Poised chromatin and bivalent domains facilitate the mitosis-to-meiosis transition in the male germline

Genome-wide transcriptional change during the mitosis-to-meiosis transition

To assess genome-wide gene expression changes during the mitosis-to-meiosis transition
in spermatogenesis, we have recently performed RNA-seq analysis at three representative
time points before, during, and after meiosis (Fig. 1a) 23]. Because purified spermatogonia consist of a heterogeneous cell population, it is
difficult to obtain a large number of homogenous cells for ChIP-seq analysis. Because
of this, we used cultured GS cells 24] as a representative stage of the mitotic phase of spermatogenesis. Our cultured GS
cells exhibited a gene expression profile similar to THY1+ undifferentiated spermatogonia
cells purified from mouse testes (Fig. 1b), confirming that the GS cells recapitulate undifferentiated spermatogonia in vivo. For meiotic and postmeiotic stages, we used purified PS and RS, respectively (Fig. 1a). To identify the unique features of germline transcriptomes during spermatogenesis,
we compared RNA-seq data from these cell types to the published RNA-seq data obtained
from THY1+ undifferentiated spermatogonia, ES cells, somatic cells, and tissues (see
Methods section). A heatmap analysis of 17,213 genes expressed (reads per kilobase
per million (RPKM) 3) in at least one condition revealed that a significant transcriptional
change occurs during the mitosis-to-meiosis transition, and that the transcriptomes
of PS and RS are largely different from that of the mitotic phase of spermatogenesis
as well as other somatic cells and tissues (Fig. 1b). Two distinct features that are common in PS and RS transcriptomes are activation
of late spermatogenesis genes, as previously described 17], 25], 26], and suppression of a large group of genes that are commonly expressed in the somatic
phase and spermatogenesis progenitor cells. Herein we will refer to the latter group
of genes as somatic/progenitor genes. This analysis suggests that there is a massive
transcriptional change at the mitosis-to-meiosis transition during differentiation
of mitotic spermatogonia into meiotic spermatocytes, and that transcriptomes during
the late stages of spermatogenesis are significantly different from that of somatic
lineages 23].

Fig. 1. The global transcriptome changes during the late stages of male germline. a Schematic of spermatogenesis. In this study, germline stem (GS) cells were used as
the representative stage of the stem cell phase. X chromosomes are depicted in green
and the Y chromosomes are depicted in orange. Barred chromosomes represent suppressed
transcription. b A heatmap showing gene expression patterns among several germ cells versus somatic
cells. All 17,213 genes that showed more than 3 RPKM in at least one cell type are
shown. RNA-seq data were obtained from published studies as described in the Methods
section. c Flow chart of grouping of each class of spermatogenesis genes, degree of overlaps,
and their expression heatmaps. d Gene ontology analysis of each class of spermatogenesis genes. e Summary table of each class of spermatogenesis genes. f Enrichment of RS active genes on the X chromosome. *P 2.2e-16, chi-square test. ES, embryonic stem cells; GS, germline stem cells; MEF,
mouse embryonic fibroblasts; PS, pachytene spermatocytes; RPKM, reads per kilobase
per million; RS, round spermatids; THY1+, THY1+ undifferentiated spermatogonia

Unique features of transcriptomes during meiosis and postmeiosis

To define lists of somatic/progenitor genes and late spermatogenesis genes, the transcriptomes
were separated into gene groups according to the following criteria (Fig. 1c,d,e): (1) four-fold change in pairwise comparisons between GS and PS, GS and RS, or PS
and RS; (2) adjusted P value ?0.05 for significance of differential expression among the cell types; and
(3) RPKM in at least one cell type ?5. Because sex chromosomes are subject to unique
epigenetic programming during meiosis and postmeiosis, we analyzed expression profiles
of genes located on autosomes and sex chromosomes separately.

On autosomes, we found that 2,826 genes were commonly active in PS and RS (abbreviated
as PS/RS active genes hereafter), and further defined the list of PS- or RS-specific
active and inactive genes (abbreviated as PS active, PS inactive, RS active, RS inactive,
respectively; Fig. 1c). Gene ontology (GO) enrichment analysis revealed that male reproduction-associated
genes are significantly enriched in PS/RS active, PS active genes, and RS active genes
(Fig. 1d). On the other hand, 2,636 autosomal genes were commonly inactive in PS and RS (abbreviated
as PS/RS inactive genes hereafter; Fig. 1c). GO enrichment analysis reveals that the PS/RS inactive gene set is enriched with
genes involved in somatic functions such as blood vessel development and tissue morphogenesis,
suggesting that they are presumably dispensable during the late stages of spermatogenesis
(Fig. 1d). We also identified 1,044 constitutively active genes and 8,910 constitutively inactive
genes on autosomes in all three cell types (see Methods).

Because of the paucity of Y-linked genes, we focused on the X chromosome for detailed
analysis. On the X chromosomes, 225 genes were significantly repressed in both PS
and RS, consistent with sex chromosome inactivation (Fig. 1c), and the GO analysis demonstrated that this group of genes was highly associated
with chromatin modification (Fig. 1d). On the other hand, 102 X-linked genes escaped sex chromosome inactivation and were
predominantly expressed in RS. Interestingly, this group of genes is specifically
expressed in the germline (Additional file 1: Figure S1), and is disproportionately
enriched on the X chromosome (102/994, 10.3 %, P 2.2e-16, chi-square test) when compared with those on the autosomes (Fig. 1f). Additionally, we identified 500 X-linked constitutively inactive genes in all three
cell types (see Methods). Taken together, our RNA-seq data are in accord with previous
gene expression studies 12], 17], 21], 25]–27], and these results confirm distinct regulation between autosomes and the X chromosome
in spermatogenesis.

Distinct epigenetic landscapes between autosomes and the X chromosome during spermatogenesis

To elucidate the epigenetic principles of mouse spermatogenesis, we performed ChIP-seq
chromatin profiling in GS, PS, and RS cells. In particular, we examined the distribution
of RNA polymerase II (RNAPII) and representative active epigenetic modifications such
as H3K4me2, H3K4me3, H4K8ac, H4K16ac, and histone lysine crotonylation (Kcr). In addition
to active modification, we examined representative silent modifications H3K27me3 and
H3K9me2.

To account for the distinct regulation of gene expression from autosomes and the X
chromosome during spermatogenesis, we first compared the average tag density (ATD)
profiles of these modifications around transcription start sites (TSSs) between all
autosomal genes and all X chromosome-linked genes during spermatogenesis (Fig. 2a,b,c). Based on the limited availability of annotated sequences on the Y chromosome, Y
chromosome data were excluded from our analysis hereafter. Consistent with the phenomena
of almost complete silencing in MSCI and postmeiotic RS-specific escape gene activation,
RNAPII was largely depleted from the X chromosome in PS and slightly increased in
RS (Fig. 2b,c). Although distribution of H3K4me2 and H3K4me3 was comparable between autosomes and
X chromosome in GS (Fig. 2a), H3K4me2 was slightly enriched on the X chromosome in PS consistent with the cytological
localization that H3K4me2 starts to accumulate on the sex chromosomes during the late
pachytene stage 21], 28], whereas H3K4me3 was enriched on autosomes at this stage (Fig. 2b). H4K8ac, Kcr, and H4K16ac were also enriched on autosomes in PS, and, curiously,
H4K16ac was continuously low on the X chromosomes compared to autosomes in all three
cell types (Additional file 1: Figure S2). In contrast to these active modifications,
H3K9me2 was slightly enriched on the X chromosome compared to autosomes in GS cells
(Fig. 2a). In PS and RS, consistent with cytological localization 17], H3K27me3 was largely depleted from the X chromosome, whereas H3K9me2 was enriched
there (Fig. 2b,c). In contrast, H3K27me3 accumulated on autosomes in PS and was highly enriched on
autosomal TSSs in RS. Therefore, these results suggest that autosomes and the X chromosomes
are subject to distinct modes of epigenetic regulation during spermatogenesis. Because
of the distinct regulation, we proceeded to analyze the epigenomes of autosomes and
sex chromosomes separately.

Fig. 2. Distinct regulation between the X chromosome and autosomes during the late stages
of male germline. a GS, b PS, and c RS are shown. Average tag density (ATD) of each histone mark was compared between
all autosomal genes and all X-linked genes. ATD, average tag density; GS, germline
stem cells; PS, pachytene spermatocytes; RS, round spermatids; ChIP-seq, chromatin
immunoprecipitation sequencing

Autosomal late spermatogenesis genes are silent, but poised in GS cells for later
activation in PS

We first focused on the events on the autosomes and investigated whether the genes
specifically regulated during spermatogenesis undergo epigenetic changes during differentiation.
Heatmap analyses revealed that, in GS cells, H3K4me2 and H3K4me3 were highly accumulated
on the active genes (both constitutively active genes and PS/RS inactive genes) (Fig. 3a). ATD analysis revealed that RNAPII and H3K4me3 were highly accumulated on TSSs of
these genes, and that H3K4me2 localization is broader than localization of RNAPII
and H3K4me3, and accumulated on the region surrounding TSSs of these genes (Fig. 3b). H4K8ac and Kcr were also accumulated around TSSs of constitutively active genes,
but were less intense on the PS/RS inactive genes that are highly expressed in GS
cells (Additional file 1: Figure S3), suggesting that, in GS cells, gene activation is distinctly regulated
between PS/RS inactive genes and constitutively active genes. Consistent with this
notion, H4K16ac accumulated on TSSs of constitutively active genes, but not on the
TSSs of PS/RS inactive genes (Additional file 1: Figure S3).

Fig. 3. Autosomal late spermatogenesis genes are poised in GS cells for activation at PS.
a A heatmap showing distribution of histone marks in GS cells. Tag density around TSS
(±5 kb) is shown. b ATD of active marks in representative groups in GS cells. c ATD of active marks at the genes activated in later stages. d ATD of silent marks in representative groups in GS cells. ATD, average tag density;
GS, germline stem cells; PS, pachytene spermatocytes; TSS, transcription start site

Notably, RNAPII, H3K4me2, and H3K4me3 were largely present on PS/RS active genes even
though these genes were silent in GS cells (Fig. 3a,b). Notably, ATD of these modifications on PS/RS active genes overlapped with that
of PS active genes in GS cells, but RS active genes did not exhibit enrichment of
active modifications in GS cells (Fig. 3c). Further, the H3K27me3 level of PS/RS active genes was lower than that of constitutively
inactive genes, whereas H3K9me2 did not show this reduction (Fig. 3d). These results suggest that the autosomal genes activated in PS are already epigenetically
poised by deposition of active modifications and RNAPII, as well as by reduction of
H3K27me3, for future activation. Similar epigenetic gene poising was observed in T
cells for genes that are inducible during T cell activation and in other systems 29], 30]. Taken together, we conclude that activation of autosomal late spermatogenesis genes
in PS is preprogrammed in GS cells.

H3K4me2 remained on somatic/progenitor genes after gene inactivation in PS

Next, we sought to examine how the meiosis-specific transcriptome is regulated for
autosomes during the PS stage. At the PS active gene synaptonemal complex protein
3 (Sycp3), active modifications such as H3K4me3, H4K8ac, H4K16ac, and Kcr were highly accumulated
at the TSS, and H3K4me2 exhibited a broader peak of enrichment near the TSS (Fig. 4a). These profiles of active modifications were common among PS/RS active genes, PS
active genes, and constitutively active genes, suggesting that PS-specific gene activation
is regulated by a similar epigenetic mechanism with that of constitutively active
genes in PS (Fig. 4b, Additional file 1: Figure S4). On the other hand, genes inactivated in PS such as vimentin (Vim) exhibited a distinct feature compared to the constitutively inactive genes: H3K4me2
largely remained in PS at PS/RS inactive genes and PS inactive genes although RNAPII
and H3K4me3 were largely depleted (Fig. 4a,c). In addition, the silent modification H3K27me3, but not H3K9me2, was highly enriched
at PS/RS inactive genes (Fig. 4a,e). These results suggest that bivalent chromatin signatures such as H3K27me3 with
H3K4me2 are associated with PS/RS inactive genes in PS.

Fig. 4. Active marks remain after the inactivation of autosomal somatic/progenitor genes in
PS. a Distribution of histone marks around PS/RS inactive Vim gene locus and PS active Sycp3 gene locus in PS. b ATD of active marks at the active genes in PS. c ATD of active marks at the silent genes in PS. d ATD of active marks in PS. These genes are activated in RS. e ATD of each silent mark in representative groups in PS. ATD, average tag density;
PS, pachytene spermatocytes; RS, round spermatids

We further investigated whether there is any epigenetic signature that predicts RS-specific
gene activation in PS. Both H3K4me2 and Kcr were broadly enriched at RS active genes
in PS, but H3K4me3 did not show enrichment compared to that of constitutively inactive
genes (Fig. 4d, Additional file 1: Figure S4). Thus, on RS active genes, H3K4me2 and Kcr, but not H3K4me3, are already
established in PS for future activation in RS.

Unique epigenetic landscape of late spermatogenesis genes on autosomes in RS

We next investigated the epigenetic signature of autosomes in postmeiotic RS specifically
focusing on the RS active genes. Interestingly, active epigenetic modifications were
present not just at the promoters of the RS active genes, such as spermatogenesis-associated
20 (Spata20), but were spread out broadly from TSSs to transcription end sites (TESs) (Fig. 5a). In the heatmap and ATP analysis, H3K4me2 was highly enriched in the gene bodies
but less enriched at the TSSs of RS active genes compared to constitutively active
genes (Fig. 5b,c). Contrastingly, H3K4me2, H4K8ac, and Kcr were highly enriched on the TSSs of RS
active genes even compared to constitutively active genes (Fig. 5c), while H4K16ac did not show such a difference (Additional file 1: Figure S5). These results reveal the unique epigenetic landscape of late spermatogenesis
genes on autosomes in RS.

Fig. 5. Epigenetic profiles for RS-specific activation and gene poising on autosomal somatic/progenitor
genes in RS. a Distribution of histone marks on PS/RS inactive Vim gene locus and RS active Spata20 gene locus in RS. b A heatmap showing distribution of histone marks in RS. Density around TSS (±5 kb)
is shown. c ATD of active marks at the active genes in RS. For H3K4me2, ATD on gene bodies from
TSS to TES and ±5 kb regions were analyzed. d ATD of active marks at the silent genes in RS. e ATD of silent marks in representative groups in RS. f Expression of each gene set in GS, PS, RS, and ES. ATD, average tag density; ES,
embryonic stem cells; GS, germline stem cells; PS, pachytene spermatocytes; RS, round
spermatids; TSS, transcription start site

Autosomal somatic/progenitor genes are silent in RS via the deposition of bivalent
epigenetic marks

We next examined the epigenetic signature of somatic/progenitor genes inactivated
in RS. H3K4me2 remained around the TSSs of PS/RS inactive genes such as Vim (Fig. 5a). RNAPII and H3K4me2/3 were present on both RS inactive genes and PS/RS inactive
genes (Fig. 5b,d). Importantly, H3K27me3 was deposited at the TSS of PS/RS inactive genes in RS compared
to PS/RS inactive genes in PS, while H3K9me2 did not exhibit this feature (Figs. 4e and 5a,e). This suggests that the deposition of bivalent marks at the TSS of somatic/progenitor
genes occurred in the transition between PS and RS without expression changes. To
determine whether somatic/progenitor genes are poised for activation after fertilization,
we compared their gene expression during spermatogenesis and in ES cells, which represent
the post-fertilization inner cell mass of blastocysts. Consistent with our global
expression analysis (Fig. 1b), somatic/progenitor genes (RS and PS/RS inactive genes) are expressed in ES cells,
whereas late spermatogenesis genes (PS/RS and RS active genes) are not expressed in
ES cells (Fig. 5f). Therefore, these results suggest that the somatic/progenitor program is suppressed
in late spermatogenesis, but poised for activation after fertilization. Importantly,
in contrast to the class of bivalent domains on developmental promoters that are consistently
silent throughout the male germline 11], 13], our analysis reveals a new class of bivalent genes that are expressed in ES and
GS cells but are temporarily suppressed in PS and RS. This suggests a novel function
of bivalent domains: suppression of the somatic/progenitor program during late spermatogenesis.

X-linked genes subject to MSCI and postmeiotic silencing are poised for activation
after fertilization without the formation of bivalent domains

Because sex chromosomes undergo MSCI and are regulated separately from autosomes in
spermatogenesis (Fig. 2), we investigated the epigenetic landscape of the X chromosomes separately from that
of autosomes. In Fig. 1, we classified X-linked genes into two major categories: the major group consists
of 225 genes that are active in GS but are subject to both MSCI and postmeiotic silencing
(PS/RS inactive); the other group consists of 102 genes that are not expressed in
GS, but are highly activated in RS (RS active). This latter group is also referred
to as escape genes because they escape chromosome-wide postmeiotic silencing in RS
and are activated 20], 21].

First, we examined the developmental changes in the epigenetic landscape of representative
genes from each group during spermatogenesis. The PS/RS inactive gene Timp1 was marked with H3K4me2 and H3K4me3 in GS, where it is transcribed (Fig. 6a). Upon entry into meiosis, Timp1 was silenced by MSCI, RNAPII disappeared from the TSS, and the H3K9me2 level increased.
However, H3K4me2 and other active modifications remained on the Timp1 locus, albeit at a lower level in PS. In agreement with the Timp1 locus, H3K4me2 remained near the TSS of PS/RS inactive X-linked genes in PS despite
the disappearance of RNAPII (Fig. 6c,d). These results suggest that MSCI and postmeiotic silencing are established without
complete removal of active modifications.

Fig. 6. Epigenetic profile of the X chromosome during the late stages of the male germline.
a Binding peaks of histone marks on PS/RS inactive Timp1 gene locus in GS, PS, and RS. b Binding peaks of histone marks on RS active Akap4 gene locus in GS, PS, and RS. ATD of histone marks in representative groups in c GS, d PS, and e RS. For H3K4me2, ATD on gene bodies from TSS to TES and ±5 kb regions were analyzed.
f Average RPKM of each group in GS, PS, RS, and ES. g, h ATD profiles of H3K4me2 on gene bodies and Kcr on TSS are compared between WT and
Rnf8 KO. Wilcoxon rank sum test was performed for read counts in the highlighted area
(H3K4me2: ?1 kb from TSS to TES; Kcr: ?500 bp to +500 bp from TSS, *P 0.05, **P 0.001). ATD, average tag density; ES, embryonic stem cells; GS, germline stem cells;
Kcr, lysine crotonylation; KO, knockout; PS, pachytene spermatocytes; RS, round spermatids;
RPKM, reads per kilobase per million; TES, transcription end site; TSS, transcription
start site; WT, wild-type

In RS, H3K4me2 and H4K16ac are still present on the Timp1 locus (Fig. 6a). Paradoxically, the H3K4me2 level at PS/RS inactive X-linked genes was higher than
that on RS active X-linked genes at the TSS, and PS/RS inactive X-linked genes had
levels of RNAPII and H3K4me3 comparable to that of RS active X-linked genes (Fig. 6e). Importantly, this group of genes was highly expressed in ES cells (Fig. 6f). These results suggest that PS/RS inactive X-linked genes are poised for activation
after fertilization, as is the case with PS/RS inactive autosomal genes. However,
H3K27me3 was largely depleted from the X chromosome in PS and RS (Fig. 2, Additional file 1: Figure S6). Therefore, PS/RS inactive X-linked genes are poised in RS without the
formation of typical bivalent domains.

Distinct epigenetic features underlie RS-specific gene activation on the X chromosome

As described above, we found that PS/RS active autosomal genes are poised in GS cells
for activation during meiosis (Fig. 3). Unlike autosomal genes, X-linked genes that are expressed at later stages possess
only a low level of active modifications in GS cells (Fig. 6c, Additional file 1: Figure S6). This result suggests that the X-linked genes are not poised in GS cells
for activation during meiosis, which is in accordance with the existence of MSCI and
which supports the notion that autosomes and the X chromosome are distinctly regulated
in GS cells prior to entry into meiosis.

Next, we examined changes in the epigenetic landscape of RS active X-linked genes
during spermatogenesis. On the RS active X-linked gene Akap4, which regulates sperm motility, active epigenetic modifications were largely absent
in GS cells (Fig. 6b). Upon entry into meiosis, active modifications started to accumulate broadly on
the Akap4 locus with the induction of modest transcription. In RS, H3K4me3, Kcr, and RNAPII
were highly accumulated around the TSS, and Akap4 was robustly expressed. Consistent with this, in ATD analysis, Kcr started to accumulate
on the TSSs of RS active X-linked genes in PS (Fig. 6d), and reached a higher level in RS (Fig. 6e). H3K4me2 became enriched on the gene bodies of RS active X-linked genes (Fig. 6e), and RS active X-linked genes were not expressed in ES cells (Fig. 6f). Unlike RS active autosomal genes (Fig. 5c), RS active X-linked genes did not gain a high level of H4K8ac accumulation (Fig. 6e). Therefore, H4K8ac is specifically associated with RS active autosomal genes.

A unique feature of the RS active X-linked genes is that this group of genes escapes
postmeiotic silencing of the sex chromosomes. To determine how this group can escape
the chromosome-wide silencing of the sex chromosomes, we investigated the profiles
of H3K9me2 on the X chromosome. H3K9me2 was consistently high in both groups of X-linked
genes compared to autosomal genes and did not exhibit a difference between active
and inactive genes in RS, whereas H3K27me3 levels were low (Additional file 1: Figure S6). This result suggests that RS active X-linked gene escape is activated
from silent X chromosomes without removing H3K9me2 and instead depends on unique profiles
of active modifications.

Previously, we have shown that RNF8 is required for the activation of a subset of
escape genes from postmeiotic silencing 21]. To elucidate the regulatory mechanism underlying expression of RS active X-linked
genes, we examined how unique profiles of active modifications are established on
the X chromosome using the testes of Rnf8 knockout (KO) mice. Both H3K4me2 and Kcr accumulate on gene bodies and TSSs of RNF8-dependent
escape genes (identified in 21]) in an RNF8-dependent manner in PS and RS (Fig. 6g,h). These results further support the conclusion that the unique localization of H3K4me2
and Kcr is important for RS-specific gene activation from the X chromosome.