Navratilova
et al. Epigenetics & Chromatin (2017) 10:3
DOI 10.1186/s13072-016-0110-4
RESEARCH
Sex-specic chromatin landscapes inan
ultra-compact chordate genome
Pavla Navratilova
1†
, Gemma Barbara Danks
1†
, Abby Long
2
, Stephen Butcher
2
, John Robert Manak
2
and Eric M. Thompson
1,3*
Abstract
Background: In multicellular organisms, epigenome dynamics are associated with transitions in the cell cycle,
development, germline specification, gametogenesis and inheritance. Evolutionarily, regulatory space has increased
in complex metazoans to accommodate these functions. In tunicates, the sister lineage to vertebrates, we examine
epigenome adaptations to strong secondary genome compaction, sex chromosome evolution and cell cycle modes.
Results: Across the 70 MB Oikopleura dioica genome, we profiled 19 histone modifications, and RNA polymerase
II, CTCF and p300 occupancies, to define chromatin states within two homogeneous tissues with distinct cell cycle
modes: ovarian endocycling nurse nuclei and mitotically proliferating germ nuclei in testes. Nurse nuclei had active
chromatin states similar to other metazoan epigenomes, with large domains of operon-associated transcription, a
general lack of heterochromatin, and a possible role of Polycomb PRC2 in dosage compensation. Testis chromatin
states reflected transcriptional activity linked to spermatogenesis and epigenetic marks that have been associated
with establishment of transgenerational inheritance in other organisms. We also uncovered an unusual chromatin
state specific to the Y-chromosome, which combined active and heterochromatic histone modifications on specific
transposable elements classes, perhaps involved in regulating their activity.
Conclusions: Compacted regulatory space in this tunicate genome is accompanied by reduced heterochromatin
and chromatin state domain widths. Enhancers, promoters and protein-coding genes have conserved epigenomic
features, with adaptations to the organization of a proportion of genes in operon units. We further identified features
specific to sex chromosomes, cell cycle modes, germline identity and dosage compensation, and unusual combina-
tions of histone PTMs with opposing consensus functions.
Keywords: Histone, Enhancer, Spermatogenesis, Polycomb, Dosage compensation, Heterochromatin, Transposable
elements, Endocycle
© The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(
http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
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and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (
http://creativecommons.org/
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) applies to the data made available in this article, unless otherwise stated.
Background
Histone proteins, which package genomic DNA, provide
multiple sites for covalent posttranslational modifications
(PTMs) by evolutionarily conserved histone modifiers
that form multimeric complexes and cooperate with non
-
histone proteins [
1, 2]. Histone PTMs are associated with
chromatin dynamics linked to transcription, replication,
DNA repair, recombination, chromosome segregation
and other mitotic and meiotic processes [
3, 4]. Impor
-
tantly, in the germ line, they help to secure correct
transgenerational inheritance, setting the stage for early
embryonic development [
5, 6]. Combinatorics of histone
PTMs, proposed to constitute a “histone code” [
7], are
part of the mechanism through which a single genome
generates a variety of cell types and states that respond
to developmental and environmental cues. Prevalent
combinations of modifications, referred to as “chromatin
states,” correlate with specific functional regions of the
genome, and many appear to be conserved among eukar
-
yotes [
8–12]. To date, however, only a few metazoan
Open Access
Epigenetics & Chromatin
*Correspondence: Eric.Thompson@uib.no
†
Pavla Navratilova and Gemma Barbara Danks contributed equally to this
work
1
Sars International Centre for Marine Molecular Biology, University
of Bergen, 5008 Bergen, Norway
Full list of author information is available at the end of the article
Page 2 of 18
Navratilova
et al. Epigenetics & Chromatin (2017) 10:3
epigenomes have been studied in detail [
10, 13–15], often
using cell lines or heterogeneous cell/tissue populations
from organisms, with the exception of invivo cell popu
-
lation studies that focused on a few histone PTMs [
16–
19]. Here, we present the germline epigenomes of the
chordate Oikopleura dioica, a member of the lineage that
comprises the closest living relatives to vertebrates [
20].
Oikopleura dioica is a semelparous pelagic tunicate
(Urochordate, Appendicularian) with a simple chordate
body plan and short, 6-day life cycle [
21]. Several major
developmental transitions are accompanied by switches
between mitotic and endocycling cell cycle modes in
both somatic tissues and the ovary [
22–24]. O. dioica
has the smallest metazoan genome sequenced to date,
organized in a haploid complement of 3 chromosomes.
At 70Mb, it is ~44-fold smaller than the human genome
despite maintaining >18,000 protein-coding genes [
25]
compared to ~20,000 in humans [
26, 27]. Introns are fre
-
quently very small (peak at 47bp; only 2.4% >1kb), as are
intergenic spaces (53% <1 kb). One quarter of the gene
complement is organized into operons [
28], and trans-
splicing of a short spliced-leader (SL) RNA occurs at
the 5′ ends of 39% of protein-coding genes [
29]. Trans
-
posable elements (TEs) form a significant proportion of
vertebrate genomes, but most vertebrate TE families are
absent in O. dioica and the density of TEs is low, with
most concentrated on the gene-poor Y-chromosome
[
25]. Major clades of non-LTR (long terminal repeat) ret
-
rotransposons are missing from the O. dioica genome,
but it has variety of LTR retrotransposons from the
Ty3/gypsy group, divergent from those found in other
organisms, as well as Dictyostelium intermediate repeat
sequence 1 (DIRS1) and Penelope-like elements [
30].
ese autonomous elements carry an env gene and are
expressed in a variety of Oikopleura tissues including
germline-associated cells [
31]. A comprehensive develop
-
mental transcriptome for O. dioica has been assembled
and includes ovary and testes samples [
32]. e full his
-
tone complement and associated PTMs have also been
characterized, showing conservation of histone variants
and a histone modification repertoire comparable to ver
-
tebrates [
33].
Regions of the O. dioica genome that have poten
-
tial regulatory function (introns and intergenic regions)
have been reduced, often to the order of one nucleo
-
some in size. How this compaction affects long-range
enhancer-mediated gene regulation [
34] and the epige
-
netic inheritance of chromatin domains through replica-
tion, mitosis and trans-generationally [
35] is unknown.
Polycomb complexes (PRC 1 and 2), via the tri-methyl
-
ation of histone 3 on lysine 27 (H3K27me3), govern core
mechanisms of metazoan epigenome heritability, organi
-
zation and developmental dynamics [
36]. Polycomb
complexes also function in sex chromosome inactivation
during dosage compensation [
37]. PRC1 is an ancient
complex and a determinant of cellular stemness [
38], but
its canonical composition has been reduced in O. dioica
and nematodes, possibly correlating with limited cel
-
lular plasticity and lack of regeneration [
39]. A number
of Polycomb complexes and modes of recruitment and
function exist, but these vary in the extent to which they
have been characterized [
40, 41]. O. dioica is an interest
-
ing model in which to investigate non-canonical Poly-
comb complexes and their functions in a rapidly evolving
lineage.
Oikopleura dioica is unusual among tunicates in that
it has genetically determined separate sexes with het
-
erogametic (XY) males and homogametic (XX) females.
Organisms with heterogametic sex chromosomes have
evolved dosage compensation mechanisms to equal
-
ize the abundance of transcripts produced by the single
X-chromosome in males and the double X-chromosome
in females [
42, 43]. In male mammals, flies and worms,
transcription of genes on the X-chromosome is upregu
-
lated. In female mammals one X-chromosome is inac-
tivated, and in hermaphrodite worms, expression from
X-chromosomes is downregulated. Different underly
-
ing components and molecular mechanisms behind the
recruitment and targeting of dosage compensation com
-
plexes as well as the resulting changes in chromatin have
been well documented [
44]. A common feature is that
complexes with other functions in the organism, such as
Polycomb in mammals, DCC in worms, or fly MSL, have
been recruited for domain regulation of X-linked genes.
It has thus far not been established that dosage compen
-
sation occurs in O. dioica, nor through what mechanism
it might be achieved, if it does occur.
Here, we sampled O. dioica testis, at early day 6 (a
few hours before germ-cell release), when it is a syncy
-
tium of mitotically proliferating, transcriptionally active,
spermatogonia nuclei (Additional file
2: Fig. S1). A pro
-
portion of active somatic genes including housekeep-
ing, self-renewal and proliferation genes are required
for mitosis and germline reprogramming. At the same
time, a testis-specific transcriptional program is required
for initiating spermatogenic gene transcription, setting
up the transmission of epigenetic memory and poising
developmental genes for expression following fertiliza
-
tion. Transitioning between these processes is rapid in
the O. dioica male germ line, but meiosis itself occurs
only in late day 6, about 2h before spawning. e day 6
O. dioica ovary consists of one single giant cell (the coe
-
nocyst), where endocycling nurse nuclei share a common
cytoplasm with meiotic nuclei arrested in prophase I [
24,
45] (Additional file2: Fig. S1). ese two populations of
nuclei occur in equivalent numbers, but the ploidy of
Page 3 of 18
Navratilova
et al. Epigenetics & Chromatin (2017) 10:3
nurse nuclei (200C) compared to that of the prophase
I meiotic nuclei (4C) [
22] means that the nurse nuclei
dominate (98% contribution) the chromatin content of
the ovary. Nurse nuclei are terminally differentiated and
help direct oocyte maturation and cellularization. A large
portion of their transcriptional output is maternal mRNA
that is subsequently stocked in the oocytes. Unlike tes
-
tis or oocyte meiotic nuclei, nurse nuclei do not traverse
mitosis and do not need to re-establish post-mitotic epi
-
genetic landscapes or undergo germline-specific gene
repression.
We extracted homogeneous nuclear populations from
testes and ovaries and profiled key histone PTMs and
nonhistone chromatin-associated proteins to explore
chromatin state landscapes in O. dioica germ lines and
their relationship to genome compaction, sex chromo
-
somes and autosomes. We found RNAPII activity-linked
signatures known from other metazoans. Chroma
-
tin domains were generally reduced in size, but we did
identify regions with histone PTMs typical of enhanc
-
ers. e ovarian, nurse nuclear epigenome consisted of
large domains of active transcription and a general lack
of repressive heterochromatin. e male germline epige
-
nome contained chromatin states specific to the spermat-
ogenic program and the X-chromosome and included an
intriguing combination of histone PTMs on the Y-chro
-
mosome, which may be involved in regulating the activ-
ity of transposable elements. is work provides the first
comprehensive view of a protochordate epigenome, pro
-
viding insight into its organization in two sex-specific tis-
sue samples.
Results
Oikopleura histone PTMs andtheir combinations
We profiled the following in maturing O. dioica testes
and ovaries: 19 histone PTMs (Additional file
3: Table
S1), using native ChIP-chip; CTCF, p300 and RNA poly
-
merase II (RNAPII) occupancy, using cross-linked ChIP-
chip; and 5-methylcytosine DNA methylation (5 mC),
using meDIP-chip. Sampled testes were in the mitotically
dividing pre-meiotic (spermatogonia) stage, whereas ova
-
ries were dominated by endocycling, transcriptionally
active nurse nuclei. We focused on histone H3 and H4
PTMs and related these profiles to gene expression lev
-
els [
32], trans-splicing status [28], chromosomal location,
and GC content of promoters (Fig.
1; Additional file 1:
Supplemental Results; Additional file
2: Fig. S2). We com
-
pared our results to those in human cells, Saccharomy-
ces cerevisiae, Drosophila melanogaster and C. elegans
(Table
1) [8, 10, 12–15, 46–52].
Combinatorial deposition of chromatin marks was ana
-
lyzed by classifying testis and ovary chromatin into 15
states (Fig.
2a; Additional file3: Tables S2 and S3), learnt
jointly across both cell types, using a multivariate hidden
Markov model (chromHMM) [
53]. ese 15 states were
reproducible when processing ovary and testes data
-
sets independently (Additional file
2: Fig. S3). Functions
were assigned to jointly learned states according to their
enrichments in an array of transcriptionally repressed
and active genomic features (Fig.
2c). Feature annota
-
tion of the separately learnt 15-state models underscored
some different uses of individual modifications in the tes
-
tis versus ovary (Additional file
2: Figs. S3 and S4). We
were also able to resolve 50 biologically meaningful chro
-
matin sub-states (Additional file
1: Supplemental Results;
Additional file
2: Fig. S5; and Additional file3: Table S4)
including a Polycomb-repressed state (state 46: enriched
for H3K27me3 and marks promoters of silent develop
-
mental genes) that was less pronounced in the 15-state
models. Unless stated otherwise, all subsequent analyses
were based on the main functional chromatin states cap
-
tured by the jointly learnt 15-state model.
Four chromatin states were specific to the ovary (1,
3, 4, 5), and four were specific to the testis (7, 8, 11, 12).
Specific chromatin states were associated with active
promoters (states 5, 8 and 9), transcription elonga
-
tion (states 3, 4, 6, 7) and silent regions (states 1, 10–12,
14–15), which included states specific to the Y-chromo
-
some (state 11) and silent transcription factors (TFs)
(state 1). State 2, which had no enrichment of any pro
-
filed modifications, covered 54% of the ovary and 40% of
the testis genomes (Fig.
2b), similar to the sum of “weak
signal” states calculated for human (45%), fly (35%) and
worm (45%) [
12]. We grouped active and silent genes by
GO terms and calculated chromatin state enrichments
on their promoters and gene bodies to reveal differential
use of chromatin states on genes with different biological
functions (Fig.
3; Additional file3: Table S5).
We compared chromatin state domain widths in O.
dioica to those found in nine human cell lines [
9] and
found that both activating and repressive domains were
significantly narrower in O. dioica (Mann–Whitney test:
W=1.189401e+12, p value <2.2e−16; Additional file
2:
Fig. S6). Domains spanning over 7 nucleosomes were
largely absent (Fig.
4a). e absence of large repressive
regions in O. dioica was notable (Fig.
4b; Additional file2:
Fig. S6) and supports previous observations of a decline
in heterochromatin coverage with decreasing genome
size [
12].
We found homologs of human histone modifier pro
-
teins and extracted gene expression values for these
homologs from a previously published transcriptomic
dataset [
32]. e expression patterns of the complement
of O. dioica histone modifiers in testes and ovaries cor
-
responded to the presence or absence of their associated
histone PTMs. Gene duplications of some modifiers and
Page 4 of 18
Navratilova
et al. Epigenetics & Chromatin (2017) 10:3
losses of others, particularly those related to DNA repair,
hormone response, Hox gene activating, RA response
and histone methyltransferases, reflected O. dioica’s
reduced NHEJ DNA repair toolkit, altered Polycomb
complex complement and dispersion of developmental
gene clusters (Fig.
5; Additional file3: Table S6; examples
and details in Additional file
1).
Transcriptionally permissive chromatin inthe ovary
e ovarian chromatin landscape was dominated by
ovary-specific states 3–5 (Fig.
2a, b). ese states were
enriched for H3K36me2 and H3K36me3 (typical of tran
-
scribed gene bodies in metazoans [
46]), covered 25%
of the genome (Fig.
2b), overlapped RNAPII-occupied
regions and correlated with high levels of transcription.
Active genes in the ovary were mostly related to house
-
keeping functions, maternal transcription and oogenesis
(Fig.
3; Additional file3: Table S5). Trans-spliced genes,
despite no significant differences in mean expression
levels compared to non-trans-spliced genes, had higher
enrichment for these two methylation marks (Additional
file
2: Fig. S2A). States 3–5 were also more enriched on
operons (a subset of trans-spliced genes) (Fig.
2c). State
3 was distinct from state 4 in its higher enrichment of
H4ac, higher prevalence in UTRs compared to gene bod
-
ies and higher enrichment in regions of RNAPII occu-
pancy and transcribed operons. Interestingly, state 3 was
also enriched at the TSS in a subset of silent genes anno
-
tated with GO terms related to nutrient response (clus-
ter 17, Fig.
3). is may reflect RNAPII pausing in genes
that regulate oocyte production in a nutrient-dependent
manner.
Promoters of operon genes were enriched for pro
-
moter state 5, characterized by H4ac, H3K27ac,
H3K4me2/3 and H3K36 methylations. e typi
-
cally active gene body marks (H3K36me and H4ac) in
these promoters may reflect the uncertainty of TSS
annotations for a subset of trans-spliced operon tran
-
scripts (since a stretch of 5′ sequence is removed from
mRNAs and replaced by the SL RNA). Enrichment
of H3K4me3, the hallmark of active promoters [
54],
was overrepresented at active promoters of operons
compared to silent operon promoters (Fisher’s test p
value=2.044×10
−10
) but not at regions surrounding
the 5′ ends of expressed downstream operon genes. is
provides evidence that these are indeed co-transcribed
Fig. 1 Percentage of genome, autosomes, X- and Y-chromosomes covered by ChIP-enriched regions for each ChIP sample, as well as percentages
of the genome covered by transcriptionally active regions (TARs) (using previously published tiling array data from [32], and their definition of a TAR
as “any stretch of consecutive positive probes in a particular sample”), in the O. dioica ovary and testis
Page 5 of 18
Navratilova
et al. Epigenetics & Chromatin (2017) 10:3
Table 1 Comparative residency of histone PTMs on genomic features of diverse eukaryotes
Histone
PTM
Human
Saccharomyces
cerevisiae
Caenorhabdis
elegans
Drosophila melanogaster
Oikopleura dioica
ovary
Oikopleura dioica
tess
H3K18a
c
Promoters, enhancers
Promoters and 5`end
of genes
Promoters and 5`end of
genes
Promoters, enhancers Promoters and 5`end of genes Promoters and 5`end of genes
H3K27a
c
Promoters, enhancer
s
Promoters and gene
bodies
Promoters, enhancers Promoters, enhancers Promoters, enhancers Promoters and gene bodies
H4ac
Promoters and gene
bodies
Transcripon start
sites
Promoters and gene
bodies
Acve and silent gene
bodies, enriched on X
Promoters and gene bodies
Silent and acve HGP genes, acve
genes on X
H3K4me
1
Promoters, enhancer
s
Transcripon end
sites
Promoters, enhancers Promoters, enhancers Promoters Promoters
H3K4me
2
Promoters, enhancer
s Gene bodies Promoters, enhancers Promoters, enhancers
Acve and silent LGP, acve
HGP
Acve and silent LGP, acve HGP
H3K4me
3
Acve and bivalent
promoters
Promoters Promoters Promoters LGP
Acve and silent LGP, acve HGP,
silent broad regions on Y
H3K9me
1
Gene bodies, esp. acve
but also silent, enhancers
not present
Gene bodies enriched on
X, enhancers
Gene bodies, esp. acve but
also silent
Gene bodies, esp. acve but
also silent
Gene bodies
enriched on X
H3K9me
2
Gene bodies, mobile DNA not present
Silent and acve genes,
mobile DNA
Silent and acve genes,
mobile DNA
Both acve and silent
promoters, esp. LGP
Acve and silent promoters, esp.
LGP, enriched on Y and mobile DNA
H3K9me
3
Silent regions not present
Silent regions, mobile
DNA
Silent regions, mobile DNA Silent regions
Silent regions, enriched on Y and
mobile DNA
H3K27me1
Gene bodies not present
Gene bodies, enriched
on X
Gene bodies Gene bodies, enriched on SL Gene bodies, enriched on X
H3K27me3
Silent, Polycomb-
repressed regions
not present Gene bodies Gene bodies
Acve and silent genes,
enriched on X
Acve and silent genes, enriched on
X
H3K36me1
5`end of highly acve HGP
genes
Gene bodies
Gene bodies
enriched on X
Gene bodies 5`end of HGP genes 5`end of HGP genes
H3K36me2
Gene bodies Gene bodies Gene bodies Gene bodies Gene bodies, enriched on SL Gene bodies
H3K36me3
Gene bodies Gene bodies Gene bodies Gene bodies Gene bodies
Acve and silent gene bodies, peaks
at silent LGC promoters, mobile
DNA, Y
H3K79me1
Gene bodies Gene bodies
Gene bodies, peaks
towards 3`
Gene bodies Gene bodies Acve and silent gene bodies
H3K79me3
Acve and silent
promoters and gene
bodies
Acve gene bodies;
Telomeres
Gene bodies, peaks
towards 5`
Acve gene bodies, peak
downstream of promoter
Acve and silent gene bodies Gene bodies
H3S2
8P
Stress response gene
promoters
NA NA NA
Acve gene bodies, esp. SL,
both silent and expressed LGP
Acve and silent genes, enriched on
X
H4K20me1
5`of acve and silent
genes. X
not present
Gene bodies
enriched on X
Gene bodies Gene bodies Acve and silent gene bodies
H4K20me3
Silent regions not present Silent regions Silent regions
Silent regions, enriched on
HGP genes
Silent regions, enriched on Y
Assignments of features marked by histone PTMs for species other than Oikopleura dioica were based on reference literature as follows: human [12, 13, 46–49];
Saccharomyces cerevisiae [
50–52]; Caenorhabditis elegans [12, 14, 15]; Drosophila melanogaster [8, 10, 12]. Associations of individual histone modication Oikopleura
dioica were assigned based on assessment of enrichment plots shown in Supplemental Figure S1. These plots show the mean signal intensity around gene start
and end sites with genes grouped according to their expression level, the GC content of their promoter, their trans-splicing status and chromosomal locations. We
used the error bars indicating 95% condence intervals on the mean to dene (by the lack of their overlap) relative enrichments between compared gene sets as
signicant. Colors indicate transcriptional activity of features marked by PTMs: green=transcribed; red=repressed; yellow=both active and silent. Esp.=especially;
X=X-chromosome; Y=Y-chromosome; HGP/LGP=high/low GC content promoter; SL genes=genes whose transcripts are subject to trans-splicing of the splice
leader (SL) sequence; N=not analyzed