DEVELOPMENTFTO mediates LINE1 m
6
A demethylation and chromatinregulation in mESCs and mouse development
Jiangbo Wei1,2†,XianbinYu1,2†,LeiYang3,4†,XuelianLiu^3 †,BoyangGao1,2, Boxian Huang^5 , Xiaoyang Dou1,2,
Jun Liu1,2‡, Zhongyu Zou1,2, Xiao-Long Cui1,2, Li-Sheng Zhang1,2, Xingsen Zhao6,7,QinzheLiu1,2,P.CodyHe1,2,
Caraline Sepich-Poore1,2,NicoleZhong1,2,WenqiangLiu^4 ,YanheLi^4 ,XiaochenKou^3 , Yanhong Zhao^3 ,YouWu^3 ,
Xuejun Cheng6,7, Chuan Chen^3 , Yiming An^3 , Xueyang Dong1,2, Huanyu Wang1,2,QiangShu^6 ,ZiyangHao1,2,
Tao Duan^4 , Yu-Ying He^8 ,XuekunLi6,7,ShaorongGao3,4*, Yawei Gao^3 *,ChuanHe1,2*N^6 -methyladenosine (m^6 A) is the most abundant internal modification on mammalian messenger RNA.
It is installed by a writer complex and can be reversed by erasers such as the fat mass and obesity-
associated protein FTO. Despite extensive research, the primary physiological substrates of FTO in
mammalian tissues and development remain elusive. Here, we show that FTO mediates m^6 A
demethylation of long-interspersed element-1 (LINE1) RNA in mouse embryonic stem cells (mESCs),
regulating LINE1 RNA abundance and the local chromatin state, which in turn modulates the
transcription of LINE1-containing genes. FTO-mediated LINE1 RNA m^6 A demethylation also plays
regulatory roles in shaping chromatin state and gene expression during mouse oocyte and embryonic
development. Our results suggest broad effects of LINE1 RNA m^6 A demethylation by FTO in mammals.N
(^6) -methyladenosine (m (^6) A) is the most
prevalent mammalian mRNA internal
modification. It is regulated by writer
and eraser proteins, thus affecting tran-
script fate through reader proteins ( 1 – 3 ).
The fat mass and obesity-associated protein
FTO was the first RNA demethylase shown to
remove mRNA m^6 A( 4 ). FTO is known to be
involved in mammalian development and hu-
man diseases; for example,Fto−/−mice display
severe developmental defects ( 5 , 6 ). Extensive
functional characterizations in human cancer
cells have shown that FTO can localize to the
cell cytoplasm and remove m^6 A from mRNA
transcripts that contribute to cancer progres-
sion ( 7 – 12 ); however, similar activity was not
apparent across mouse and human tissues,
where FTO tends to exhibit nuclear localiza-
tion ( 13 ). Another form of m^6 A, m^6 Am, which
is enriched at the cap of a portion of mRNA
and certain small nuclear RNAs, was also iden-
tified as being a substrate of FTO ( 14 – 16 ).
However, depletion of the cap-m^6 Ammethyl-
transferase PCIF1 only causes mild cellular ef-
fects ( 17 – 19 ) and has negligible impacts on
mouse viability or fertility ( 20 ). These discor-
dant findings indicate that the functionally rel-
evant substrates of FTO during mammalian
development remain elusive.
UnlikePcif1knockout (KO),Mettl3depletion
in mice causes early embryonic lethality ( 21 ).
We recently found that chromatin-associated
regulatory RNAs (carRNAs) can be m^6 A methyl-
ated by METTL3, which regulates chromatin
state and transcription in mouse embryonic
stem cells (mESCs) ( 22 ). Independent reports
confirmed the chromatin-regulating role of
carRNA m^6 A and further showed notable ef-
fects of m^6 A on the expression of endogenous
retroviruses (ERVs) and heterochromatin for-
mation ( 23 – 25 ). Thus, we speculated that a
subset of m^6 A-marked carRNAs could be the
physiological substrates of FTO and that FTO-
mediated m^6 A demethylation may regulate
chromatin state in mammalian tissues and
during development.
Long-Interspersed Element-1 (LINE1) RNA is a
major substrate of FTO in mESCs
To uncover the major substrates of FTO in
these contexts, we derivedFto−/−and control
wild-type (WT) mESCs (fig. S1, A to C). We
quantified m^6 A-level changes of RNAs isolated
from different subcellular fractions between
Fto−/−and WT mESCs (fig. S1D). The m^6 A levels
of RNA isolated from chromatin-associated
and soluble nuclear fractions were increased
(fig. S1, E to G), consistent with the nuclear
localization of FTO (fig. S1H). We performed
m^6 A methylated RNA immunoprecipitation se-
quencing (MeRIP-seq) to examine the chromatin-
associated RNA (caRNA) methylome of WT
andFto−/−mESCs (fig. S1, I to L) and detected
more hypermethylated peaks withFtodeple-
tion (fig. S1M), accompanied by an increased
overall caRNA m^6 A level (fig. S1N).
We annotated carRNAs as promoter-associated
RNA, enhancer RNA, and RNA transcribed
from transposable elements (repeat RNA) ( 22 ).
Most carRNAs exhibited more hypermethyl-
ated m^6 A peaks (fig. S2A) and elevated m^6 A
levels (fig. S2B) uponFto KO. Compared with
other carRNAs,Fto depletion led to more pro-
nounced hypermethylation of repeat RNAs
(Fig. 1A), more down-regulated m^6 A-marked
repeat RNAs (fig. S2C), and greater down-
regulation of hypermethylated repeat RNAs
(Fig. 1B). UponFtoKO, carRNA expression
changes negatively correlated with m^6 A-level
changes (fig. S2D), with repeat RNAs showing
the strongest correlation between transcript
down-regulation and m^6 A hypermethylation
(fig. S2, E and F).
Among m^6 A-marked repeat RNAs, LINE1
RNA emerged as a major substrate of FTO in
mESCs. It showed the highest number of hy-
permethylated peaks, the most increased m^6 A
levels, the most reduced abundance (Fig. 1C),
and a reduced overall expression (fig. S3, A
and B) uponFto KO. LINE1 RNA mainly asso-
ciates with chromatin in mESCs (fig. S3, C and
D) ( 26 , 27 ). We observed colocalization of
LINE1 RNA and FTO protein (fig. S3D) and
validated the binding of LINE1 RNA by FTO
(fig. S3E). The m^6 A level and expression of
whole-cell LINE1 RNA exhibited changes sim-
ilar to those on chromatin uponFtoKO (fig.
S3, F and G), accompanied by reduced L1ORF1p
expression (fig. S3H). Treating WT mESCs with
an FTO inhibitor ( 9 ) recapitulated the effect of
Fto KO on LINE1 RNA (fig. S3I).
UponFtoKO, we observed that greater
m^6 A level increases correlated with greater
LINE1 RNA abundance reductions (Fig. 1D).
Across published mouse and human tissue
m^6 Amethylomes( 13 ), LINE1 RNA m^6 A level
also negatively correlated with its expression,
and high FTO expression was associated with
low m^6 A level and high LINE1 RNA expres-
sion (fig. S4), supporting LINE1 RNA as a sub-
strate of FTO in most tissues.
LINE elements are one of the most abun-
dant retrotransposons in mammalian genomes,
and LINE1 RNA is known to play critical
roles during mammalian early development
( 26 , 27 ). In mESCs, LINE1 RNA can function as
a nuclear RNA scaffold fortrans-regulation,
with LINE1 RNA knockdown by morpholino
antisense oligo (ASO) causing activated two-
cell (2C) program and repressed ESC-high
genes ( 27 , 28 ). Fto KO largely recapitulated
these transcriptomic changes (Fig. 1E), with
RESEARCH
Weiet al., Science 376 , 968–973 (2022) 27 May 2022 1of6
(^1) Department of Chemistry, Department of Biochemistry and
Molecular Biology, and Institute for Biophysical Dynamics,
The University of Chicago, Chicago, IL 60637, USA.^2 Howard
Hughes Medical Institute, The University of Chicago,
Chicago, Chicago, IL 60637, USA.^3 Institute for Regenerative
Medicine, Shanghai East Hospital, Shanghai Key Laboratory
of Signaling and Disease Research, Frontier Science Center
for Stem Cell Research, School of Life Sciences and
Technology, Tongji University, Shanghai 200120, China.
(^4) Clinical and Translation Research Center of Shanghai First
Maternity & Infant Hospital, Shanghai Key Laboratory of
Signaling and Disease Research, Frontier Science Center for
Stem Cell Research, School of Life Sciences and Technology,
Tongji University, Shanghai 200092, China.^5 State Key
Laboratory of Reproductive Medicine, Suzhou Affiliated
Hospital of Nanjing Medical University, Suzhou Municipal
Hospital, Gusu School, Nanjing Medical University, Suzhou
215002, China.^6 The Children's Hospital, School of Medicine,
Zhejiang University, National Clinical Research Center for
Child Health, Hangzhou 310052, China.^7 The Institute of
Translational Medicine, School of Medicine, Zhejiang
University, Hangzhou 310029, China.^8 Department of
Medicine, Section of Dermatology, University of Chicago,
Chicago, IL 60637, USA.
*Corresponding author. Email: [email protected] (C.H.);
[email protected] (Y.G.); [email protected] (S.G.).
†These authors contributed equally to this work.
‡Present address: State Key Laboratory of Protein and Plant Gene
Research, School of Life Sciences, Peking-Tsinghua Center for Life
Sciences, Peking University, Beijing 100871, China.