Science - USA (2022-05-27)

RNA were also reversed in the rescued em-
bryos (fig. S18, Q and R).

Discussion

In this work, we have shown functionally rel-
evant substrates and proposed mechanisms
of RNA m^6 A demethylation through FTO in
mammalian development. Our findings sup-
port LINE1 RNA as a major substrate of FTO
in mESCs, although FTO may additionally me-
diate m^6 A demethylation of other carRNAs
to affect gene expression (supplementary text
and figs. S19 to S21). In contrast to certain
cancer cells in which FTO can be hijacked to
mediate mRNA m^6 A demethylation ( 7 – 12 ),
which may dominate chromatin state regula-
tion ( 34 ) (supplementary text and fig. S22), we
foundthatFTO-mediatedm^6 A demethylation
maintained LINE1 RNA abundance in mESCs,
which contributes to promoting local chroma-
tin openness and activating LINE1-containing
genes. We further showed that the FTO-LINE1
RNA axis is functionally relevant in mouse
oocyte and embryonic development. How FTO
achieves target selectivity in different cellular

contexts still needs future investigation. In

addition to m^6 A, RNA 5-methylcytosine oxi-

dation is also known to affect transcription

of ERVL and ERVL-associated genes in mESCs

( 35 ), suggesting a potential widespread pres-

ence of regulation through retrotransposon

RNA modifications ( 36 ).

Materials and methods summary

Fto−/−and control WT mESCs were derived

from the inner cell mass of E3.5 blastocysts. m^6 A

immunoprecipitation was performed for non-

ribosomal RNA isolated from the chromatin-

associated fraction or from whole cell, as

indicated, using the EpiMarkN^6 -Methyladenosine

Enrichment Kit (New England Biolabs). All

RNA-seq libraries were prepared using SMARTer

Stranded Total RNA-Seq Kit version 2, Pico

Input Mammalian (TaKaRa). For most sam-

ples, libraries were sequenced on an Illumina

NovaSeq 6000 in a 100-base-pair paired-end

mode. For RNA-seq data, trimmomatic trim-

med reads were aligned to the mm10 refer-

ence genome using HISAT2. Read counts were

calculated by featureCounts, and differential

expression was analyzed using DESeq2. For

the generation of preimplantation embryos,

MII oocytes were subjected to intracytoplas-

mic sperm injection and embryo culture. De-

tailed materials and methods are available in

the supplementary materials.

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ACKNOWLEDGMENTS

We thank P. Zhang forFto−/−mice and S. Liu, H.-L. Sun, and

H. Shi for discussions.Funding:This work was supported by the

National Institutes of Health (grants R01 ES030546 and RM1

HG008935 to C.H.); the National Key R&D Program of China

(grants 2020YFA0113200 and 2018YFA0108900 to Y.G.); and the

National Natural Science Foundation of China (grants 31922022,

31721003, 31820103009, and 82071720 to Y.G., S.G., and B.H.).

C.S.-P. is supported by Medical Scientist Training Program grant

T32GM007281 and National Cancer Institute fellowship F30

CA253987. C.H. is an investigator of the Howard Hughes Medical

Institute.Author contributions:C.H., J.W., and Y.G. conceived the

original idea and designed original studies. J.W. performed most

experiments with help from B.G., J.L., L.Y., Z.Z, L.-S.Z., Q.L., P.C.H.,

N.Z., X. Dong, H.W., and Z.H. X.Y. performed most bioinformatics

analyses with input from J.W., X.-L.C., X. Dou, Y.G. and C.H.

Supervised by S.G. and Y.G., X. Liu and L.Y. performed oocyte- and

embryonic development-related experiments and chimera

generation with help from B.H., W.L., Y.L., X.K., Y.Z., Y.W., C.C.,

Y.A., and T.D. X.Z., X.C., Q.S., and X. Li helped with mouse brain studies.

Y.-Y.H. provided Mel624 cells and participated in discussions. J.W. and

C.H. wrote the manuscript with input from Y.G., C.S.-P., B.G., X.Y.,

Z.Z., and N.Z. All authors approved the final manuscript.Competing

interests:C.H. is a scientific founder and a scientific advisory board

member of Accent Therapeutics, Inc., Inferna Green, Inc., and

AccuaDX, Inc. The remaining authors declare no competing interests.

Data and materials availability:Sequencing data are available at the

Gene Expression Omnibus (GEO accession nos. GSE151704 and

GSE151780). All other data are available in the manuscript or the

supplementary materials.License information:Copyright © 2022

the authors, some rights reserved; exclusive licensee American

Weiet al., Science 376 , 968–973 (2022) 27 May 2022 5of6

Fig. 5. The FTO-LINE1 RNA
axis plays critical roles
during early development.
(A) Number of GV oocytes from
4-week-old WT andFto−/−mice.
Error bars and means ± SEM
are shown forn = 4 WT mice
andn =6Fto−/−mice. (B) Ratio
of surrounded nucleolus (SN)/
nonsurrounded nucleolus
(NSN) oocytes from 4-week-old
WT mice (n = 5) andFto−/−
mice (n = 4). (C) Relative LINE1
RNA expression measured by
reverse transcription qPCR
in WT andFto−/−oocytes.
(D) Left: DNase I-TUNEL
assay showing more closed
chromatin in oocytes upon
FtoKO. Scale bars, 50mm. The
nucleus was counterstained
by DAPI. Representative images
were selected from three
independent experiments.
Right: relative TUNEL intensity
in WT andFto−/−oocytes (n =
12 each). pSN, partly sur-
rounded nucleolus. (E) Implan-
tation rate (left) and E7.5
embryo rate (right) ofFtoP+/M+,
FtoP+/M–, FtoP–/M+, and
FtoP–/M–zygotes. (F) Relative
LINE1 RNA expression
measured by reverse
transcription qPCR inFtoP+/M+andFtoP–/M–morulae. For (A), (C), (D), and (F),Pvalues were determined
using unpaired two-tailedt tests; error bars and means ± SD are shown forn = 3 experiments in (C) and
(F). For (B) and (E),Pvalues were determined using two-tailedz tests.

AB

E

DAPI TUNEL

pSN

DAPI TUNEL

NSN

KO

WT

0.0

0.5

1.0

1.5

< 0.0001

WTFto

-/-

Normalized TUNEL signal

D

Number of GV oocytes

0.0005

WTFto

-/-

0

20

40

60

80

100

GV SN/NSN ratio

0.001

0

30

60

90

(^120) SN
NSN
WTFto
-/-
F
Implantation rate% E7.5 embryo rate%
Completed Failed
Fto
P+M

• Fto
  P+M-
  Fto
  P-M+
  Fto
  P-M-
  Fto
  P+M+
  Fto
  P+M-
  Fto
  P-M+
  Fto
  P-M
  (^0) -
  30
  60
  90
  120
  0
  30
  60
  90
  120
  n = 24n = 27n = 30n = 30
  0.04 0.2 0.02
  n = 24n = 27n = 30n = 30
  0.09 0.4 0.006
  C
  Relative LINE1 expression
  in oocytes
  GV MII
  0.0
  0.5
  1.0
  1.5
  0.0003 0.0001
  WT
  Fto-/-
  Relative LINE1 expression
  in morulae
  Fto
  P+M


• 
  

  Fto
  P-M-
  0.0
  0.4
  0.8
  1.2 0.003
  RESEARCH | RESEARCH ARTICLE