Science - USA (2022-05-27)

responsive (fig. S13, F and G). We further ex-
plored thecis -regulatory role of intragenic
LINE1 RNAs suppressed byFtoKO and ob-
served significantly decreased expression and
reduced transcription rate for genes contain-
ing down-regulated LINE1 RNA compared with
other genes (Fig. 4C). Accordingly, reduced
H3K4me3 and H3K27ac levels and Pol II
binding, accompanied by elevated H3K9me3
levels, were observed for these gene loci (Fig.
4D) and those containing FTO-targeted LINE1
subfamilies (fig. S13H).
Most genes containing down-regulated LINE1
RNA were also down-regulated uponFtoKO
(fig. S14A), and these genes enriched pathways
involved in differentiation and development
(fig. S14B). We examined several key genes in
ESC pluripotency and early development (Esrrb,
Nek5, Phf3,andZfp982)andfoundthatallof
them contained intragenic LINE1 RNA with
increased m^6 A level and decreased expression
uponFtoKO, associated with more closed
local chromatin and reduced gene transcrip-
tion rate (fig. S14, C to G), which could all be
reversed by targeting LINE1 RNA with dCas13b-
wtFTO (fig. S15, A to I).
Certain 2C genes, such asDub1andZscan4,
do not contain LINE1 RNA. These genes and
MERVL RNA, a 2C-specific transposon, could
be repressed by LINE1 RNA throughtrans-
regulation ( 27 ). For these transcripts, we ob-
served reduced expression when applying
dCas13b-wtFTO to LINE1 RNA inFto−/−mESCs
(fig. S15J).

The FTO-LINE1 RNA axis is functionally
relevant in mouse development

We examined the mouse cerebellum, hippo-
campus, and adult neural stem cells because

Fto is highly expressed in mouse brain tissues

( 31 ). We observed increased LINE1 RNA m^6 A

levels, decreased LINE1 RNA expressions, and

more closed chromatin from samples derived

fromFto−/−mice compared with WT controls

(fig. S16, A to J). Similar trends ofFtoand

LINE1 RNA abundance during mESC differ-

entiation were also observed for adult neural

stem cells (fig. S16, K and L).

We next investigated the effects of FTO

during early development.Fto−/−pups were

born at a lower rate than the expected Men-

delian ratio (fig. S17A).Fto−/−female mice

showed ovarian defectsand impaired fertility

(fig. S17, B and C). Previous studies revealed

that LINE1 is activated before the sex deter-

mination of primordial germ cells and the

meiotic entry of oocytes ( 32 , 33 ). Indeed, both

Ftoand LINE1 RNA expression decreased

from primordial germ cell to the metaphase II

(MII) stage (fig. S17D). The number of germi-

nal vesicle (GV) oocytes and the ratio of sur-

rounded nucleolus GV oocytes fromFto−/−

mice were lower compared with WT controls

(Fig. 5, A and B).Ftodepletion led to sig-

nificantly reduced LINE1 RNA expression in

GV and MII oocytes (Fig. 5C and fig. S17E),

with down-regulated LINE1 subfamilies large-

ly resembling those observed in mESCs (fig.

S17F). We observed more closed chromatin for

GV oocytes fromFto−/−mice compared with

WT controls (Fig. 5D).Fto−/−GV oocytes could

mature to the MII stage (fig. S17G), butFto−/−

MII oocytes showed increased chromosome

misalignment and spindle collapse (fig. S17H).

RNA-sequencing (RNA-seq) results revealed

greater expression decreases of genes contain-

ing down-regulated LINE1 RNA compared

with other genes inFto−/−GV and MII oocytes,

respectively (fig. S17, I and J). GO analysis sug-

gested thatFto KO caused the observed defects

in oocyte development, likely through LINE1

RNA, which regulates LINE1-containing genes

(fig. S17K).

We further generated WT (FtoP+/M+), mater-

nal Fto -deficient (FtoP+/M–), paternalFto -deficient

(FtoP–/M+), and homozygous KO (FtoP–/M–)em-

bryos (fig. S18, A and B). Embryos from all four

groups could reach the blastocyst stage [em-

bryonic day 3.5 (E3.5)] and hatch out of the zona

pellucida at E4.5, butFto-deficient embryos

showedaslightlyweakenedabilitytodoso(fig.

S18C). Moreover, maternal loss ofFtoseverely

impeded decidua formation and the generation

of E7.5 embryos, with no E7.5 embryos produced

upon homozygousFto depletion (Fig. 5E and fig.

S18D). We examined the transcriptome differ-

ences betweenFtoP–/M–andFtoP+/M+morulae,

in which we detected repressed LINE1 RNA

and down-regulated LINE1 subfamilies sim-

ilar to those in mESCs (Fig. 5F and fig. S18, E

and F). We again observed induced expres-

sion ofZscan4and MERVL RNA (fig. S18, G

and H) and greater expression decreases of

genes containing down-regulated LINE1 RNA,

including regulators essential during early em-

bryonic development, such asLin28b, Tet2,and

Gsk3b(fig.S18,ItoL).

Finally, we applied dCas13b-wtFTO inFto−/−

MII oocytes and fertilized them withFto−/−

sperm. Embryos were developed in vitro to the

morula stage for subsequent analyses (fig. S18,

M and N). Induced LINE1 RNA associated with

more open chromatin was observed inFtoP–/M–

morulae by targeting dCas13b-wtFTO to LINE1

RNA inFto−/−MII oocytes (fig. S18, O and P).

The expression ofZscan4,MERVLRNA,and

selected genes containing down-regulated LINE1

Weiet al., Science 376 , 968–973 (2022) 27 May 2022 4of6

Fig. 4.FtoKO deactivates LINE1-
containing genes by repressing intra-
genic LINE1 RNA.(A) Boxplots showing
gene expression fold changes uponFtoKO
from caRNA sequencing. Genes were
categorized according to their genomic
distance to the nearest LINE1 RNA
with at least 10 reads.Pvalue was
determined using Wilcoxon rank sum test.
(B) Scatter plot showing the negative
correlation of fold changes between
expression of LINE1-containing genes and
m^6 A level of corresponding intragenic
LINE1 RNA uponFtoKO. Intragenic LINE1
RNAs were categorized into 100 bins on
the basis of their ranked expression
fold changes uponFtoKO.r refers to
Pearson’s correlation coefficient.P value
was calculated based ont distribution.
(C) Boxplots showing fold changes of gene
expression from caRNA sequencing (left)
and gene transcription rate (right) uponFtoKO. Genes were categorized into three groups: genes containing down-regulated LINE1 RNA, genes near (<1 Mb) down-regulated
LINE1 RNA, and genes containing other LINE1 RNA.P values were determined using Wilcoxon rank sum tests. (D) Profiles of H3K4me3, H3K9me3, H3K27ac, and Pol II levels
on loci of genes containing down-regulated LINE RNA from 3.0 kb upstream of the TSS and 3.0 kb downstream of the TES in WT andFto−/−mESCs.

1.0

1.5

< 2e-16

< 2e-16

0.0

-0.5

0.5

Log

FC exp 2

Fto

KO/WT

A C

< 2e-16

0 kb

< 10 kb< 100 kb< 1 MbOthers

-1.0

0.0

-0.5

0.5

1.5

1.0

Log

FC exp 2

Fto

KO/WT

To the nearest LINE1

D

-0.15

-0.10

-0.05

0.00

-1 2 -012

r = -0.5087

p = 6.5e-8

Log 2 FC m^6 A Fto KO/WT

of intragenic LINE1 RNA

Log

FC exp 2

Fto

KO/WT

of LINE1-containing gene

B

< 2e-16

< 2e-16

0.0

-0.4

-0.6

-0.2

0.2

Δ transcription

Fto

KO/WT

Down-LINE1

Containing

Coverage (RPKM)on down-LINE1-containing gene

H3K27ac

10

20

30

40

(^50) Pol II
6
8
10
12
14
0
20
40
60
80
H3K4me3
H3K9me3
1
2
3
4
5
-3.0 TSS TES 3.0-3. 0 TSS TES 3.0-3.0 TSS TES 3.0-3.0 TSS TES 3.0
WT
FtoKO
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