BACKGROUND
in eukaryotes, chromatin is the interface through which genetically encoded information is read to orchestrate a diversity of cellular and organismal functions.
chromosomal packaging of nuclear dna is achieved through small basic histone proteins.
the highly conserved core histones, h <dig>  h <dig>  h2a and h2b form an octamer of two h2a/h2b dimers flanking a central h3/h <dig> tetramer that wrap ~ <dig> bp of dna in  <dig>  turns to form the nucleosome core particle   <cit> .
tetramer formation is common to archaeal and eukaryotic histones.
the eukaryotic origin of h2a-h2b dimers, doubling the dna wrap capacity, has been discussed in light of demands of increased genome size  <cit>  and kinetic constraints of dna compaction imposed by eukaryotic mitosis  <cit> .
interactions between the histone octamer and dna are further modulated by linker histones h1/h <dig>  which associate with dna at its entry/exit site on the ncp surface  <cit> .
roles of histones in structural compaction must be balanced with regulatory mechanisms that permit selective access to dna to enable functions such as transcription, replication and dna repair.
three major strategies contribute to regulatory remodeling of chromatin: atp-dependent complexes that act on nucleosomes to modify accessibility of wrapped dna sequences to trans-acting factors  <cit> , the deployment of histone variants that alter nucleosome dynamics, and covalent posttranslational histone modifications  including acetylation, methylation, phosphorylation, ubiquitylation, citrullination, adp-ribosylation, glycoslylation and sumoylation  <cit> .
one current view is that eukaryotic genomes are indexed locally, and over broader regions, through a combination of histone variants and their diverse ptms  <cit> .
main lineages of canonical core histones, constituting the bulk of histone proteins, are assembled into chromatin during dna replication.
these replication dependent  genes lack introns, are typically organized in gene clusters and their mrnas possess a conserved stem-loop  in the 3'utr coupling gene expression to dna replication.
in contrast, histone variants are often transcribed from orphan genes that contain introns, lack the sl and their expression is not restricted to s-phase.
consequently, they are referred to as replacement or replication-independent  variants.
from an evolutionary perspective, histone variants are classified as universal or lineage-specific variants, where the term variant refers to non-allelic sequence variation.
universal variants have ancient functions common to eukaryotic cells, whereas lineage-specific variants appear specialized to certain organismal requirements.
core histones clearly differ in their evolutionary propensity to diversify as distinct variants.
h <dig> is highly constrained as it makes contacts with the other  <dig> core histones and its n-terminal tail residues are subject to extensive ptms.
this is reflected in its nearly invariant stature.
no h <dig> amino acid sequence variants have been reported in any multicellular organism to date and diversified h4s are known only in trypanosomes and ciliates  <cit> .
despite h2bs showing substantially less evolutionary sequence constraint than h <dig> and h <dig> they, like h <dig>  exhibit little specialization  <cit> , with the exception of gametogenesis variants.
on the other hand, h <dig> and h2a are much richer in their genealogy with diversification of a number of functionally essential variants.
the two h <dig> molecules interact with each other in the nucleosome, as do the two h2a molecules, whereas h <dig> and h2b do not.
two-fold nucleosomal symmetry is organized along the c-terminal four-helix bundle dimerization interface of the two h3s  <cit> .
interaction between the two h2as is mediated by their loop  <dig>  domains.
specializations have evolved in the h <dig> lineage with at least two h <dig> variants present in most eukaryotes.
the replacement variant h <dig>  can undergo rd or ri assembly and deposition is primarily in transcribed regions of euchromatin  <cit> .
h <dig>  differs from canonical h <dig> at few positions, predominantly, one in the n-terminal tail and three in the α <dig> helix of the histone fold domain , the latter three required for ri assembly  <cit> .
all eukaryotes possess h <dig>  though asomycetes lack canonical h <dig>  phylogenetically, the two proteins appear not to be separate, early-branching lineages, as distinct h <dig> and h <dig>  histones have arisen multiple times  <cit> .
a second common h <dig> variant is centromeric h <dig>  essential for recruitment of kinetochore components vital to mitosis.
cenh3s retain only ~50% identity to h3s in the hfd and have divergent n-terminal tails from  <dig> to  <dig> residues.
cenh <dig> phylogeny is poorly resolved.
they appear to have arisen many times with rapid divergence under positive selection.
there are also lineage-specific meiotic and spermatogenesis h <dig> variants  <cit> .
among core histones, h2as have evolved the greatest variant diversity.
there are two nearly universal variants, h2ax and h2a.z.
h2ax plays an important role in the maintenance of eukaryotic genome integrity by participating in double-strand dna-break  repair by nonhomologous end joining .
h2ax has arisen multiple times during evolution but similar constraints have led to convergent acquisition of the h2ax-specific phosphorylation motif sqe/dΦ   <cit> .
phosphorylation of the s residue in this c-terminal motif forms γh2ax, which assists in the recruitment of histone modifiers, chromatin remodelers and dna repair complexes.
h2ax has entirely replaced canonical h2a in fungi and giardia  <cit>  but is absent in c. elegans and protozoan parasites, plasmodium and trypanosomes.
h2a.z had a single evolutionary origin, and has remained distinct from canonical h2a  <cit> .
it is essential for viability in a range of species,  <cit>  and is implicated in gene activation, chromosome segregation, heterochromatin silencing, and cell cycle progression  <cit> .
a common thread in a number of these disparate roles may be the proclivity of h2a.z to form more stably positioned nucleosomes  <cit>  combined with differential use of ptms.
other h2a variants include the c-terminally extended macroh2a, the c-terminally truncated mammalian h2a.bbd and an array of more poorly studied lineage-specific variants.
linker h <dig> variants form a complex family and subtypes are classified as canonical rd  or ri linker histones .
they display tissue and developmental specificity, with vertebrates commonly expressing testes-specific  and oocyte-specific  variants  <cit> .
structurally, metazoan h1s are divided into three domains: a short, flexible n-terminal, a globular domain containing a winged-helix fold and a long, lysine rich c-terminal tail.
h <dig> variants differ in biophysical properties, association with repressed or active chromatin and their ability to increase or decrease transcription when over-expressed.
the above picture is derived from histone sequence comparisons in a wide array of eukaryotes, but to date, very few full histone complements have been analyzed more comprehensively and information is principally restricted to universal variants as well as some lineage-specific variants in mammals and plants.
modification of metazoan histone variant lineages occurs on a background of genome architecture that shows global similarities from sponges to vertebrates  <cit>  but the urochordate, oikopleura dioica , a member of the closest sister group to vertebrates, demonstrates that this ancestral genome architecture can be profoundly modified  <cit> .
in this fast-evolving, pan-global zooplankton, a repertoire of > <dig> genes has been extraordinarily compacted into a genome of only  <dig> mb, with sizes of introns and intergenic regulatory regions greatly reduced.
whereas other chordates generally grow through cell proliferation, rapid growth of o. dioica over a short, 6-day life cycle is achieved principally through endoreduplication  <cit> .
here, we characterize the highly diverse histone complement that has emerged in concert with evolution of this intriguing lineage at the invertebrate-vertebrate transition.
RESULTS
histone gene organization in oikopleura dioica
histone genes were dispersed throughout the o. dioica  genome with most in clusters up to quintets.
in total,  <dig> histone genes  encoding  <dig> different histone proteins  were identified .
clustered genes showed typical rd features.
they were intron-less and most shared divergent promoters.
primary transcripts of these genes contain both a conserved sl and a downstream polyadenylation  signal in their 3' utrs but lack the histone downstream element  <cit> .
a second set of histone genes were present as orphan genes:  <dig> h <dig> ,  <dig> h <dig>  and  <dig> h2a isoforms .
these genes have introns  and the sl is degenerate or absent , suggesting they are ri variants.
to assess phylogeny among histone families, unrooted maximum likelihood trees were generated.
as expected, genes encoding the same histone protein were more closely related and the most divergent histones exhibited the longest branch lengths .
clusters i, ix and xi, had the same h2a and h2b phylogenies and likely result from more recent gene duplications.
to compare the o. dioica histone complement to that of other urochordates we extended genome searches to the histone genes of two ascidians, ciona intestinalis and ciona savignyi.
surprisingly, we found that the histone complements of the two ascidians differed significantly in terms of histone gene number and organization.
the c. savignyi genome  contains a high number  of histone genes partly organized in large clusters, whereas the c. intestinalis genome  encoded only  <dig> histone genes, mostly interspersed among non-histone genes.
a locus within the c. savignyi genome was found where histone genes are arranged similarly to the tandem-arrays of sea urchin and drosophila.
table  <dig> shows that histone gene complements and their organization can differ considerably even over modest phylogenetic distances.
dispersed
h3t.2
h2bfwt
h2bt.1
h2bt.2
h2at.2
h2at.3
h2asq.1
h2asq.2
h <dig>
h <dig>
h <dig>
h <dig> *4
h <dig> *5
h1a
h1t2
§+ isoforms = additional isoforms in the genome.
† = differentiation h <dig> variant.
expression: peaks at *1d3/d4; * <dig> peaks in early tadpoles and d <dig>  where it is enriched in the testes; *3commences  <dig> h pf and decreases after d4; * <dig> peaks during organogenesis and *5peaks at metamorphosis.
the nomenclature of odh1s does not imply direct orthology with human h <dig> -h <dig>
expression of histone gene clusters is developmentally co-regulated
we assessed expression profiles of all histone genes throughout development by qrt-pcr, including maturing males and females.
o. dioica histone genes were co-regulated in clusters and most genes belonging to the same locus showed the same developmental expression profiles .
histone clusters could be assigned to seven expression patterns: 1) throughout the life cycle  2) exclusively during organogenesis , 3) male-specific expression in mature d <dig> animals , 4) expression peaking in early tadpoles and male d <dig> animals , 5) expression peaking at metamorphosis , 6) expression primarily in d <dig> and d <dig> animals  and 7) transcripts predominantly present from  <dig> h post-fertilization  to d <dig> .
histone isoforms exhibiting multiple changes in amino acid sequence were commonly male-specific.
however, some divergent core histone variants displayed expression profiles that preceded gonad maturation in d3/d <dig> adults  , from  <dig> h pf to d <dig>   or after organogenesis and in late adults at d <dig>  .
to improve resolution of germline-specific expression of different histone proteins, maturing d5/d <dig> animals were dissected and cdnas prepared separately from testes, ovaries and trunks.
expression ratios were up to 4300-fold higher in testes and 330-fold higher in ovaries for certain isoforms .
extrapolation of ovary-specific transcripts to oogenic function is complicated by maternal mrna storage in oocytes during vitellogenesis.
taking this into account, only h2a.
<dig> was clearly significantly enriched in the ovary .
the potential combinatorial possibilities of histone core and linker isoforms based on their transcript levels throughout development are schematically summarized in figure  <dig>  during organogenesis, transcript levels of all canonical histones were increased by the activation of additional, organogenesis-specific histone gene loci.
at least one male specific isoform exists in each of the five histone families, including a male-specific h4t isoform.
mass spectrometry of o. dioica core histones from mature sperm and whole animals
o. dioica core histones were prepared for mass spectrometry from d <dig> and d <dig> animals, which include endocycling nuclei of the epithelium, the mitotic nuclei of d <dig> male gonads and female and male meiotic nuclei of d <dig> animals .
histone sequence coverage by lc-ms/ms ranged from 51%  to 90%  and most peptides were identified several times .
many conserved ptms associated with transcriptional activity and silent chromatin in other species were identified.
the n- and to a lesser extent, c-termini, of o. dioica h <dig>  h <dig> and h2b contained numerous acetylated lysine residues .
conserved sites for mono-, di-, and trimethylations such as h4k <dig>  h3k <dig>  h3k <dig>  h3k <dig> and ubiquitylation of h2bk <dig>  were observed, though trimethylation of h4k <dig> and h3k <dig> were not detected.
peptides were found for canonical h2b.
<dig> and/or h2b.
<dig> monomethylated at r <dig> and dimethylated at their last c-terminal k residues as well as the dimethylation of h <dig> at r <dig>  we found few or no peptides unique to the divergent variants h2b.
<dig>  h2b.
<dig>  h2a.
<dig>  h2a.
<dig> and cenh <dig>  possibly due to low abundance.
the large number of histone variants expressed in testes led us to investigate their retention in mature sperm.
mass spectrometry of histones isolated from mature sperm revealed many peptides specific for canonical and male-specific o. dioica histone isoforms .
o. dioica core h4s
whereas other multicellular organisms express a single h <dig> isoform, o. dioica expresses two histone h4s, canonical h <dig>  and a male-specific h4t.
the major h <dig>  subtype was expressed from five genes, with three expressed throughout development and genes at loci ii and xii peaking at organogenesis .
the h4t gene, located in a male-specific histone gene cluster  was specifically expressed in d <dig> males, with a > <dig> enrichment in testes compared to ovaries .
h4t-specific peptides were found by mass spectrometry in o. dioica sperm histones .
within the h4t amino acid sequence v <dig> and l <dig> are replaced by i <dig> and m <dig>  in a region of the h <dig> n-terminal tail that interacts with the acidic patch of the adjacent h2a-h2b dimer .
there is also a third residue-change within the α <dig> helix, where a <dig> is replaced by s. based on h <dig> structure, s <dig> is not solvent accessible, facing towards the alpha  <dig> helix of h <dig>
o. dioica h2b isoforms are expressed before onset and during spermatogenesis
no replacement variants have been identified among h2bs and all odh2b genes are intron-less and contain the sl in their 3'utr .
odh2b.
<dig> and h2b.
<dig>  were expressed throughout development , while the other  <dig> h2b isoforms showed different levels of enrichment in testes.
whereas h2bt.
<dig>  h2bt.
<dig> and h2bt.
<dig> were exclusively expressed in testes, with an increase up to >3500-fold compared to ovaries , the divergent h2b.
<dig> gene was expressed at d3/d <dig>  preceding spermatogenesis  and divergent h2b.
<dig> expression peaked in early tadpoles and d <dig> .
intriguingly, these two divergent h2b isoforms display complementary expression patterns  centered around the d3/d <dig> transition when germline differentiation commences.
h2b.
<dig> and h2b.
<dig> have reduced sequence identity to somatic o. dioica h2b.
<dig>  but do not show significant similarities to testes-specific h2bfwt of mammals, which also shows reduced sequence identity  to mammalian somatic h2b  <cit> .
mammalian testes-specific h2bs of the type tsh2b/th2b have few residue changes compared to their canonical counterparts, but all share three substitutions within their hfd, i <dig>  g <dig> and n <dig>  interestingly, canonical odh2bs share these same residue substitutions and therefore have slightly higher sequence similarity to testes-specific rather than somatic h2bs of mammals .
structurally, all odh2b proteins  have short n-terminal tails compared to other chordate h2bs and none of the isoforms contain k/r-rich motifs characteristic of h2bs retained in echinoderm sperm  <cit> .
o. dioica core h3s
the o. dioica h <dig> complement comprises all universal h <dig> variants but lacks mammalian-specific h <dig> .
odh <dig>  and h <dig>  are 100% identical in amino acid sequence to vertebrate orthologs .
additionally, o. dioica expressed three male-specific h <dig> isoforms  highly enriched in testes .
h3t.
<dig> and h3t.
<dig> contained a fully conserved sl and were co-regulated within a male-specific histone gene cluster , whereas h3t.
<dig> was expressed from an orphan gene with a degenerate sl , indicative of a replacement variant.
within chordates, testes-specific h <dig> variants have only been reported from mammals.
odh3ts, however, are more divergent than mammalian h3ts when compared to canonical h <dig> /h <dig> .
odh3t.
<dig> has three residue substitutions in the n-terminal tail and ten in the histone fold, whereas mammalian h3ts exhibit only one and two substitutions, respectively.
odh3ts and mammalian h3ts share no common residue changes and phylogenetic analyses indicate that the h3ts of o. dioica are more closely related to other odh3s than to mammalian h3ts .
we did not find similar h3t genes within the genomes of the two ciona species, suggesting these isoforms might be specific to the appendicularian lineage.
a notable substitution within the n-terminus of h3t.
<dig> is the change of a <dig> to a phosphorylatable t residue.
the same position in h <dig>  is s, which is phosphorylated in mitosis and meiosis and is the only known h <dig> -specific modification  <cit> .
within the histone fold, the most apparent sequence changes of the odh3ts are exposed on the disc surface of the nucleosome, surrounding k <dig>  a conserved site for methylation which was present in o. dioica canonical h3s .
further residue changes occur at positions relevant for intranucelosomal interactions.
within loop  <dig>  and α-helix  <dig>  i <dig> and i <dig>  which make contact with loop  <dig>  and α-helix  <dig> of h <dig>  respectively, are replaced by v <dig> and e <dig>
another intron-containing h <dig> gene lacking the sl encodes a divergent h <dig> replacement variant that we identified as the o. dioica centromeric h <dig>  odcenh <dig> shows characteristic cenh <dig> features: a very divergent n-terminal tail, its l <dig> loop is extended and f <dig> in l <dig>  which interacts with the l <dig> of h <dig>  is replaced by w. when expressed as an egfp fusion protein, odcenh <dig> specifically localized to the centromeres of o. dioica chromosomes  <cit> .
o. dioica h2as: h2a.z-splice variants but no h2ax
similar to vertebrates, the h2a family is the largest and most diverse group of core histones in o. dioica.
h2a.
<dig>  encoded by  <dig> genes, exhibits the highest identity to canonical vertebrate h2as.
h2a.
<dig> and h2a.
<dig>  both expressed throughout development, differ by a single amino acid change from a to s at position  <dig> .
o. dioica also expresses six male-specific isoforms  and two divergent replacement variants, h2a.
<dig> and h2a.
<dig>  lacking the sl .
comparing the h2asq and h2at proteins with each other and with h2a.
<dig>  they mainly differ in their n-terminal tails and their docking domain, which interacts with the h3-h <dig> tetramer .
nevertheless, all male-specific h2as share residue changes in structurally important regions such as: the h2a self-dimerization domain, within l <dig> that interacts with the h2b l <dig>  dna contacts, and residues in the α <dig> helices that interact within the h2a-h2b dimer.
odh2as have short c-terminal tails compared to vertebrate h2as.
no macroh2a gene was identified in o. dioica.
more surprising was the finding that no odh2as contained the pikk substrate sqe/dΦ phosphorylation motif that characterizes all h2axs.
three male-specific o. dioica h2a isoforms  contain a putative phosphorylation sq-motif, adjacent to the c-terminal alpha helix, as a result of a residue change from a <dig> to s .
although this sq-motif could present an alternative phosphorylation site for pikks, its location in the docking domain makes it less solvent accessible than the typical position of the sqe/dΦ-motif at the end of the c-terminal tail.
we conclude there is no h2ax homolog in o. dioica.
the odh2a.z variant exhibits the specific divergent characteristics with respect to h2a.
<dig> described for universal h2a.zs, including a highly positive n-terminal tail, a divergent l <dig>  and divergent self-dimerization and docking domains.
further, h2a.z-specific substitutions are present, including the replacement of n <dig> with d, which extends the acidic patch, a substitution of e <dig> by g and the presence of two solvent accessible histidines .
odh2a.z differs from consensus h2a.z sequences at several residues that are invariant in higher eukaryotes and similar in position to residue changes in tetrahymena h2a.z .
these include three non-conservative amino acid substitutions in the self-dimerization domain , two s-residues  replacing two a-residues within the α <dig> helix and substitution of s by m at position  <dig>  adjacent to the acidic patch.
similar to tetrahymena h2a.z, the odh2a.z c-terminal tail lacks one of two conserved k-residues that are mono-ubiquitylated in mammalian heterochromatin.
another atypical feature of odh2a.z is extension of a comparatively short h2a.z c-terminal tail by  <dig> amino acids containing a "ppqppq" motif.
a further similarity to tetrahymena h2a.z is the long n-terminal tail of odh2a.z, extended by  <dig>  mainly g, k, and a residues.
h2a.z n-terminal tails of higher eukaryotes harbor several gk-repeats that can be acetylated .
intriguingly, we found that o. dioica is able to modify the number of gk-motifs within its h2a.z n-terminal tail by alternative splicing.
alternative splice acceptor sites within the second exon of the odh2a.z gene, result in shortening of the n-terminus by precisely one  or two  of the first three gk-pairs .
full length odh2a.za was predominantly expressed, but all three isoforms were present throughout development.
we also identified two additional h2a variants that did not resemble any universal or lineage-specific h2a variants.
h2a.
<dig> and h2a.
<dig> showed 78% amino acid sequence identity to each other but ≤55% identity with other odh2as.
from the n-terminal tail to the α <dig> helix, h2a.
<dig> and h2a.
<dig> show almost no amino acid sequence conservation to canonical h2a.
<dig>  similar to h2a.z, both variants differed significantly in the self-dimerization domain, within l <dig> and in their docking domains, but do not show typical features in primary structure described for the mammalian h2a.bbd.
as for the odh2b subtype h2b.
<dig>  h2a.
<dig> was predominantly restricted to d3/d <dig> animals  and transcripts were enriched in the ovary .
expression of h2a.
<dig> commenced after zygotic gene activation and decreased significantly when animals began to mature after d <dig> .
o. dioica linker h1s
we identified  <dig> odh <dig> genes encoding  <dig> different h <dig> proteins.
with the exception of d <dig> males, h <dig>  was expressed at every stage of development , while h <dig>  and h <dig>  were expressed from  <dig> h pf to d <dig>  peaking during metamorphosis .
h <dig> , h <dig>  and h <dig>  were the dominant h1s throughout development , while h <dig>  and h1t were restricted to organogenesis and testes, respectively .
the h <dig> , h <dig>  and h1t genes are intron-less, located within histone clusters, and contain the sl .
variants h <dig>  and h <dig>  were expressed from orphan genes that contain one intron and a degenerate sl, suggesting replacement variant function.
increased content of positively charged residues and tail length have been reported to strengthen binding affinity, whereas phosphorylation at s/tpxk motifs weakens h <dig> binding and destabilizes chromatin structures  <cit> .
the n-terminal tails of o. dioica h1s are short in comparison to h <dig> subtypes of mammals with h <dig> , h <dig>  and h <dig>  also having short c-terminal tails .
odh <dig>  contains the most extended c-terminus and has the highest content of positively charged residues .
it is also the only o. dioica linker histone with two phosphorylation motifs within the c-terminus, while h <dig> , h <dig> , h <dig>  have one.
this may suggest that among o. dioica linker histones, h <dig> , restricted to early development, has the widest range of binding affinities, a feature that may facilitate chromatin remodeling events during embryogenesis.
in contrast, odh1t, the shortest linker histone, lacks the conserved phosphorylation motif, suggesting a more restricted binding affinity range.
discussion
analyses of histone complements of higher eukaryotes have been published for metazoans such as drosophila  <cit> , human and mouse  <cit> , chicken  <cit> , c. elegans  <cit> xenopus  <cit>  and sea urchin  <cit> .
comprehensive developmental expression data is only available for the sea urchin histone complement.
here we show that the genome of the urochordate o. dioica encodes a high diversity of histone isoforms.
as in other organisms, the canonical histone genes are organized as clusters and share divergent promoters, though in contrast to drosophila, sea urchin and mammals, the histone genes have not remained physically linked in one or two large clusters or tandem repeats.
instead they are dispersed in small groups throughout the genome, similar to the c. elegans histone complement.
organization of the o. dioica histone complement exhibited limited affinity with the sister urochordate ascidian species, c. intestinalis or c. savignyi, which in turn, differed significantly from each other.
therefore, developmental mode , genome size, and phylogenetic relationship do not appear to be overriding determinants of histone gene organization.
histone modification patterns and genome size
a current view is that histone modifications become more complex from unicellular eukaryotes to mammals  <cit> .
lysine acetylation of h <dig> and h <dig> n-terminal tails, as well as methylated h3k <dig>  h3k <dig> and h3k <dig> are considered active marks, while methylated h3k <dig>  h3k <dig> and h4k <dig> mark silent chromatin.
combined with previous studies  <cit>  the mass spectrometry data now indicate that all conserved "on" and "off" marks are present on o. dioica histones.
whereas unicellular eukaryotes, such as yeast, tetrahymena and plasmodium principally exhibit modifications associated with transcriptional activation, mammals also employ a wider range of marks involved in gene silencing  <cit> .
the observation that hallmarks of silenced chromatin such as h3k <dig> and h3k <dig> methylation are low in yeast and tetrahymena  <cit>  has been explained by the fact that the majority of the genome in unicellular eukaryotes is transcriptionally competent, whereas more than 60% of the mammalian genome is permanently silenced and only ~3% of its dna encodes structural genes  <cit> .
considering the high gene density of the o. dioica genome  and the short intergenic regions, we also expect the proportion of the o. dioica genome that is permanently silenced to be relatively low compared to that of human.
however, our analysis revealed no obvious lack of silent heterochromatic marks in o. dioica, since methylation of h3k <dig>  and h4k <dig> were detected by mass spectrometry and methylation of h3k <dig> has been confirmed by immunofluorescence  <cit> .
in the unicellular eukaryotes, plasmodium, tetrahymena and s. cerevisiae, protein coding sequence accounts for ~ <dig>   <dig>  and 70%, of the respective genomic sequences  <cit>  whereas they account for ~44% of the genome in the multicellular neurospora crassa  <cit>  and o. dioica  <cit> .
despite a higher proportion of protein coding sequence than tetrahymena, the latter two organisms do not exhibit the same reduction in repressive chromatin marks as the former three, suggesting that augmentation of repressive marks is more associated with increased developmental complexity/cell type specification than with the global percentage of transcriptionally inactive genomic regions per se.
a developmental cell cycle switch in linker h <dig> variants
the number of linker histone subtypes identified across multicellular organisms is quite variable compared to core histone families.
at least  <dig> linker histones are expressed in human and mice, the c. elegans genome encodes  <dig> h <dig> isoforms, but only one linker histone is known in drosophila  <cit> .
the  <dig> odh <dig> proteins contain comparatively short n- and c-terminal tails.
the only linker histone type with similarly short c- and n-termini in vertebrates is h <dig> , a replacement variant present mainly in terminally differentiated cells but also found in cells that have entered a prolonged inter-mitotic period  <cit> .
odh <dig>  and the replacement variants odh <dig>  and odh <dig>  have the shortest c-termini and we found them to be the only h <dig> isoforms present in the endocycling stages of o. dioica, when the majority of cell fates are definitively determined and these cells no longer undergo mitosis.
furthermore, they contain only one s/tpxk motif, while odh <dig> , which is restricted to the mitotic stages of o. dioica, has two.
endocycling chromatin may not require the same extent of dynamic modulation as is necessary to regulate a mitotic cell cycle.
this may result in the deployment of linker histones with a less dynamic range of binding affinities expected from the short odh <dig> , odh <dig>  and odh <dig>  proteins.
absence and innovation in the o. dioica histone h2a complement
the absence of h2ax raises interesting questions about genome integrity and the double-strand break -repair pathway in o. dioica.
rapid phosphorylation of h2ax generating γh2ax  <cit>  is central to both of the alternative dna repair pathways, nonhomologous end-joining  and homologous recombination , which compete for dsb in eukaryotic cells  <cit> .
nevertheless, h2ax is dispensable for hr during meiosis in h2ax-deficient mice  <cit>  and the mre11-rad50-nbs <dig> repair complex is able to recruit repair and signaling proteins to dsb sites in the absence of h2ax  <cit> .
hr efficiency is influenced by template accessibility, causing an up regulation of hr during s and g <dig> phases of the cell cycle when sister chromatids are available  <cit> .
the challenge of locating a homologous template for hr repair may not be an obstacle in the compact genome of o. dioica, where up to several hundred copies of each locus  <cit>  are available for recombination in endocycling cells.
it is therefore possible that dsbs arising in endocycling cells of o. dioica that do not traverse mitosis, could be repaired by an h2ax-independent hr pathway.
in support of this, the o. dioica genome lacks many key components of the nhej pathway  <cit> , including dna-pk, a key protein that facilitates alignment of non-complementary ends and regulates end-processing.
the observation that both h2ax and dna-pk are present in the sister class ascidian c. intestinalis, indicates secondary loss of h2ax in the appendicularian lineage as opposed to urochordates as a whole having failed to evolve an h2ax variant.
h2a.z is one of the best studied universal core histone variants and, with the exception of cenh <dig>  the only variant found to be essential in several species  <cit> .
h2a.z can be found at active gene loci and the acetylation mark is erased at the onset of mitosis  <cit> , suggesting that acetylation of its n-terminus may be required to lower affinity for dna and facilitate access for essential competing factors at promoters or to prevent higher order folding.
studies on h2a.z  acetylation in tetrahymena revealed that acetylation of the n-terminus works to modulate an essential charge patch  <cit>  and viable transformants could be obtained when all of the six k residues within the n-terminus of h2a.z were mutated to r as long as a charge reducing mutation was also included.
this suggests that the function of h2a.z acetylation in tetrahymena, in contrast to a site-specific histone code, is to alter the charge of the n-terminal domain.
it is therefore intriguing that o. dioica uses alternative splicing to control the number of acetylatable k residues in the n-terminus of h2a.z.
deployment of h2a.z splice variants with altered numbers of acetylatable k residues would seem less dynamic than simply modulating the acetylation status of a fixed number of k-residues.
however, in the endoreduplicative cell cycle variant used predominantly for growth in o. dioica, genes are disrupted transiently by passage of the replication fork but do not undergo the extensive condensation cycles occurring during the proliferative mitotic cell cycles characteristic of growth in most chordates.
on the other hand, a challenge during non-polytene endocycles is to coordinately regulate hundreds of copies of a given gene dispersed in a single interphase nucleus  <cit> .
in this situation, more fixed stepped levels of charge, with a reduced dynamic modulation range may be advantageous.
the existence of two non-redundant h2a.z variants in vertebrates  <cit>  displaying subtle differences in their association with post translationally modified canonical histones and chromatin localization  <cit>  suggests that generation of different h2a.z variants might not be an uncommon strategy in animals to further extend the functional repertoire of h2a.z proteins.
as several metazoan h2a.zs contain a conserved intron directly following the start atg and possess multiple alternative splice acceptors within their second exon, it will be of interest to determine whether generation of alternative splice h2a.z variants is not unique to o. dioica.
variant male nucleosomes in o. dioica
o. dioica has a particularly high number of histone isoforms expressed exclusively or highly enriched in testes, including  <dig> h2at,  <dig> h2bt,  <dig> h3t and  <dig> h4t.
testes-specific histones are poorly studied but are thought to substitute for canonical histones in meiotic and post-meiotic cells prior to replacement of histones by protamines  <cit> .
analyses of mammalian testes-specific h2as, h2bs  <cit>  and h3s  <cit>  indicate that nucleosomes containing these variants are less stable than those composed of canonical histones.
o. dioica histone isoforms exclusively expressed in testes  might affect intranucleosomal interactions.
o. dioica h2at and h2bt histones exhibit alterations in regions that promote stabilization of interactions with the h3-h <dig> tetramer and within the h2a-h2b dimer.
furthermore, several of the odh2bts have shortened n-terminal tails, lacking the first 14- <dig> n-terminal residues , predicted to form an α-helix in vertebrate h2bs   <cit> .
the n-terminal tails of mammalian h2bs are involved in interactions with dna, internucleosomal histone-dna interactions  <cit>  and are important for the mitotic and apoptotic condensation of chromosomes.
very divergent or short tails may be compromised in some of these functions and result in further loss of nucleosome stability.
these features might provide the basis for structural transitions and assembly of genomic sub-regions into altered dna-packaging structures and/or facilitate histone mobilization during male pronuclear reprogramming.
is there a testes-specific histone code in o. dioica?
the massive synthesis of histone variants, including testes-specific members, and the stage-specific posttranslational modification of histones during spermiogenesis have led to proposal of a testes-specific "histone code" generated by combining both histone variants and ptms  <cit> .
in zebrafish sperm, genes with embryonic functions are embedded in distinctive, complex and atypical chromatin structures  <cit> .
hyperacetylation of histones is associated with histone replacement in early elongating spermatids  <cit>  and has been reported for some testes-specific variants such as rat th2b  <cit> .
consistent with this, we found several k residues within the n- and c-termini of odh2bt.
<dig> and h2bt.
<dig> to be acetylated.
moreover, o. dioica tiling array data confirm expression of a bromodomain-containing testes-specific factor   <cit>  that induces chromatin remodeling in the presence of histone hyperacetylation in mammals  <cit> , possibly mediating nuclear reorganization during spermiogenesis.
residues exposed on the nucleosome disc surface may in part account for the extreme evolutionary constraints on histone sequences, since they interact with nuclear factors and mediate internucleosomal interactions.
dot <dig> is the methyltransferase responsible for mono-, di-, and trimethylation of h3k <dig>  a residue exposed on the disc surface within the globular domain of h <dig>  h3k <dig> methylation  is found in euchromatin  <cit>  but the modification also plays an important role in heterochromatin formation in mice and yeast  <cit> .
interestingly, the residues surrounding k <dig> are substituted in all male-specific h3ts of o. dioica.
in h3t.
<dig>  not only have the surrounding residues been modified, but k <dig> has been replaced by r, raising the possibility that the odh3t isoforms lack k <dig> methylation.
these residues are nearly invariant across species, though substitutions are present in pollen-specific h3s of arabidopsis and rice, c. elegans h <dig>  variants, and all h3s identified in plasmodium and tetrahymena .
the fact that both dot <dig> and k <dig> methylation are absent in arabidopsis  <cit>  and plasmodium  <cit>  may suggest that dot <dig> binding could partially account for evolutionary constraints on these residues.
these residues likely create a specialized nucleosome surface for dot <dig> binding, as single mutations centered directly around h3k <dig> distinctly affected the three typical forms of yeast silencing .
a single r <dig> substitution, as in odh3t.
<dig>  weakened silencing of rdna and the silent mating loci but enhanced silencing at telomeres.
together with the surrounding mutations found in the other odh3t variants this may indicate an inability of dot <dig> to methylate h3k <dig> in odh3ts.
no h <dig> sequence variant has been described in higher metazoans and the fact that o. dioica males express a h <dig> sequence variant appears unique.
expression of a male-specific h <dig> gene has been reported in rats  <cit> , but this transcript encodes an invariant h <dig> protein sequence.
two of the residue changes in odh4t occur within the n-terminal tail adjacent to the h4k <dig> methylation site, a core histone modification exposed on the nucleosome disc surface.
h4k20me <dig> affects internucleosomal interactions resulting in more compact higher order chromatin structure  <cit> .
in view of the extreme conservation of the h <dig> amino acid sequence across metazoans, also preserved in the other  <dig> o. dioica h <dig>  genes, a neutral function of these mutations is questionable.
CONCLUSIONS
the traditional view of histones as slow evolving proteins has changed considerably within the last decade.
it is now clear that histone variants have diversified in many species to assume crucial roles in transcriptional activation, dna repair, chromosome segregation and other processes.
recent data from more exotic model organisms such as trypanosomes  <cit>  and rotifers  <cit>  increasingly link histone complement evolution to specific life history traits of organisms.
the results here show that there is considerable plasticity in histone gene organization and variation within histone families even within the chordate lineage.
of particular note among the high diversity of histone variants in the fast evolving appendicularian lineage, is a concentration of variation in all core and linker histone families associated with differentiation of the male germline, raising intriguing questions regarding the testes as an evolutionary playground for the innovation of histone variants.

 in eukaryotes, chromatin is the interface through which genetically encoded information is read to orchestrate a diversity of cellular and organismal functions. chromosomal packaging of nuclear dna is achieved through small basic histone proteins. the highly conserved core histones, h h h2a and h2b form an octamer of two h2a/h2b dimers flanking a central h3/h tetramer that wrap ~ bp of dna in turns to form the nucleosome core particle. tetramer formation is common to archaeal and eukaryotic histones. the eukaryotic origin of h2a-h2b dimers, doubling the dna wrap capacity, has been discussed in light of demands of increased genome size and kinetic constraints of dna compaction imposed by eukaryotic mitosis. interactions between the histone octamer and dna are further modulated by linker histones h1/h which associate with dna at its entry/exit site on the ncp surface. roles of histones in structural compaction must be balanced with regulatory mechanisms that permit selective access to dna to enable functions such as transcription, replication and dna repair. three major strategies contribute to regulatory remodeling of chromatin: atp-dependent complexes that act on nucleosomes to modify accessibility of wrapped dna sequences to trans-acting factors, the deployment of histone variants that alter nucleosome dynamics, and covalent posttranslational histone modifications including acetylation, methylation, phosphorylation, ubiquitylation, citrullination, adp-ribosylation, glycoslylation and sumoylation. one current view is that eukaryotic genomes are indexed locally, and over broader regions, through a combination of histone variants and their diverse ptms. main lineages of canonical core histones, constituting the bulk of histone proteins, are assembled into chromatin during dna replication. these replication dependent genes lack introns, are typically organized in gene clusters and their mrnas possess a conserved stem-loop in the 3'utr coupling gene expression to dna replication. in contrast, histone variants are often transcribed from orphan genes that contain introns, lack the sl and their expression is not restricted to s-phase. consequently, they are referred to as replacement or replication-independent variants. from an evolutionary perspective, histone variants are classified as universal or lineage-specific variants, where the term variant refers to non-allelic sequence variation. universal variants have ancient functions common to eukaryotic cells, whereas lineage-specific variants appear specialized to certain organismal requirements. core histones clearly differ in their evolutionary propensity to diversify as distinct variants. h is highly constrained as it makes contacts with the other core histones and its n-terminal tail residues are subject to extensive ptms. this is reflected in its nearly invariant stature. no h amino acid sequence variants have been reported in any multicellular organism to date and diversified h4s are known only in trypanosomes and ciliates. despite h2bs showing substantially less evolutionary sequence constraint than h and h they, like h exhibit little specialization, with the exception of gametogenesis variants. on the other hand, h and h2a are much richer in their genealogy with diversification of a number of functionally essential variants. the two h molecules interact with each other in the nucleosome, as do the two h2a molecules, whereas h and h2b do not. two-fold nucleosomal symmetry is organized along the c-terminal four-helix bundle dimerization interface of the two h3s. interaction between the two h2as is mediated by their loop domains. specializations have evolved in the h lineage with at least two h variants present in most eukaryotes. the replacement variant h can undergo rd or ri assembly and deposition is primarily in transcribed regions of euchromatin. h differs from canonical h at few positions, predominantly, one in the n-terminal tail and three in the α helix of the histone fold domain, the latter three required for ri assembly. all eukaryotes possess h though asomycetes lack canonical h phylogenetically, the two proteins appear not to be separate, early-branching lineages, as distinct h and h histones have arisen multiple times. a second common h variant is centromeric h essential for recruitment of kinetochore components vital to mitosis. cenh3s retain only ~50% identity to h3s in the hfd and have divergent n-terminal tails from to residues. cenh phylogeny is poorly resolved. they appear to have arisen many times with rapid divergence under positive selection. there are also lineage-specific meiotic and spermatogenesis h variants. among core histones, h2as have evolved the greatest variant diversity. there are two nearly universal variants, h2ax and h2a.z. h2ax plays an important role in the maintenance of eukaryotic genome integrity by participating in double-strand dna-break repair by nonhomologous end joining. h2ax has arisen multiple times during evolution but similar constraints have led to convergent acquisition of the h2ax-specific phosphorylation motif sqe/dΦ. phosphorylation of the s residue in this c-terminal motif forms γh2ax, which assists in the recruitment of histone modifiers, chromatin remodelers and dna repair complexes. h2ax has entirely replaced canonical h2a in fungi and giardia but is absent in c. elegans and protozoan parasites, plasmodium and trypanosomes. h2a.z had a single evolutionary origin, and has remained distinct from canonical h2a. it is essential for viability in a range of species, and is implicated in gene activation, chromosome segregation, heterochromatin silencing, and cell cycle progression. a common thread in a number of these disparate roles may be the proclivity of h2a.z to form more stably positioned nucleosomes combined with differential use of ptms. other h2a variants include the c-terminally extended macroh2a, the c-terminally truncated mammalian h2a.bbd and an array of more poorly studied lineage-specific variants. linker h variants form a complex family and subtypes are classified as canonical rd or ri linker histones. they display tissue and developmental specificity, with vertebrates commonly expressing testes-specific and oocyte-specific variants. structurally, metazoan h1s are divided into three domains: a short, flexible n-terminal, a globular domain containing a winged-helix fold and a long, lysine rich c-terminal tail. h variants differ in biophysical properties, association with repressed or active chromatin and their ability to increase or decrease transcription when over-expressed. the above picture is derived from histone sequence comparisons in a wide array of eukaryotes, but to date, very few full histone complements have been analyzed more comprehensively and information is principally restricted to universal variants as well as some lineage-specific variants in mammals and plants. modification of metazoan histone variant lineages occurs on a background of genome architecture that shows global similarities from sponges to vertebrates but the urochordate, oikopleura dioica, a member of the closest sister group to vertebrates, demonstrates that this ancestral genome architecture can be profoundly modified. in this fast-evolving, pan-global zooplankton, a repertoire of > genes has been extraordinarily compacted into a genome of only mb, with sizes of introns and intergenic regulatory regions greatly reduced. whereas other chordates generally grow through cell proliferation, rapid growth of o. dioica over a short, 6-day life cycle is achieved principally through endoreduplication. here, we characterize the highly diverse histone complement that has emerged in concert with evolution of this intriguing lineage at the invertebrate-vertebrate transition.  histone gene organization in oikopleura dioica histone genes were dispersed throughout the o. dioica genome with most in clusters up to quintets. in total, histone genes encoding different histone proteins were identified. clustered genes showed typical rd features. they were intron-less and most shared divergent promoters. primary transcripts of these genes contain both a conserved sl and a downstream polyadenylation signal in their 3' utrs but lack the histone downstream element. a second set of histone genes were present as orphan genes: h, h and h2a isoforms. these genes have introns and the sl is degenerate or absent, suggesting they are ri variants. to assess phylogeny among histone families, unrooted maximum likelihood trees were generated. as expected, genes encoding the same histone protein were more closely related and the most divergent histones exhibited the longest branch lengths. clusters i, ix and xi, had the same h2a and h2b phylogenies and likely result from more recent gene duplications. to compare the o. dioica histone complement to that of other urochordates we extended genome searches to the histone genes of two ascidians, ciona intestinalis and ciona savignyi. surprisingly, we found that the histone complements of the two ascidians differed significantly in terms of histone gene number and organization. the c. savignyi genome contains a high number of histone genes partly organized in large clusters, whereas the c. intestinalis genome encoded only histone genes, mostly interspersed among non-histone genes. a locus within the c. savignyi genome was found where histone genes are arranged similarly to the tandem-arrays of sea urchin and drosophila. table shows that histone gene complements and their organization can differ considerably even over modest phylogenetic distances. dispersed h3t.2 h2bfwt h2bt.1 h2bt.2 h2at.2 h2at.3 h2asq.1 h2asq.2 h h h h *4 h *5 h1a h1t2 §+ isoforms = additional isoforms in the genome. † = differentiation h variant. expression: peaks at *1d3/d4; * peaks in early tadpoles and d where it is enriched in the testes; *3commences h pf and decreases after d4; * peaks during organogenesis and *5peaks at metamorphosis. the nomenclature of odh1s does not imply direct orthology with human h -h expression of histone gene clusters is developmentally co-regulated we assessed expression profiles of all histone genes throughout development by qrt-pcr, including maturing males and females. o. dioica histone genes were co-regulated in clusters and most genes belonging to the same locus showed the same developmental expression profiles. histone clusters could be assigned to seven expression patterns: 1) throughout the life cycle 2) exclusively during organogenesis, 3) male-specific expression in mature d animals, 4) expression peaking in early tadpoles and male d animals, 5) expression peaking at metamorphosis, 6) expression primarily in d and d animals and 7) transcripts predominantly present from h post-fertilization to d. histone isoforms exhibiting multiple changes in amino acid sequence were commonly male-specific. however, some divergent core histone variants displayed expression profiles that preceded gonad maturation in d3/d adults, from h pf to d or after organogenesis and in late adults at d. to improve resolution of germline-specific expression of different histone proteins, maturing d5/d animals were dissected and cdnas prepared separately from testes, ovaries and trunks. expression ratios were up to 4300-fold higher in testes and 330-fold higher in ovaries for certain isoforms. extrapolation of ovary-specific transcripts to oogenic function is complicated by maternal mrna storage in oocytes during vitellogenesis. taking this into account, only h2a. was clearly significantly enriched in the ovary. the potential combinatorial possibilities of histone core and linker isoforms based on their transcript levels throughout development are schematically summarized in figure during organogenesis, transcript levels of all canonical histones were increased by the activation of additional, organogenesis-specific histone gene loci. at least one male specific isoform exists in each of the five histone families, including a male-specific h4t isoform. mass spectrometry of o. dioica core histones from mature sperm and whole animals o. dioica core histones were prepared for mass spectrometry from d and d animals, which include endocycling nuclei of the epithelium, the mitotic nuclei of d male gonads and female and male meiotic nuclei of d animals. histone sequence coverage by lc-ms/ms ranged from 51% to 90% and most peptides were identified several times. many conserved ptms associated with transcriptional activity and silent chromatin in other species were identified. the n- and to a lesser extent, c-termini, of o. dioica h h and h2b contained numerous acetylated lysine residues. conserved sites for mono-, di-, and trimethylations such as h4k h3k h3k h3k and ubiquitylation of h2bk were observed, though trimethylation of h4k and h3k were not detected. peptides were found for canonical h2b. and/or h2b. monomethylated at r and dimethylated at their last c-terminal k residues as well as the dimethylation of h at r we found few or no peptides unique to the divergent variants h2b. h2b. h2a. h2a. and cenh possibly due to low abundance. the large number of histone variants expressed in testes led us to investigate their retention in mature sperm. mass spectrometry of histones isolated from mature sperm revealed many peptides specific for canonical and male-specific o. dioica histone isoforms. o. dioica core h4s whereas other multicellular organisms express a single h isoform, o. dioica expresses two histone h4s, canonical h and a male-specific h4t. the major h subtype was expressed from five genes, with three expressed throughout development and genes at loci ii and xii peaking at organogenesis. the h4t gene, located in a male-specific histone gene cluster was specifically expressed in d males, with a > enrichment in testes compared to ovaries. h4t-specific peptides were found by mass spectrometry in o. dioica sperm histones. within the h4t amino acid sequence v and l are replaced by i and m in a region of the h n-terminal tail that interacts with the acidic patch of the adjacent h2a-h2b dimer. there is also a third residue-change within the α helix, where a is replaced by s. based on h structure, s is not solvent accessible, facing towards the alpha helix of h o. dioica h2b isoforms are expressed before onset and during spermatogenesis no replacement variants have been identified among h2bs and all odh2b genes are intron-less and contain the sl in their 3'utr. odh2b. and h2b. were expressed throughout development, while the other h2b isoforms showed different levels of enrichment in testes. whereas h2bt. h2bt. and h2bt. were exclusively expressed in testes, with an increase up to >3500-fold compared to ovaries, the divergent h2b. gene was expressed at d3/d preceding spermatogenesis and divergent h2b. expression peaked in early tadpoles and d. intriguingly, these two divergent h2b isoforms display complementary expression patterns centered around the d3/d transition when germline differentiation commences. h2b. and h2b. have reduced sequence identity to somatic o. dioica h2b. but do not show significant similarities to testes-specific h2bfwt of mammals, which also shows reduced sequence identity to mammalian somatic h2b. mammalian testes-specific h2bs of the type tsh2b/th2b have few residue changes compared to their canonical counterparts, but all share three substitutions within their hfd, i g and n interestingly, canonical odh2bs share these same residue substitutions and therefore have slightly higher sequence similarity to testes-specific rather than somatic h2bs of mammals. structurally, all odh2b proteins have short n-terminal tails compared to other chordate h2bs and none of the isoforms contain k/r-rich motifs characteristic of h2bs retained in echinoderm sperm. o. dioica core h3s the o. dioica h complement comprises all universal h variants but lacks mammalian-specific h. odh and h are 100% identical in amino acid sequence to vertebrate orthologs. additionally, o. dioica expressed three male-specific h isoforms highly enriched in testes. h3t. and h3t. contained a fully conserved sl and were co-regulated within a male-specific histone gene cluster, whereas h3t. was expressed from an orphan gene with a degenerate sl, indicative of a replacement variant. within chordates, testes-specific h variants have only been reported from mammals. odh3ts, however, are more divergent than mammalian h3ts when compared to canonical h /h. odh3t. has three residue substitutions in the n-terminal tail and ten in the histone fold, whereas mammalian h3ts exhibit only one and two substitutions, respectively. odh3ts and mammalian h3ts share no common residue changes and phylogenetic analyses indicate that the h3ts of o. dioica are more closely related to other odh3s than to mammalian h3ts. we did not find similar h3t genes within the genomes of the two ciona species, suggesting these isoforms might be specific to the appendicularian lineage. a notable substitution within the n-terminus of h3t. is the change of a to a phosphorylatable t residue. the same position in h is s, which is phosphorylated in mitosis and meiosis and is the only known h -specific modification. within the histone fold, the most apparent sequence changes of the odh3ts are exposed on the disc surface of the nucleosome, surrounding k a conserved site for methylation which was present in o. dioica canonical h3s. further residue changes occur at positions relevant for intranucelosomal interactions. within loop and α-helix i and i which make contact with loop and α-helix of h respectively, are replaced by v and e another intron-containing h gene lacking the sl encodes a divergent h replacement variant that we identified as the o. dioica centromeric h odcenh shows characteristic cenh features: a very divergent n-terminal tail, its l loop is extended and f in l which interacts with the l of h is replaced by w. when expressed as an egfp fusion protein, odcenh specifically localized to the centromeres of o. dioica chromosomes. o. dioica h2as: h2a.z-splice variants but no h2ax similar to vertebrates, the h2a family is the largest and most diverse group of core histones in o. dioica. h2a. encoded by genes, exhibits the highest identity to canonical vertebrate h2as. h2a. and h2a. both expressed throughout development, differ by a single amino acid change from a to s at position. o. dioica also expresses six male-specific isoforms and two divergent replacement variants, h2a. and h2a. lacking the sl. comparing the h2asq and h2at proteins with each other and with h2a. they mainly differ in their n-terminal tails and their docking domain, which interacts with the h3-h tetramer. nevertheless, all male-specific h2as share residue changes in structurally important regions such as: the h2a self-dimerization domain, within l that interacts with the h2b l dna contacts, and residues in the α helices that interact within the h2a-h2b dimer. odh2as have short c-terminal tails compared to vertebrate h2as. no macroh2a gene was identified in o. dioica. more surprising was the finding that no odh2as contained the pikk substrate sqe/dΦ phosphorylation motif that characterizes all h2axs. three male-specific o. dioica h2a isoforms contain a putative phosphorylation sq-motif, adjacent to the c-terminal alpha helix, as a result of a residue change from a to s. although this sq-motif could present an alternative phosphorylation site for pikks, its location in the docking domain makes it less solvent accessible than the typical position of the sqe/dΦ-motif at the end of the c-terminal tail. we conclude there is no h2ax homolog in o. dioica. the odh2a.z variant exhibits the specific divergent characteristics with respect to h2a. described for universal h2a.zs, including a highly positive n-terminal tail, a divergent l and divergent self-dimerization and docking domains. further, h2a.z-specific substitutions are present, including the replacement of n with d, which extends the acidic patch, a substitution of e by g and the presence of two solvent accessible histidines. odh2a.z differs from consensus h2a.z sequences at several residues that are invariant in higher eukaryotes and similar in position to residue changes in tetrahymena h2a.z. these include three non-conservative amino acid substitutions in the self-dimerization domain, two s-residues replacing two a-residues within the α helix and substitution of s by m at position adjacent to the acidic patch. similar to tetrahymena h2a.z, the odh2a.z c-terminal tail lacks one of two conserved k-residues that are mono-ubiquitylated in mammalian heterochromatin. another atypical feature of odh2a.z is extension of a comparatively short h2a.z c-terminal tail by amino acids containing a "ppqppq" motif. a further similarity to tetrahymena h2a.z is the long n-terminal tail of odh2a.z, extended by mainly g, k, and a residues. h2a.z n-terminal tails of higher eukaryotes harbor several gk-repeats that can be acetylated. intriguingly, we found that o. dioica is able to modify the number of gk-motifs within its h2a.z n-terminal tail by alternative splicing. alternative splice acceptor sites within the second exon of the odh2a.z gene, result in shortening of the n-terminus by precisely one or two of the first three gk-pairs. full length odh2a.za was predominantly expressed, but all three isoforms were present throughout development. we also identified two additional h2a variants that did not resemble any universal or lineage-specific h2a variants. h2a. and h2a. showed 78% amino acid sequence identity to each other but ≤55% identity with other odh2as. from the n-terminal tail to the α helix, h2a. and h2a. show almost no amino acid sequence conservation to canonical h2a. similar to h2a.z, both variants differed significantly in the self-dimerization domain, within l and in their docking domains, but do not show typical features in primary structure described for the mammalian h2a.bbd. as for the odh2b subtype h2b. h2a. was predominantly restricted to d3/d animals and transcripts were enriched in the ovary. expression of h2a. commenced after zygotic gene activation and decreased significantly when animals began to mature after d. o. dioica linker h1s we identified odh genes encoding different h proteins. with the exception of d males, h was expressed at every stage of development, while h and h were expressed from h pf to d peaking during metamorphosis. h, h and h were the dominant h1s throughout development, while h and h1t were restricted to organogenesis and testes, respectively. the h, h and h1t genes are intron-less, located within histone clusters, and contain the sl. variants h and h were expressed from orphan genes that contain one intron and a degenerate sl, suggesting replacement variant function. increased content of positively charged residues and tail length have been reported to strengthen binding affinity, whereas phosphorylation at s/tpxk motifs weakens h binding and destabilizes chromatin structures. the n-terminal tails of o. dioica h1s are short in comparison to h subtypes of mammals with h, h and h also having short c-terminal tails. odh contains the most extended c-terminus and has the highest content of positively charged residues. it is also the only o. dioica linker histone with two phosphorylation motifs within the c-terminus, while h, h, h have one. this may suggest that among o. dioica linker histones, h, restricted to early development, has the widest range of binding affinities, a feature that may facilitate chromatin remodeling events during embryogenesis. in contrast, odh1t, the shortest linker histone, lacks the conserved phosphorylation motif, suggesting a more restricted binding affinity range. discussion analyses of histone complements of higher eukaryotes have been published for metazoans such as drosophila, human and mouse, chicken, c. elegans xenopus and sea urchin. comprehensive developmental expression data is only available for the sea urchin histone complement. here we show that the genome of the urochordate o. dioica encodes a high diversity of histone isoforms. as in other organisms, the canonical histone genes are organized as clusters and share divergent promoters, though in contrast to drosophila, sea urchin and mammals, the histone genes have not remained physically linked in one or two large clusters or tandem repeats. instead they are dispersed in small groups throughout the genome, similar to the c. elegans histone complement. organization of the o. dioica histone complement exhibited limited affinity with the sister urochordate ascidian species, c. intestinalis or c. savignyi, which in turn, differed significantly from each other. therefore, developmental mode, genome size, and phylogenetic relationship do not appear to be overriding determinants of histone gene organization. histone modification patterns and genome size a current view is that histone modifications become more complex from unicellular eukaryotes to mammals. lysine acetylation of h and h n-terminal tails, as well as methylated h3k h3k and h3k are considered active marks, while methylated h3k h3k and h4k mark silent chromatin. combined with previous studies the mass spectrometry data now indicate that all conserved "on" and "off" marks are present on o. dioica histones. whereas unicellular eukaryotes, such as yeast, tetrahymena and plasmodium principally exhibit modifications associated with transcriptional activation, mammals also employ a wider range of marks involved in gene silencing. the observation that hallmarks of silenced chromatin such as h3k and h3k methylation are low in yeast and tetrahymena has been explained by the fact that the majority of the genome in unicellular eukaryotes is transcriptionally competent, whereas more than 60% of the mammalian genome is permanently silenced and only ~3% of its dna encodes structural genes. considering the high gene density of the o. dioica genome and the short intergenic regions, we also expect the proportion of the o. dioica genome that is permanently silenced to be relatively low compared to that of human. however, our analysis revealed no obvious lack of silent heterochromatic marks in o. dioica, since methylation of h3k and h4k were detected by mass spectrometry and methylation of h3k has been confirmed by immunofluorescence. in the unicellular eukaryotes, plasmodium, tetrahymena and s. cerevisiae, protein coding sequence accounts for ~ and 70%, of the respective genomic sequences whereas they account for ~44% of the genome in the multicellular neurospora crassa and o. dioica. despite a higher proportion of protein coding sequence than tetrahymena, the latter two organisms do not exhibit the same reduction in repressive chromatin marks as the former three, suggesting that augmentation of repressive marks is more associated with increased developmental complexity/cell type specification than with the global percentage of transcriptionally inactive genomic regions per se. a developmental cell cycle switch in linker h variants the number of linker histone subtypes identified across multicellular organisms is quite variable compared to core histone families. at least linker histones are expressed in human and mice, the c. elegans genome encodes h isoforms, but only one linker histone is known in drosophila. the odh proteins contain comparatively short n- and c-terminal tails. the only linker histone type with similarly short c- and n-termini in vertebrates is h, a replacement variant present mainly in terminally differentiated cells but also found in cells that have entered a prolonged inter-mitotic period. odh and the replacement variants odh and odh have the shortest c-termini and we found them to be the only h isoforms present in the endocycling stages of o. dioica, when the majority of cell fates are definitively determined and these cells no longer undergo mitosis. furthermore, they contain only one s/tpxk motif, while odh, which is restricted to the mitotic stages of o. dioica, has two. endocycling chromatin may not require the same extent of dynamic modulation as is necessary to regulate a mitotic cell cycle. this may result in the deployment of linker histones with a less dynamic range of binding affinities expected from the short odh, odh and odh proteins. absence and innovation in the o. dioica histone h2a complement the absence of h2ax raises interesting questions about genome integrity and the double-strand break -repair pathway in o. dioica. rapid phosphorylation of h2ax generating γh2ax is central to both of the alternative dna repair pathways, nonhomologous end-joining and homologous recombination, which compete for dsb in eukaryotic cells. nevertheless, h2ax is dispensable for hr during meiosis in h2ax-deficient mice and the mre11-rad50-nbs repair complex is able to recruit repair and signaling proteins to dsb sites in the absence of h2ax. hr efficiency is influenced by template accessibility, causing an up regulation of hr during s and g phases of the cell cycle when sister chromatids are available. the challenge of locating a homologous template for hr repair may not be an obstacle in the compact genome of o. dioica, where up to several hundred copies of each locus are available for recombination in endocycling cells. it is therefore possible that dsbs arising in endocycling cells of o. dioica that do not traverse mitosis, could be repaired by an h2ax-independent hr pathway. in support of this, the o. dioica genome lacks many key components of the nhej pathway, including dna-pk, a key protein that facilitates alignment of non-complementary ends and regulates end-processing. the observation that both h2ax and dna-pk are present in the sister class ascidian c. intestinalis, indicates secondary loss of h2ax in the appendicularian lineage as opposed to urochordates as a whole having failed to evolve an h2ax variant. h2a.z is one of the best studied universal core histone variants and, with the exception of cenh the only variant found to be essential in several species. h2a.z can be found at active gene loci and the acetylation mark is erased at the onset of mitosis, suggesting that acetylation of its n-terminus may be required to lower affinity for dna and facilitate access for essential competing factors at promoters or to prevent higher order folding. studies on h2a.z acetylation in tetrahymena revealed that acetylation of the n-terminus works to modulate an essential charge patch and viable transformants could be obtained when all of the six k residues within the n-terminus of h2a.z were mutated to r as long as a charge reducing mutation was also included. this suggests that the function of h2a.z acetylation in tetrahymena, in contrast to a site-specific histone code, is to alter the charge of the n-terminal domain. it is therefore intriguing that o. dioica uses alternative splicing to control the number of acetylatable k residues in the n-terminus of h2a.z. deployment of h2a.z splice variants with altered numbers of acetylatable k residues would seem less dynamic than simply modulating the acetylation status of a fixed number of k-residues. however, in the endoreduplicative cell cycle variant used predominantly for growth in o. dioica, genes are disrupted transiently by passage of the replication fork but do not undergo the extensive condensation cycles occurring during the proliferative mitotic cell cycles characteristic of growth in most chordates. on the other hand, a challenge during non-polytene endocycles is to coordinately regulate hundreds of copies of a given gene dispersed in a single interphase nucleus. in this situation, more fixed stepped levels of charge, with a reduced dynamic modulation range may be advantageous. the existence of two non-redundant h2a.z variants in vertebrates displaying subtle differences in their association with post translationally modified canonical histones and chromatin localization suggests that generation of different h2a.z variants might not be an uncommon strategy in animals to further extend the functional repertoire of h2a.z proteins. as several metazoan h2a.zs contain a conserved intron directly following the start atg and possess multiple alternative splice acceptors within their second exon, it will be of interest to determine whether generation of alternative splice h2a.z variants is not unique to o. dioica. variant male nucleosomes in o. dioica o. dioica has a particularly high number of histone isoforms expressed exclusively or highly enriched in testes, including h2at, h2bt, h3t and h4t. testes-specific histones are poorly studied but are thought to substitute for canonical histones in meiotic and post-meiotic cells prior to replacement of histones by protamines. analyses of mammalian testes-specific h2as, h2bs and h3s indicate that nucleosomes containing these variants are less stable than those composed of canonical histones. o. dioica histone isoforms exclusively expressed in testes might affect intranucleosomal interactions. o. dioica h2at and h2bt histones exhibit alterations in regions that promote stabilization of interactions with the h3-h tetramer and within the h2a-h2b dimer. furthermore, several of the odh2bts have shortened n-terminal tails, lacking the first 14- n-terminal residues, predicted to form an α-helix in vertebrate h2bs. the n-terminal tails of mammalian h2bs are involved in interactions with dna, internucleosomal histone-dna interactions and are important for the mitotic and apoptotic condensation of chromosomes. very divergent or short tails may be compromised in some of these functions and result in further loss of nucleosome stability. these features might provide the basis for structural transitions and assembly of genomic sub-regions into altered dna-packaging structures and/or facilitate histone mobilization during male pronuclear reprogramming. is there a testes-specific histone code in o. dioica? the massive synthesis of histone variants, including testes-specific members, and the stage-specific posttranslational modification of histones during spermiogenesis have led to proposal of a testes-specific "histone code" generated by combining both histone variants and ptms. in zebrafish sperm, genes with embryonic functions are embedded in distinctive, complex and atypical chromatin structures. hyperacetylation of histones is associated with histone replacement in early elongating spermatids and has been reported for some testes-specific variants such as rat th2b. consistent with this, we found several k residues within the n- and c-termini of odh2bt. and h2bt. to be acetylated. moreover, o. dioica tiling array data confirm expression of a bromodomain-containing testes-specific factor that induces chromatin remodeling in the presence of histone hyperacetylation in mammals, possibly mediating nuclear reorganization during spermiogenesis. residues exposed on the nucleosome disc surface may in part account for the extreme evolutionary constraints on histone sequences, since they interact with nuclear factors and mediate internucleosomal interactions. dot is the methyltransferase responsible for mono-, di-, and trimethylation of h3k a residue exposed on the disc surface within the globular domain of h h3k methylation is found in euchromatin but the modification also plays an important role in heterochromatin formation in mice and yeast. interestingly, the residues surrounding k are substituted in all male-specific h3ts of o. dioica. in h3t. not only have the surrounding residues been modified, but k has been replaced by r, raising the possibility that the odh3t isoforms lack k methylation. these residues are nearly invariant across species, though substitutions are present in pollen-specific h3s of arabidopsis and rice, c. elegans h variants, and all h3s identified in plasmodium and tetrahymena. the fact that both dot and k methylation are absent in arabidopsis and plasmodium may suggest that dot binding could partially account for evolutionary constraints on these residues. these residues likely create a specialized nucleosome surface for dot binding, as single mutations centered directly around h3k distinctly affected the three typical forms of yeast silencing. a single r substitution, as in odh3t. weakened silencing of rdna and the silent mating loci but enhanced silencing at telomeres. together with the surrounding mutations found in the other odh3t variants this may indicate an inability of dot to methylate h3k in odh3ts. no h sequence variant has been described in higher metazoans and the fact that o. dioica males express a h sequence variant appears unique. expression of a male-specific h gene has been reported in rats, but this transcript encodes an invariant h protein sequence. two of the residue changes in odh4t occur within the n-terminal tail adjacent to the h4k methylation site, a core histone modification exposed on the nucleosome disc surface. h4k20me affects internucleosomal interactions resulting in more compact higher order chromatin structure. in view of the extreme conservation of the h amino acid sequence across metazoans, also preserved in the other o. dioica h genes, a neutral function of these mutations is questionable.  the traditional view of histones as slow evolving proteins has changed considerably within the last decade. it is now clear that histone variants have diversified in many species to assume crucial roles in transcriptional activation, dna repair, chromosome segregation and other processes. recent data from more exotic model organisms such as trypanosomes and rotifers increasingly link histone complement evolution to specific life history traits of organisms. the results here show that there is considerable plasticity in histone gene organization and variation within histone families even within the chordate lineage. of particular note among the high diversity of histone variants in the fast evolving appendicularian lineage, is a concentration of variation in all core and linker histone families associated with differentiation of the male germline, raising intriguing questions regarding the testes as an evolutionary playground for the innovation of histone variants. 
modification of metazoan histone variant lineages occurs on a background of genome architecture that shows global similarities from sponges to vertebrates, but the urochordate, oikopleura dioica, a member of the sister group to vertebrates, exhibits profound modification of this ancestral architecture. histone variants alter the composition of nucleosomes and play crucial roles in transcription, chromosome segregation, dna repair, and sperm compaction. these results show that histone gene complements and their organization differ considerably even over modest phylogenetic distances. no obvious reduction in silent heterochromatic marks was observed despite high gene density in this extraordinarily compacted chordate genome. universal histone variants h, cenh and h2a.z are present but o. dioica lacks homologs of macroh2a and h2ax. mass spectrometry identified acetylation, methylation and ubiquitylation posttranslational modifications and showed that hallmark ptms of "active" and "repressive" chromatin were present in o. dioica. substantial innovation among all core and linker histone variants has evolved in concert with adaptation of specific life history traits in this rapidly evolving chordate lineage. we show that a histone complement of gene loci encodes histone variants, grouped in distinct sets of developmental expression profiles throughout the life cycle. a particularly diverse array of male-specific histone variants was uncovered, including a testes-specific h4t, the first metazoan h sequence variant reported.
 histone variants alter the composition of nucleosomes and play crucial roles in transcription, chromosome segregation, dna repair, and sperm compaction. modification of metazoan histone variant lineages occurs on a background of genome architecture that shows global similarities from sponges to vertebrates, but the urochordate, oikopleura dioica, a member of the sister group to vertebrates, exhibits profound modification of this ancestral architecture.  we show that a histone complement of gene loci encodes histone variants, grouped in distinct sets of developmental expression profiles throughout the life cycle. a particularly diverse array of male-specific histone variants was uncovered, including a testes-specific h4t, the first metazoan h sequence variant reported. universal histone variants h, cenh and h2a.z are present but o. dioica lacks homologs of macroh2a and h2ax. the genome encodes many h2a and h2b variants and the repertoire of h2a.z isoforms is expanded through alternative splicing, incrementally regulating the number of acetylatable lysine residues in the functionally important n-terminal "charge patch". mass spectrometry identified acetylation, methylation and ubiquitylation posttranslational modifications and showed that hallmark ptms of "active" and "repressive" chromatin were present in o. dioica. no obvious reduction in silent heterochromatic marks was observed despite high gene density in this extraordinarily compacted chordate genome.  these results show that histone gene complements and their organization differ considerably even over modest phylogenetic distances. substantial innovation among all core and linker histone variants has evolved in concert with adaptation of specific life history traits in this rapidly evolving chordate lineage. histone complementdna repairurochordateposttranslational modificationendocyclegametogenesistestesh2a.zalternative splicing 