論文の公開元へTranscription of MERVL retrotransposons is required for preimplantation embryo development (本文)
概要
nature genetics
Article
https://doi.org/10.1038/s41588-023-01324-y
Transcription of MERVL retrotransposons
is required for preimplantation embryo
development
Received: 22 March 2022
Accepted: 26 January 2023
Published online: 2 March 2023
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Akihiko Sakashita1,3, Tomohiro Kitano 1,3, Hirotsugu Ishizu1, Youjia Guo 1,
Harumi Masuda1, Masaru Ariura1, Kensaku Murano 1 & Haruhiko Siomi 1,2
Zygotic genome activation (ZGA) is a critical postfertilization step that
promotes totipotency and allows different cell fates to emerge in the
developing embryo. MERVL (murine endogenous retrovirus-L) is transiently
upregulated at the two-cell stage during ZGA. Although MERVL expression
is widely used as a marker of totipotency, the role of this retrotransposon
in mouse embryogenesis remains elusive. Here, we show that full-length
MERVL transcripts, but not encoded retroviral proteins, are essential for
accurate regulation of the host transcriptome and chromatin state during
preimplantation development. Both knockdown and CRISPRi-based
repression of MERVL result in embryonic lethality due to defects in
differentiation and genomic stability. Furthermore, transcriptome and
epigenome analysis revealed that loss of MERVL transcripts led to retention
of an accessible chromatin state at, and aberrant expression of, a subset of
two-cell-specific genes. Taken together, our results suggest a model in which
an endogenous retrovirus plays a key role in regulating host cell fate potential.
Fertilization and early preimplantation development are processes in
which unipotent gametes unite and acquire totipotency (Fig. 1a). After
fertilization, embryos undergo zygotic genome activation (ZGA), a
process that is widely conserved in vertebrates1–3. ZGA involves a transcriptional burst of hundreds to thousands of two-cell-specific genes.
At this point, gene expression switches from a maternal to zygotic program2,4. ZGA occurs in two distinct waves called minor and major ZGA5.
In mice, minor ZGA occurs from S phase in the zygote to G1 phase in the
early two-cell stage embryo, whereas the major wave occurs during the
second round of DNA replication at the middle-to-late two-cell stage6,7.
Both waves of ZGA are critical for the embryo to acquire developmental competence6,8. However, the molecular events that drive ZGA and
lead to acquisition of totipotency and developmental competence are
still enigmatic.
Approximately 40% of the mouse genome is occupied by transposable elements (TEs), mobile genetic elements of which ~10% are endogenous retrovirus (ERV)9. Notably, the expression of murine endogenous
retrovirus with leucine transfer RNA primer binding site (MERVL) is
specifically activated at the two-cell stage concomitant with ZGA10–12.
Recently, the transcription factor DUX, which is expressed during
minor ZGA, was documented as an upstream regulator that activates
two-cell genes and MERVL13–15. Furthermore, the MERVL long terminal
repeat (LTR) promoter drives a subset of two-cell genes and generates
chimeric transcripts with the host genes12. The above findings suggest
that DUX/MERVL may activate an early transcriptional network that is
required for ZGA and totipotency.
In 2012, Macfarlan et al. found that a rare transient cell population
(~1%) in mouse embryonic stem cell (ESC) and induced pluripotent stem
cell cultures expresses high levels of MERVL and two-cell genes without
expression of pluripotent inner cell mass (ICM) maker genes, such as
Pou5f1 (also known as Oct4), Sox2 and Nanog12. MERVL expression has
been used as a marker for totipotent cells, as MERVL+ cells can commit
to both embryonic and extraembryonic lineages after injection into
recipient embryos at the eight-cell and morula stages12,16,17.
Department of Molecular Biology, Keio University School of Medicine, Tokyo, Japan. 2Human Biology Microbiome Quantum Research Center
(WPI-Bio2Q), Keio University, Tokyo, Japan. 3These authors contributed equally: Akihiko Sakashita, Tomohiro Kitano. e-mail: awa403@keio.jp
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Nature Genetics | Volume 55 | March 2023 | 484–495
484
Article
https://doi.org/10.1038/s41588-023-01324-y
a
Preimplantation development
Fertilization
Germ cells
0.5 dpc
1.5 dpc
Zygote
2-cell
2.5 dpc
4-cell
3.5 dpc
8-cell
Morula
4.5 dpc
Blastocyst
TE
Differentiated
Totipotent
Lineage commitment
b
ICM
Pluripotent
c
MERVL-int transcripts
MT2_Mm transcripts
6
4-cell
Early 2-cell
8-cell
Middle 2-cell
Late 2-cell
Morula
Blastocyst
Log2 RPKM
Log2 RPKM
6
Zygote
0
0
Preimplantation development
Preimplantation development
d
MERVL transcript
Early
2-cell
Middle
2-cell
Zygote
(PN stage)
Early
2-cell
Middle
2-cell
Late
2-cell
4-cell
8-cell~
morula
4-cell
8-cell~
morula
MERGE with DAPI
Zygote
(PN stage)
e
MERVL-Gag protein
MERGE with DAPI
Late
2-cell
Fig. 1 | MERVL RNA exhibits dynamic nuclear-cytoplasmic expression
during early stages of mouse preimplantation development. a, Schematic
of mouse preimplantation development. Totipotency is restricted to earlystage development (that is, zygote and two-cell stages). Blastomeres gradually
transition to a pluripotent state from the four-cell stage onward and develop
into a blastocyst consisting of inner cell mass (ICM) and trophectoderm (TE)
before implantation in the uterus at 4.5-days postcoitum (dpc). b,c, Violin plots
showing the log2-transformed reads per kilobase of exon per million reads
mapped (log2RPKM) values of MERVL-int (b) and its LTR promoter, MT2_Mm (c)
during preimplantation development. Each plot encompasses box plot; central
bars represent medians, box edges indicate 50% of data points and the whiskers
show 90% of data points. d, Representative images of smFISH for MERVL RNA
with 4,6-diamidino-2-phenylindole (DAPI) counterstain during preimplantation
development, from four independent experiments. Scale bars, 20 µm. ♀, female
pronucleus (PN); ♂, male PN. e, Representative images of immunofluorescence
staining for MERVL-Gag protein with DAPI counterstain during preimplantation
development, from six independent experiments. Scale bars, 20 µm. ♀, female
PN; ♂, male PN. Data for panels in b and c are available as source data.
Despite the above findings, the function of MERVL itself remains
unclear. Here, we overcome technical limitations in interrogating TE
functions and analyze the role of MERVL in preimplantation development. We found that depletion of MERVL transcripts resulted in
embryonic lethality due to defects in early lineage specification and
genome stability, demonstrating that MERVL is essential for mouse
preimplantation development.
from each blastomere at eight representative stages of preimplantation
development18 (Fig. 1a). To define regions of nonredundant MERVLs in
mouse genome, we used RepeatMasker to annotate the genome for
unique interspersed internal regions of MERVL (MERVL-int, n = 1,426)
and LTR promoters of the MERVL (MT2_Mm, n = 2,366). The expression
of MERVL and its LTR promoter culminated in the middle of the two-cell
stage and then gradually decreased until blastocyst stage (Fig. 1b,c).
We also set out to investigate the expression and localization of
MERVL transcripts in preimplantation embryos using single-molecule
fluorescence in situ hybridization (smFISH). Interestingly, smFISH
revealed that MERVL expression is detectable in the nuclei from zygotes
and early two-cell stage embryos in which polyadenylated MERVL
Results
MERVL exhibits distinct localization in mouse embryos
To understand the dynamics of MERVL expression, we first analyzed
publicly available single-cell RNA-sequencing (scRNA-seq) datasets
Nature Genetics | Volume 55 | March 2023 | 484–495
485
Article
mRNA cannot be detected (Fig. 1b,d). Afterwards, MERVL RNA gradually translocated from the nucleus at middle two-cell stage onward
and was highly restricted to the cytoplasm by late two-cell stage
(Fig. 1d). These changes in MERVL transcript localization were consistent with increased MERVL protein levels during the middle two-cell
stage (Fig. 1e). These observations raise the possibility that nuclear
MERVL transcript has distinct roles in gene regulation in the early
stages of preimplantation development compared to cytoplasmic
MERVL transcript, leading us to investigate MERVL function further.
MERVL-KD results in embryonic lethality
Inconsistencies regarding the early embryonic phenotypes of MERVL
knockdown (KD) in previous studies11,19,20, led us to re-examine the
KD effects of MERVL on preimplantation development. To this end,
we developed specific antisense oligonucleotides (ASOs) that target interspersed MERVL copies (Fig. 2a and Extended Data Fig. 1a,b).
After predicting the genome-wide target sites of individual ASOs using
BLASTn, we confirmed that 46.9% (n = 669/1,426) of MERVL copies
were targeted by at least one ASO with up to two mismatches allowed
(Extended Data Fig. 1c). Subsequently, we confirmed that our ASO
sequences efficiently targeted full-length MERVL (≥5 kb, n = 377/556,
67.8%), by combining three independent anti-MERVL ASOs (Extended
Data Fig. 1d). We experimentally validated the KD efficiency of each
ASO using a recently developed ESC-based in vitro system (Extended
Data Fig. 1e and Methods)21 in which MERVL expression was drastically reduced at both the mRNA and protein levels (Extended Data
Fig. 1f–h). Injection of each ASO into the male pronucleus of zygotes
also leads to substantial reduction of MERVL RNA signal at the late
two-cell stage (Extended Data Fig. 2a). Because we noted that cocktail
of three independent ASOs increased MERVL-KD efficiency (Fig. 2b
and Extended Data Fig. 2), mixed ASOs (1:1:1 = 20 µM) were used in
subsequent experiments.
Next, we monitored the effects of MERVL-KD using ASOs on preimplantation development (Fig. 2c,d). MERVL-KD embryos displayed a
significant developmental delay from 2.5 dpc (Fig. 2d). ...
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