their interaction with DNA. One attractive hypothesis stipulates that certain combinations of these histone modifications may function, alone or together, as a part of a predictive histone code to provide ground rules for chromatin folding. We consider four features that relate histone modifications to chromatin folding: charge neutralisation, molecular specificity, robustness and evolvability. Next, we present evidence for the association among different histone modifications at various levels of chromatin organisation and show how these relationships relate to function such as transcription, replication and cell division. Finally, we propose a model where the histone code can set critical checkpoints for chromatin to fold reversibly between different orders of the organisation in response to a biological stimulus.

DNA | nucleosomes | histone modifications

Introduction

The genetic information within chromosomes of eukaryotes is packaged into chromatin, a long and folded polymer of double-stranded DNA, histones and other structural and nonstructural proteins. The repeating units of the polymer, the nucleosomes, are 147 base-pairs (1.75 turn) of DNA wrapped around an octamer of 4 histone proteins [START_REF] Luger | Crystal structure of the nucleosome core particle at 2.8 å resolution[END_REF][START_REF] Andrews | Nucleosome structure (s) and stability: variations on a theme[END_REF]. Nucleosomes are thought to be further compacted into a higher order 30 nm chromatin fibre by linker histone H1 [START_REF] Wong | Distinct requirements for chromatin assembly in transcriptional repression by thyroid hormone receptor and histone deacetylase[END_REF]. The structure of nucleosomes can be altered post-translationally by the small chemical modifications of histone protein [START_REF] Grunstein | Histone acetylation in chromatin structure and transcription[END_REF][START_REF] Lanctôt | Dynamic genome architecture in the nuclear space: regulation of gene expression in three dimensions[END_REF]. Subsequently, one can characterise the organisation of chromatin into three interrelated categories: (1) the basic building blocks, (2) the functional structure of chromatin and (3) the higher order spatial arrangement of chromatin.

The two classical building blocks (Figure 1A, first column): beads-on-a-string and 30 nm chromatin fibre have been extensively studied previously [START_REF] Luger | Crystal structure of the nucleosome core particle at 2.8 å resolution[END_REF][START_REF] Kornberg | Chromatin structure: oligomers of the histones[END_REF][START_REF] Richmond | Structure of the nucleosome core particle at 7 a resolution[END_REF][START_REF] Kornberg | Twenty-five years of the nucleosome, fundamental particle of the eukaryote chromosome[END_REF]. Regarding the intermediary level of compaction, chromatin can display several configurations (active, repressed, inactive) depending upon enrichment of a particular histone mark (Figure 1A, second column). At a higher order (Figure 1A, third column), chromatin can be either described as a bimodal heterochromatin/euchromatin model (condensed and open regions, respectively), chromosome territories [START_REF] Markaki | Functional nuclear organization of transcription and dna replication a topographical marriage between chromatin domains and the interchromatin compartment in Cold Spring Harbor symposia on quantitative biology[END_REF] or as a very condensed structure in the The spatial organization of chromatin can be studied at three levels: at the lowest orders, which include the beads-on-a-string model and the chromatin fibre (left column), at the level of functional chromatin domains (middle column) and high order chromatin patterns (right column). The organization of chromatin domains can be modelled using various post-translational histone modifications. Depending upon the kind of histone modification a nucleosome is enriched with, chromatin can be either highly condensed, in an open conformation or switch between these two extreme forms. The higher order chromatin patterns can either be viewed from: the chromosome territory model of DNA organization, the over-simplified bimodal classification of condensed and open chromatin as hetero-and euchromatin respectively, and the highly condensed configuration of metaphase chromosomes. (B) The components of the histone code. On the left, the input system A, made of combinations of histone modifications (coloured pins). One unique combination can be found at three different positions 1,2,3, in the genome. The combinations of the input system A is translated into components of the output system B, with a black and white colour-scale. The translation happens via adaptors such as transcription factors or epigenetic regulators that recognise specific histone modifications to change chromatin state.

case of the metaphase chromosome. Chromatin fibres indeed present a variety of sizes [START_REF] Prakash | Structure, function and dynamics of chromatin in Chromatin Architecture[END_REF] and of shapes ( [START_REF] Dubochet | Cryo-electron microscopy of vitrified sv40 minichromosomes: the liquid drop model[END_REF] shows a solenoid model of chromatin), while recent studies attempt to challenge their existence [START_REF] Fussner | Living without 30nm chromatin fibers[END_REF], hinting that the hierarchy beadson-a-string/fibres/domains might be much more complicated and diverse than we currently think.

On the functional side, it has been shown that biochemical changes made to specific histones tails are associated with different condensation levels of chromatin. For instance, trimethylaton of lysine 9 on histone 3 (H3K9me3) is usually associated with condensed chromatin and centromeric regions [START_REF] Fischle | Regulation of hp1-chromatin binding by histone h3 methylation and phosphorylation[END_REF][START_REF] Ekwall | Epigenetic control of centromere behavior[END_REF]. On the other hand, trimethylaton of lysine 4 on histone 3 (H3K4me3) is strongly enriched at promoter regions of active genes where chromatin exists in an open conformation [START_REF]Active genes are tri-methylated at k4 of histone h3[END_REF][START_REF] Ruthenburg | Methylation of lysine 4 on histone h3: intricacy of writing and reading a single epigenetic mark[END_REF]. Chromatin can also exist in a switchable state between these two extreme forms if histone H3 is trimethylated at lysine 27 (H3K27me3) [START_REF] Chandra | Independence of repressive histone marks and chromatin compaction during senescent heterochromatic layer formation[END_REF]. H3K27me3 is a histone mark characteristic of repressed genes.

The number of ways histone proteins can be modified is quite extensive and can explain the vast majority of possible combinations that can lead to various functional outcomes.

The relationship between these combinations and the function they perform is referred to as the "histone code" [START_REF] Strahl | The language of covalent histone modifications[END_REF][START_REF] Jenuwein | Translating the histone code[END_REF][START_REF] Turner | Cellular memory and the histone code[END_REF]. Previously, biological codes have been thoroughly described [START_REF] Barbieri | The organic codes: an introduction to semantic biology[END_REF][START_REF] Barbieri | Biosemiotics: a new understanding of life[END_REF][START_REF] Kühn | Is the "histone code" an organic code?[END_REF]; they comprise of input system made of signs with no biological function that are translated into organic output functions via adaptor molecules. In the example of the genetic code, the inputs are codons, the adaptor is the translation machinery

Outlook

The urge to discover secrets is deeply ingrained in human nature; even the least curious mind is roused by the promise of sharing knowledge withheld from others. Most of us are driven to sublimate this urge by the solving of artificial puzzles devised for our entertainment. Detective stories or crossword puzzles cater for the majority; the solution of secret codes may be the hobby of the few. J. Chadwick, The Decipherment of Linear B [START_REF] Chadwick | The decipherment of Linear B[END_REF]. Citation inspired from The Code Book by Simon Singh [START_REF] Singh | The code book: the secret history of codes and code-breaking[END_REF].

From the day/night dichotomy to the genetic code, nature is full of symmetric, antagonistic exemplars and patterns. One such example is the organisation of structurally distinct chromatin states (active, inactive) on a single chromosome. In this article, we try to show how simple combinations of essential elements such as histone modifications can participate in sophisticated cellular features such as the structure of the genome. Here a code is identified, where an input system (histone modifications) is translated into an output system (chromatin states) via adaptors (epigenetic regulators or transcription factors). Such a code has a distinct importance in gene regulation and consequently for the cellular phenotype. and the outputs are amino acids. In the case of the histone code, combinations of histone modifications at a given position on the genome constitute the input system, the adaptors are the epigenetic regulators (for instance, the enzymatic complex Ezh2) that bind the modifications and outputs are chromatin features such as the level of chromatin compaction or gene expression (Figure 1B).

Here, we present supportive evidence for a combinatorial occurrence of histone modifications and its consequence for higher-order chromatin folding. We start with a brief historical introduction to chromatin biology. We then show the correlation among histone modifications at the level of nucleosomes, regulatory regions, TAD and chromosomes. Finally, we propose a model showing that these distinct chromatin domains can co-exist on a single chromosome.

Histone modifications, a robust but flexible matrix for evolution

Histones were first identified as a fundamental component of the nucleus in the early days of molecular biology [START_REF] Stedman | The basic proteins of cell nuclei[END_REF][START_REF] Wilkins | Physical studies of the molecular structure of deoxyribose nucleic acid and nucleoprotein in Cold Spring Harbor symposia on quantitative biology[END_REF] and were described as circular structures responsible for compacting DNA [START_REF] Luger | Crystal structure of the nucleosome core particle at 2.8 å resolution[END_REF][START_REF] Kornberg | Chromatin structure: oligomers of the histones[END_REF][START_REF] Finch | Solenoidal model for superstructure in chromatin[END_REF]. They were soon associated with DNA periodicity and compaction [START_REF] Kornberg | Chromatin structure: oligomers of the histones[END_REF][START_REF] Busch | The cell nucleus[END_REF][START_REF] Langridge | Molecular structure of deoxyribonucleic acid (dna)[END_REF][START_REF] Wilkins | X-ray diffraction studies of the molecular structure of nucleohistone and chromosomes[END_REF][START_REF] Pardon | X-ray diffraction studies on oriented nucleohistone gels in Cold Spring Harbor symposia on quantitative biology[END_REF], where DNA coils around an octamer of four core histone proteins (H2A/H2B dimer and H3/H4 tetramer, Figure 2A) [START_REF] Luger | Crystal structure of the nucleosome core particle at 2.8 å resolution[END_REF][START_REF] Oudet | Electron microscopic and biochemical evidence that chromatin structure is a repeating unit[END_REF]. The histone core with DNA was termed nucleosome and is known to be the first level of chromatin compaction. Early experiments showed that histones, via their N-terminal tails, can experience modifications such as acetylation and methylation, that can further alter the compaction of chromatin [START_REF] Grunstein | Histone acetylation in chromatin structure and transcription[END_REF]. The nomenclature of histone modification were named the following way: in the case of H3K9me3, H3 refers to the core histone protein, K refers to the amino acid, the number 9 indicates the position of lysine residue from N-terminal end of the amino acid tail of histone protein and me3 refers to the type of modification on the lysine residue (Figure 2B).

Over the course of the last decade, amino acids have been shown to experience several modifications, of at least twelve types: acetylation (lysine), methylation (lysine and arginine), phosphorylation (serine and threonine), sumoylation (lysine), ubiquitylation (lysine), ADP ribosylation, butyrylation, citrullination, crotonylation, formylation, proline isomerization, propionylation [START_REF] Delange | Histone iii. sequence studies on the cynaogen bromide peptides; complete amino acid sequence of calf thymus histone iii[END_REF][START_REF] Adamietz | Adp-ribosylation of nuclear proteins in vivo. identification of histone h2b as a major acceptor for mono-and poly (adp-ribose) in dimethyl sulfate-treated hepatoma ah 7974 cells[END_REF][START_REF] Liebich | Non-enzymatic glycation of histones[END_REF][START_REF] Hymes | Biotinylation of histones by human serum biotinidase: assessment of biotinyl-transferase activity in sera from normal individuals and children with biotinidase deficiency[END_REF][START_REF] Wondrak Gt | Histone carbonylation in vivo and in vitro[END_REF][START_REF] Tan | Identification of 67 histone marks and histone lysine crotonylation as a new type of histone modification[END_REF].

At a fundamental level, most of these modifications are neutralizing the net charge between DNA and histone proteins, and much of the gene regulation is a by product of effective net charge in the environment [START_REF] Allfrey | Acetylation and methylation of histones and their possible role in the regulation of rna synthesis[END_REF][START_REF] Wade | Histone acetylation: chromatin in action[END_REF]. Hence a combination of marks must work in synergy to provide sufficient proof reading so that no gene is accidentally turned on or off [46-Fig. 2. Histone modifications are major biochemical features of chromatin. Histone can experience various post-translational modifications in their protruding N-terminal tails but also within the C-terminal regions. These changes affect not only the overall compaction of chromatin but also gene expression. (A) The principal modifications on the four core histones: H2A, H2B, H3, H4. Two patterns worth noting here are the arrangement of lysines (K4, K9, K14, K18, K23, K27) and occurrence of Lysine (K), Serine (S) and Arginine (A) at [START_REF] Markaki | Functional nuclear organization of transcription and dna replication a topographical marriage between chromatin domains and the interchromatin compartment in Cold Spring Harbor symposia on quantitative biology[END_REF][START_REF] Prakash | Structure, function and dynamics of chromatin in Chromatin Architecture[END_REF][START_REF] Dubochet | Cryo-electron microscopy of vitrified sv40 minichromosomes: the liquid drop model[END_REF] and [START_REF] Wilkins | Physical studies of the molecular structure of deoxyribose nucleic acid and nucleoprotein in Cold Spring Harbor symposia on quantitative biology[END_REF][START_REF] Finch | Solenoidal model for superstructure in chromatin[END_REF][START_REF] Busch | The cell nucleus[END_REF] position on the amino acid tail of histone H3. Modified from [START_REF] Rodríguez-Paredes | Cancer epigenetics reaches mainstream oncology[END_REF] with permission. (B) Nomenclature of histones post-transcriptional modifications. The different features are the type of histone (H3 in our example), the amino acid and its position (Lysine at position 27) and the chemical modification, here a trimethylation, or triple methylation of the lysine 27 from the Nterminal end of the amino acide tail. (C) Different enzymes lead to writing, erasing and reading of chemical groups on the tails of histone proteins. Writers are enzymes that introduce various chemical modifications to histones. There are about 100 different enzymes that can write acetyl, methyl and phosphoryl groups (circles) to individual amino acid residues. Erasers are the enzymes that remove histone modifications and perform the opposite task of writers. Readers are enzymes that recognise specific chemical groups on the amino acid residues. For instance, bromodomains engage with acetylated lysines (modified from [START_REF] Tarakhovsky | Tools and landscapes of epigenetics[END_REF] with permission). Ac, acetylation; Me, methylation; P, phosphorylation; Ub, ubiquitination.

48]

. Lastly, the combination of histone modifications are stably maintained across many species and protein motifs have evolved to recognise histone modifications [START_REF] Winston | The bromodomain: a chromatin-targeting module?[END_REF].

We consider four measures which characterize the function of histone modifications 1. charge neutralisation 2. molecular specificity 3. robustness

evolvability

Charge neutralisation: Histone modifications are usually modified via enzymes that can neutralise the excess of charge on the DNA [START_REF] Biswas | Atomistic simulations of nucleosomes[END_REF][START_REF] Norton | Histone acetylation reduces nucleosome core particle linking number change[END_REF]. A nucleosome core particle has an overall residual charge of -150e (DNA contributes -294e, and histones contribute +144e) and is therefore electrically not neutral [START_REF] Cortini | The physics of epigenetics[END_REF]. Tails can attract negatively remote DNA molecules (the negatively charged due to phosphate oxide groups) to induce regional DNA compaction. Subsequently, the folding of DNA is also highly dependent on the positive counterions in the environment [START_REF] Bertin | Structure and phase diagram of nucleosome core particles aggregated by multivalent cations[END_REF][START_REF] Yang | The divalent cations ca2+ and mg2+ play specific roles in stabilizing histone-dna interactions within nucleosomes that are partially redundant with the core histone tail domains[END_REF].

Early experiments showed that acetylation (CH3-CH2-) or methylation (CH3-) of nucleosomes occur respectively in 50-60% and 40% of histones and can lead to increase and decrease of polymerase activity, respectively [START_REF] Allfrey | Acetylation and methylation of histones and their possible role in the regulation of rna synthesis[END_REF]. Methyl groups are produced during the metabolism of methionine, a subpart of the B12 vitamin circuit while acetylation is generated from the acetyl-Coenzyme A [START_REF] Kuo | Roles of histone acetyltransferases and deacetylases in gene regulation[END_REF][START_REF] Wang | Nutrition, epigenetics, and metabolic syndrome[END_REF]. Histone acetylation impairs the affinity of histones to DNA via charge antagonism, reduction of compaction and subsequent recruitment of certain factors such as SIR at telomeres or TAF1 at promoters, inducing activation of transcription [START_REF] Mathis | Effect of histone acetylation on structure and in vitro transcription of chromatin[END_REF][START_REF] Hong | Studies of the dna binding properties of histone h4 amino terminus. thermal denaturation studies reveal that acetylation markedly reduces the binding constant of the h4" tail" to dna[END_REF][START_REF] Morinière | Cooperative binding of two acetylation marks on a histone tail by a single bromodomain[END_REF]. This property is accompanied by the higher mobility of histones along DNA [START_REF] Cosgrove | Regulated nucleosome mobility and the histone code[END_REF]. Differently, methylation is associated to compaction of chromatin and so to reduced transcription, with reduced histone mobility [START_REF] Allfrey | Acetylation and methylation of histones and their possible role in the regulation of rna synthesis[END_REF][START_REF] Cosgrove | Regulated nucleosome mobility and the histone code[END_REF][START_REF] Prakash | A condensed history of chromatin research in Chromatin Architecture[END_REF].

Molecular specificity:

The histone modification sequence is orchestrated by the enzymes that either deposit, read or remove the marks, and serve as the adaptors molecules essential to biological codes [START_REF] Barbieri | The organic codes: an introduction to semantic biology[END_REF]. Enzymes that deposit marks are diverse and reflect the large spectrum of modifications available in nature (Figure 2C). Well-known examples are of histone acetyl-transferases (HAT) which transfer the acetyl group from acetyl-Coenzyme A to the tail of histones, and histone deacetylases (HDAC) which remove the acetyl groups, most notably upon chromatin compaction. Other complexes involved in histone regulation include the Trithorax complex (Trx) which is involved in methylation of histones and Polycomb (PcG) with module PRC2 involved in the deposition of H3K27me3 mark via methyltransferase Ezh2 [START_REF] Cao | Role of histone h3 lysine 27 methylation in polycomb-group silencing[END_REF][START_REF] Czermin | Drosophila enhancer of zeste/esc complexes have a histone h3 methyltransferase activity that marks chromosomal polycomb sites[END_REF].

Additionally, the H3K27me3 mark is bound by the PRC1 complex that can mono-ubiquitinate histone H2A on lysine 119 (H2AK119Ub1). This process is known to silence genes and is an excellent example of a combination of marks found at a genomic site with an impact on the function. In contrast, transcription factors with bromodomains are known to bind acetylated lysines [START_REF] Dhalluin | Structure and ligand of a histone acetyltransferase bromodomain[END_REF] and induce gene activation via acetyltransferase activity [START_REF] Santillan | Bromodomain and histone acetyltransferase domain specificities control mixed lineage leukemia phenotype[END_REF].

Robustness: Robustness is the capacity of living systems to preserve a given function during evolutionary times. Many important core features of organisms are robust and found in a wide variety of species; for instance, the skeleton is found in all vertebrates and can be considered a robust feature. Small or big structural variations are present in all groups, but the template remains the same. Robustness seems to imply two features: cooperation and redundancy; cooperation of the different basic molecular elements of the system, including quality control checkpoints [START_REF] Wagner | Robustness and evolvability: a paradox resolved[END_REF] and a high redundancy of the molecular elements, in order to resist to small changes. In the example of the skeleton, many different genes are involved, each of them participating to build the scaffold (cooperation).

Nevertheless, many genes perform the same function so if one gene fails or fluctuates, several other ones can take over to perform the same function (redundancy).

Overall, robustness is a requirement for all fundamental systems of life, including all the core biological codes such as the genetic code, which can produce proteins despite occasional changes [START_REF] Barbieri | Active and poised promoter states drive folding of the extended hoxb locus in mouse embryonic stem cells[END_REF]. Similarly, the histone code seems to feature properties favouring robustness. As we will see later, very similar histone modifications such as acetylations or methylations act in concert to form the histone code (cooperation), which potentially means that the loss of one mark does not necessarily affect the output of the code very strongly (redundancy).

For instance, for a given genomic region, active promoters, different combinations are possible, with small variations (Fig- ure 4). Such a redundancy stems in the high similarity of nucleosome structure across kingdoms archaea and eukaryotes [START_REF] Mattiroli | Structure of histone-based chromatin in archaea[END_REF] and the fact that most well-known histone modifications are found across eukaryotes, including human, mouse, fly and C.elegans [START_REF] Ho | Comparative analysis of metazoan chromatin organization[END_REF]. For instance, H3K9me3 is found to be present in vertebrates, invertebrates and yeast [START_REF] Consortium | The encode (encyclopedia of dna elements) project[END_REF][START_REF] Consortium | An integrated encyclopedia of dna elements in the human genome[END_REF]. Functions of enzymes associated with histone modifications remodelling seem to be very conserved across eukaryotes [START_REF] Laible | Mammalian homologues of the polycomb-group gene enhancer of zeste mediate gene silencing in drosophila heterochromatin and at s. cerevisiae telomeres[END_REF][START_REF] Luense | Comprehensive analysis of histone post-translational modifications in mouse and human male germ cells[END_REF]. Subsequently, the histone code provides a robust system that is not prone to mistakes and delivers a reliable answer to stimuli, and therefore seems to fill all requirements of a robust system.

Evolvability: A consequence of robustness is that small variations are usually harmless, a property that opens doors to small changes through evolution, with various outcomes.

Evolvability of the histone code indeed does not operate at the level of histone sequence itself, as histone sequences are found to be very conserved. Nevertheless, minute changes in histone associations are found between relatively close species such as chimpanzee and human, or primates and rodents [START_REF] Ho | Comparative analysis of metazoan chromatin organization[END_REF][START_REF] Shulha | Human-specific histone methylation signatures at transcription start sites in prefrontal neurons[END_REF].

Other marks such as H3K4me3 seem to have different patterns in complex organisms compared to rather simple distribution in yeast.

As a result of these different features, the histone code can be viewed as a stable system (notion of robustness) with enough flexibility that provides a basic ground to generate new functions by accumulation of slight changes of its elements (notion of evolvability).

Histone code dictates combinatorial associations between histone modifications at the nucleosome level

Many histone modifications have been proven to function in a concerted manner with other marks. Theory by Jenuwein and Allis hypothesizes that these combinations serve as elementary signs which are translated into instructions by chromatin protein complexes to regulate the genome [START_REF] Jenuwein | Translating the histone code[END_REF]. Genomic approaches such as ChIP-Seq have identified the precise localization of proteins on DNA, and even more strikingly, have been able to distinguish between different modifications due to highly specific antibodies against mono-di-or trimethylation, among others (see [START_REF] Barski | High-resolution profiling of histone methylations in the human genome[END_REF], a pioneering example). The findings in these studies show that histone modifications of different classes are found simultaneously at the same genomic positions, id est in the same nucleosome. This is the result of an amplification process, for example, H4K20me1 can be recognized by enzyme Suv4-20h1 which triggers a second methylation event on the same lysine, generating a H4K20me2 mark [START_REF] Pei | Mmset regulates histone h4k20 methylation and 53bp1 accumulation at dna damage sites[END_REF][START_REF] Kuo | Orc1 bah domain links h4k20me2 to dna replication licensing and meier-gorlin syndrome[END_REF].

Another example is the PcG complex involved in gene silencing, which is made of the association of complexes PRC1 and PRC2. PRC2 is specialized in depositing the H3K27me3 mark [START_REF] Cao | Role of histone h3 lysine 27 methylation in polycomb-group silencing[END_REF][START_REF] Czermin | Drosophila enhancer of zeste/esc complexes have a histone h3 methyltransferase activity that marks chromosomal polycomb sites[END_REF], while PRC1 recognizes H3K27me3 [START_REF] Müller | Histone methyltransferase activity of a drosophila polycomb group repressor complex[END_REF] and subsequently ubiquitinates lysine 119 of histone H2A via protein Ring 1B E3 ubiquitin ligase [START_REF] De Napoles | Polycomb group proteins ring1a/b link ubiquitylation of histone h2a to heritable gene silencing and x inactivation[END_REF], amplifying the signal.

More recent studies have demonstrated that variants of the PRC1 complex can come in first to deposit H2A ubiquitination, which is thereafter recognised by PRC2 to finally deposit H3K27me3 [START_REF] Cooper | Targeting polycomb to pericentric heterochromatin in embryonic stem cells reveals a role for h2ak119u1 in prc2 recruitment[END_REF][START_REF] Blackledge | Variant prc1 complex-dependent h2a ubiquitylation drives prc2 recruitment and polycomb domain formation[END_REF]. Histone modifications do not need to be exactly on the same histone protein to act synergistically or on neighbor nucleosomes, providing that the different modifications acting synergistically are close together in 3D space [START_REF] Fraser | Nuclear organization of the genome and the potential for gene regulation[END_REF]. The combinations of histone marks affect transcription and so indirectly cellular events which are important for cell and organism identity. For instance, during interphase, phosphorylation at H3S10 associates with H3K9 and H3K14 acetylation and induces chromatin relaxation [START_REF] Prigent | Phosphorylation of serine 10 in histone h3, what for?[END_REF]. During mitosis, DNA needs to be packed and H3S10 phosphorylation heavily triggers compaction. Two combinations with the same mark participate to two different cellular outcomes.

The amount of information carried by the overall collection of modifications can be probed by checking the correlation of marks between individual histones, using data from ChIP-Seq [START_REF] Prakash | Histone code and higher-order chromatin folding: A hypothesis[END_REF]. The data reveals a global redundancy factor of about 1:7 with five different clusters using 39 different marks (Figure 3A). As a result, the number of possible functions that can be directed by the system, even if the number of marks is high, is relatively low, which is confirmed by genomic studies [START_REF] Rao | A 3d map of the human genome at kilobase resolution reveals principles of chromatin looping[END_REF]. An insightful model has also shown that a simple two-marks histone code can already provide a wide panel of function, hinting that few core elements can lead to a large combination of genomic functions [START_REF] Dodd | Theoretical analysis of epigenetic cell memory by nucleosome modification[END_REF][START_REF] Dodd | Barriers and silencers: a theoretical toolkit for control and containment of nucleosome-based epigenetic states[END_REF].

Direct experimental evidence for associations of histone modifications was brought by studies using co-marking of histone modifications [START_REF] Bernstein | A bivalent chromatin structure marks key developmental genes in embryonic stem cells[END_REF] and genomic experiments associated to microscopy (co-occurring iChIP in [START_REF] Shema | Single-molecule decoding of combinatorially modified nucleosomes[END_REF]). The singlenucleosome experiments confirm very well-known histone code rule to happen on isolated nucleosomes, such as the bivalent state (H3K27me3/H3K4me3), which is typical of embryonic stem cells. An example of a cis and trans crosstalk within a nucleosome is shown in Figure 3B.

Advance microscopy techniques [START_REF] Betzig | Imaging intracellular fluorescent proteins at nanometer resolution[END_REF][START_REF] Rust | Stochastic optical reconstruction microscopy (storm) provides sub-diffraction-limit image resolution[END_REF][START_REF] Schermelleh | Subdiffraction multicolor imaging of the nuclear periphery with 3d structured illumination microscopy[END_REF][START_REF] Prakash | High-density superresolution microscopy with an incoherent light source and a conventional epifluorescence[END_REF] have been powerful at seeing the distribution of histone modifications in the nucleus [START_REF] Żurek-Biesiada | Localization microscopy of dna in situ using vybrant® dyecycle violet fluorescent probe: A new approach to study nuclear nanostructure at single molecule resolution[END_REF][START_REF] Żurek-Biesiada | Quantitative super-resolution localization microscopy of dna in situ using vybrant® dyecycle violet fluorescent probe[END_REF][START_REF] Szczurek | Single molecule localization microscopy of the distribution of chromatin using hoechst and dapi fluorescent probes[END_REF]. In particular, single molecule localisation microscopy can discriminate between regions painted with H3K4me3 and H3K27me3 [START_REF] Prakash | Superresolution imaging reveals structurally distinct periodic patterns of chromatin along pachytene chromosomes[END_REF][START_REF] Kirmes | A transient ischemic environment induces reversible compaction of chromatin[END_REF]. These marks are highly associated with the general compaction level of chromatin (Figure 3C). Overall, one usually distinguishes between euchromatin and heterochromatin, the first being opened, in an "active" state prompt for genes to be transcribed, while the latter is in a repressed state [START_REF] Popken | Remodeling of the nuclear envelope and lamina during bovine preimplantation development and its functional implications[END_REF][START_REF] Zinner | Biochemistry meets nuclear architecture: multicolor immuno-fish for co-localization analysis of chromosome segments and differentially expressed gene loci with various histone methylations[END_REF][100].

Histone code associations with genome function and structure

Historically, the association of histone modifications with transcription is known since long [START_REF] Allfrey | Acetylation and methylation of histones and their possible role in the regulation of rna synthesis[END_REF]103]. Regarding modification associations, studies have revealed that an increase in the complexity of the histone make-up results in progressive changes in the gene expression [104], with acetylation to be globally associated to increase in transcription and deacetylation with decrease in transcription. To our knowledge, H3K36 methylation by the SET2 complex (co-transcriptionally binding to elongating Pol II) targets Rpd3S deacetylases to suppress cryptic transcription start sites within genes [105, 106].

ChIP-seq experiments have helped to draw a more refined picture of the relationships between histone modifications and gene function [101]. Hidden Markov models were employed to guess where a histone modification combination (or "chromatin state") starts and ends. After testing many combinations of parameters, the model was found to compromise 51 states, which is a surprisingly large number. Most of these states are found to be associated with specific genomic regions. 11 out of the 51 states fall in promoter regions, which is a significant enrichment when considering the low amount of promoter regions in the genome compared to non-coding regions (Figure 4A).

Promoter regions are associated with acetylated marks, all of which show a similar bimodal profile around transcription start sites (TSS) (Figure 4B). Transcribed regions are associated with particular kinds of methylation, entirely different from The impact of histone modifications on transcription and other cellular functions is largely based on the influence of chromatin structure. At a nanoscale, methylation or deacetylation of histone tails ensure the stability of nucleosomes [START_REF] Biswas | Atomistic simulations of nucleosomes[END_REF]. At a gene scale, the makeup is thought to be locally uniform, guiding folding properties, for instance in the region of Hox genes [START_REF] Barbieri | Active and poised promoter states drive folding of the extended hoxb locus in mouse embryonic stem cells[END_REF]. Areas with similar epigenetic make-up may fold together while repelling other parts of different make-up, thus contributing to the formation of locally coherent topological chromatin domains in the size range of 0.5-1 Mb, or TAD described by recent chromatin conformation capturing approaches [START_REF] Rao | A 3d map of the human genome at kilobase resolution reveals principles of chromatin looping[END_REF]111]. Indeed, we have recently shown that histone modifications cluster at genome scale using localisation microscopy [112].

Previously, it was shown with the confocal microscope, that H3K4me3-rich, H3K27me3-rich and H3K9me3-rich regions partially segregate (for instance in [113]). On our localisation images, interphase and M-phase chromatin could also be roughly divided into H3K4me3-rich, H3K27me3-rich and H3K9me3-rich regions of highly different patterns. These marks are known to be respectively associated with active genes, inactive genes and deeply repressed chromatin [114].

Along with microscopy, genomics has been the most powerful at characterising the relationship between modifications of histones and structure. The HiC method has helped to identify regions of structurally complex DNA folding, whether loose or compacted, that are thought to be functional units [111].

ChIP-seq of histone modifications has shown that chromatin domains described with the HiC method can be associated with combinations of histones (Figure 5A). In mammals, domains related to active chromatin usually show H3K4me3 and acetylated histones H3 and H4 at the promoters of genes, while inactive domains display genes silenced and mark H3K27me3.

Differently, H3K27me3-rich domains have the potential to be activated on demand, depending on the tissue, and so are more open than H3K9me-rich domains [115]. Finally, domains associated to H3K9me2/H3K9me3 are very compact and constitute a significant portion of chromatin that is never active and is structurally very condensed. Figure 5B shows localisation microscopy images with the clusters of active, inactive and repressed chromatin, painted with H3K14ac, H3K9me3 and H3K27me3 respectively [START_REF] Prakash | Structure, function and dynamics of chromatin in Chromatin Architecture[END_REF]. From these different findings, we propose a model for chromatin domain formation (Figure 5C).

Histone code across the cell cycle

During the cell cycle, the genome structure is heavily modified via epigenetic mechanisms. Nevertheless, activity and regulation of genes have to continue to a certain extent. Keeping the epigenetic makeup of chromatin during DNA condensation at the end of metaphase to switch to mitosis is relatively costly in terms of energy, so some optimal regulatory mechanisms have to happen to "memorise" the epigenetic status of the dividing cell. So far, we have considered cells in G1 or G0 phase, but the picture is rather different during mitosis, es-Fig. 6. Histone code segregates functional compartments in the mieotic chromosomes. (A) Super-resolution microscopy reveals higher-order clusters of chromatin patterns along the pachytene chromosome. The DNA map of meiotic chromosomes at pachytene stage shows different condensation levels. These levels were found to be constrained by anti-correlating clusters of histone modifications (pink) along the synaptonemal complex proteins 3 (SYCP3) (green). Histone modifications associated with transcription (H3K4me3) emanate radially in loop-like structures while the histone modifications associated with repressive chromatin (H3K27me3) are confined to axial regions of the SYCP3. The histone modifications associated with centromeric chromatin (H3K9me3) are found at one end of the SYCP3 (figure and caption modified from [START_REF] Prakash | Histone code and higher-order chromatin folding: A hypothesis[END_REF]114]). Secondly, besides the epigenetic mechanisms that accompany replication and mitosis, another kind of epigenetic regulation is devoted to preserving the transcriptional status of genes so that activity can be restored in daughter cells at the end of mitosis. The view gets even more complex when considering symetric and asymetric division [123,124] Despite costly at first sight, epigenetic inheritance may be relatively cheap to maintain with the recruitment of proper readers and writers in the vicinity of replication sites to stochastically recreate the "parental" epigenetic make-up in daughter strands. In the future, integration of modelling, genomic and microscopy features will be key to understanding the mechanisms of epigenetic inheritance.

The association of histone code with cell identity and dynamics

Overall, genomic studies have shown that there is a substantial reshuffling of histone modifications during the cell cycle [START_REF] Rao | A 3d map of the human genome at kilobase resolution reveals principles of chromatin looping[END_REF].

To show these associations, careful choice of FISH probes associated with H3K4me3/H3K27me3/H3K9me3 were found to demonstrate that chromatin domain stained with any of these marks anti-localise (Figure 6 Another key event of cell differentiation is the increase of the proportion of densely repressed chromatin, usually associated with telomeres and nucleoli. These regions are associated with H3K9me3 and eventually H3K27me3 (Figure 6B). Cells become slowly more specialised during development, which means that their specialised metabolism focuses on expressing of only a small subset of genes with a majority of genes being put in a repressed state. A lot of genomic locations indeed become repressed, histone combination usually turns to a deacetylated state with repressed marks, such as H3K27me3. Some tissues experience bigger changes than others, for instance, T With time, Hox genes are de-repressed and activated by methylation of lysine 4 of histone three while H3K27me3 is dissociated. Progression is also spatial, with Hox genes being activated from one end of the cluster to the other [140]. Furthermore, both "active" and "inactive" parts of the Hox genes cluster form separate chromatin domains (Figure 5C). Differently, in tissues with no Hox gene expression, such as brain, most of clusters are silenced and fold according to cluster positions, instead of active/inactive status, showing a deep connection between histone code, dynamics of transcriptional regulation, high-order DNA folding and body-wide phenotype.

Conclusion

We have presented arguments for the existence of a spectrum of epigenetic features related to chromatin structure, epitomized by the term histone code. A consequence of the intrinsic redundancy of the code is robustness to small changes and therefore the message it delivers will not be very much altered if, for instance, an enzyme depositing one mark is not fully functional. Nevertheless, the position themselves where the histone modifications occur, are not prone to variations. As an example, the alteration of lysine H3K27 into a methionine is found in a certain type of glioma [141]. We can only hypothesize that similar mutations in other important modifications may have other dramatic effects for cellular phenotype and life span of organisms with either cancer or developmental defects. However, most importantly, redundancy of the code is a way to increase the probability for factors to be brought to the nucleosomes, for instance in a case of a protein complex where one protein binds to one histone modification, and another protein binds to a different histone modification. This is shown by the fact that factors from the same complex usually bind similar marks or associated marks either on the same histone or close positions. The free energy of the compound/nucleosome system will increase, increasing the probability of binding [142].

Redundancy of information provided by the histone code is an essential feature of robustness of cellular processes. As the number of histone modifications defining a given chromatin state increases, so does the probability to recruit specific factors that will change or modulate the state of chromatin.

Overall, the complexity of histone modulation helps to ensure that events will happen at the right time in the right place.

Though not fully understood yet, these processes are certainly a key point to understand the mechanisms that connect gene regulation and chromatin folding.

ACKNOWLEDGMENTS. D.F. thanks the Center for Computa-

tional Sciences in Mainz for the financial support. K.P. thanks Wioleta Dudka for proofreading the manuscript.

Fig. 1 .

 1 Fig. 1. Chromatin spatial organization and relationship with the histone code. (A)The spatial organization of chromatin can be studied at three levels: at the lowest orders, which include the beads-on-a-string model and the chromatin fibre (left column), at the level of functional chromatin domains (middle column) and high order chromatin patterns (right column). The organization of chromatin domains can be modelled using various post-translational histone modifications. Depending upon the kind of histone modification a nucleosome is enriched with, chromatin can be either highly condensed, in an open conformation or switch between these two extreme forms. The higher order chromatin patterns can either be viewed from: the chromosome territory model of DNA organization, the over-simplified bimodal classification of condensed and open chromatin as hetero-and euchromatin respectively, and the highly condensed configuration of metaphase chromosomes. (B) The components of the histone code. On the left, the input system A, made of combinations of histone modifications (coloured pins). One unique combination can be found at three different positions 1,2,3, in the genome. The combinations of the input system A is translated into components of the output system B, with a black and white colour-scale. The translation happens via adaptors such as transcription factors or epigenetic regulators that recognise specific histone modifications to change chromatin state.

Fig. 3 .

 3 Fig. 3. A binary combinatorial histone code at the level of nucleosomes. (A). Different clusters of histone modifications is revealed by hierarchical clustering. Correlation between modifications are displayed with a color scheme, red to blue from negative to positive correlation coefficient. Clusters are identified by black boxes (figure from [42], courtesy Kernel Press, Mainz). (B) Different cis and trans patterns of histone crosstalk (figure from [43], reprinted with permission). (C) The impact of three primary histone modifications on chromatin compaction. H3K4me3-rich nucleosomes are associated with active chromatin in an open conformation, H3K27me3-rich nucleosomes are associated with repressed chromatin, while H3K9me3-rich nucleosomes are associated with highly-compacted, inactive chromatin.

Fig. 4 .

 4 Fig. 4. The histone code at the level of genomic features. (A) The combinations of histone modifications and their associations to various genomic states (figure modified from [101], reprinted with permission). Here, the genome is partitioned into five broad regions: promoter states, transcribed states, active intergenic, repressive and repetitive state. A combination of histone marks is associated with each genomic state. The relative degree of association is indicated by a colour scale spanning frequency values between 0 and 1. (B) Profile of histone modifications around the transcription start site (TSS). On top panel, profile for acetylation marks; below panel: methylations. X-axes: Distance to TSS. The negative values are upstream from the gene and the positive values downstream, irrespective of the strandness of the gene. Y-axes: Normalized read counts. Profiles were generated by us with a R script [102].

Fig. 5 .

 5 Fig. 5. The histone code predicts the degree of folding of chromatin domains. (A) A section of chromosome 19 shows different subcompartments based on histone modifications. Here, chromatin domains with high contact frequencies correlate with histone marks associated with inactive chromatin such as H3K9me3 and H4K20me3. Such relation between H3K9me3 and H4K20me3 was also found at the nucleosomes level based on the entirely different method and cell line. This shows that relationship between histone modifications holds at various length scales. Figure reprinted from [84], with permission. (B) Imaging of histone modifications further reveals the distribution of domains associated to different histone modifications. The different histone modifications have distinct molecular signatures, probably due to different folding rules and roles in gene regulatory mechanisms. While H3K9me3 seems to be mostly enriched at chromocenters as expected, H3K27me3 is distributed more in speckle-like patterns. The acetylation patterns (H3K14ac) vary in density and are more diverse in distribution. Figure adapted from [107]. Scale bar: 1 µm. (C) A model portraying three stages of a Hox gene activation, with progressive suppression of a silent chromatin domain made up of H3K27me3-and H3K9me3-rich histones toward the installation of an active domain rich with H3K4me3. The change of domain happens literally at the same place, with inhibitory marks being progressively evicted from the region to be replaced by the active domain. Note the region of inhibitory marks remain at the bottom, to keep the two extremities of the loop in a condensed fashion and leave the loose active region in a relatively consistent open shape.

  Fig. 6. Histone code segregates functional compartments in the mieotic chromosomes. (A) Super-resolution microscopy reveals higher-order clusters of chromatin patterns along the pachytene chromosome. The DNA map of meiotic chromosomes at pachytene stage shows different condensation levels. These levels were found to be constrained by anti-correlating clusters of histone modifications (pink) along the synaptonemal complex proteins 3 (SYCP3) (green). Histone modifications associated with transcription (H3K4me3) emanate radially in loop-like structures while the histone modifications associated with repressive chromatin (H3K27me3) are confined to axial regions of the SYCP3. The histone modifications associated with centromeric chromatin (H3K9me3) are found at one end of the SYCP3 (figure and caption modified from [42, 114]). (B) A model for co-existence of different chromatin domains on a single chromosome. The two strands of the SYCP3 are displayed in black. In purple, active chromatin marked by H3K4me3 covers the entire surface of chromosomes (shown in blue). Symmetrical and periodic clusters of repressed chromatin H3K27me3 are shown with yellow spots. Inactive polar chromatin marked by H3K9me3 appears as an orange spiral pattern. The plot in the middle shows the distance distribution of DNA, active (H3K4me3) and inactive (H3K27me3, H3K9me3) regions of the genome from the central axis of SYCP3.

  . The two marks which participate the most in this kind of remodelling are H3K27me3 and H3K9me3 [116], driving gene silencing and high chromatin compaction, respectively. A fundamental biological question is whether the epigenetic marks and so the histone code rules are preserved through replication, a mechanism named epigenetic inheritance [125-127]. In order replication to happen, the DNA becomes naked, and the nucleosomes need to be reassembled so chromatin keeps its functions in later stages. Many mechanisms are known, some involving a de novo "random" refolding which is independent of the DNA status before S-phase, and one mechanism dependent on the previous status, involving an epigenetic memory. In one model, all histone modifications are preserved and memorised on each DNA strand of the two double helices after replication. Differently, histone modifications can be partially transmitted to the daughter DNA molecules, but via copy mechanisms involving histone modifications, readers and writers recruited locally. In this case, all gaps can be filled to restore an epigenetic state very close to the one of the parent DNA molecule. Another important question is the recycling of nucleosomes and histones. Are nucleosomes de-assembled and reconstructed in the newly synthesised DNA molecules or are they mostly transmitted in an intact form, with missing nucleosomes being de novo assembled? The answer is that in most cases, each nucleosome is transmitted intact to one of the two daughter strands, so the histone code is preserved [123, 128]. The equivalent position on the other strand is possibly filled with information coming from adjacent redundant nucleosomes via enzymes CAF-1 and ASF1 [129].

  ), a fact confirmed in Drosophila more recently, with overlaps [115]. The model of domains that we described previously is static, and in reality, chromatin domains experience a large change in embryonic development and differentiation processes of adult tissues (Figure 7). According to microscopy and genomic data, during early stages of development, a heavy remodelling of chromatin structure happens. Most of the chromatin is relatively loose at first, and becomes more compacted in later stages, with associated silencing of many genes. In embryonic stem cells, a relatively high number of genes are expressed, including genes for maintenance of stem cell status, such as cMyc or Sox2 [130]. These genes are usually painted with H3K4me3 and H3K27ac marks at their promoters, prone to recruit transcription factors such as Pou5f1, Sox2, cMyc and Klf4 [131]. Many specialised genes are on the other hand showing poised promoters. These promoters have an ambivalent epigenetic status, comprising both active and inactive marks H3K4me3 and H3K27me3, respectively, and have the potential to either be activated or repressed during differentiation.

Fig. 7 .

 7 Fig. 7. Histone code during developmental processes. The model depicts the rules of the histone code associated to major developmental and regulatory processes happening in the human body. The different colours of circle feature different kinds of histone modifications combinations with the most significant modifications displayed. Blue: Euchromatin (H3K4me2/3 and H3K27ac, see Hawkins et al. 2010 [132]). Brown: repressed chromatin (H3K9me2/3, Ibid). Red: Inactive chromatin (H3K27me3, Ibid). Purple: Poised transcription (H3K4me3 and H3K27me3, see Gapp et al. 2014 [133]). Black: Quiescent chromatin (H4K20me2/3, see Evertts et al. 2013 and Onodera and Nakayama 2015 [134, 135]). Pathways from one genome status to another are figured by pale grey lines associated with names of chromatin modifiers or readers involved.