Science - 31 January 2020

Liuet al.,Science 367 , 580–586 (2020) 31 January 2020 5of6

A

-1.00 -0.67 -0.33 0.00 0.33 0.67 1.00 -3.00 -2.00 -1.00 0.00 1.00 2.00 3.00

0.0 0.5 1.0 1.5

TSS TTS TSS TTS 0.0 1.0 2.0 3.0TSS TTS TSS TTS

TSS TTS

0.0 0.5 1.0 1.5

TSS TTS 0.0 1.0 2.0 3.0TSS TTS TSS TTS

H3K27ac level changes

m^6 A_Mettl3-/--1

non−m^6 A_Mettl3-/--1

m^6 A_Mettl3-/--2

non−m^6 A_Mettl3-/--2

Mettl3-/--1 Mettl3-/--2

H3K4me3 level changes

TSS TTSTSS TTS TSS TTSTSS TTS

EP300 binding 02468

−1kb−500 0 500 1kb −1kb −500 0 500 1kb

2 4 6 8 10 12

Binding DNA loci

YY1 binding

WT Mettl3-/-

m^6 A Level

High Medium Low

0

1

2

3

−1.0 −0.8 −0.6

R^2 = 0.62

p = 5.4e−23

Mettl3-/- vs WT

m^6 A log 2 FC

EP300 binding log

FC 2

−1

0

1

2

−0.9 −0.8 −0.7 −0.6

YY1 binding log

FC 2

R^2 = 0.37

p = 1.3e−11

m^6 A log 2 FC

Binding DNA loci

Mettl3-/- vs WT

WT Mettl3-/-

C

E

B

D

FG

H3K27ac level changes

m^6 A only

EP300/YY1 only

m^6 A + EP300/YY1

H Mettl3-/- vs WT

-10^30103104105

TUNEL_488nm

0

20

40

60

80

100

Normalized to mode

I

0

2

4

6

Relative level

m

6 A

Half lifetime

Transcription rate

H3K4me3H3K27ac

p = 0.0003

p = 0.035

p = 0.0019

p = 0.0014

p = 0.0002

Arntl gRNA

J Control

Gene transcription

H3K27ac/H3K4me3

m^6 A

Histone

K

0.1 0.3 0.5 0.7

−2.5kb 0 2.5kb

0.05 0.10 0.15 0.20

−2.5kb 0 2.5kb

EP300 binding

YY1 binding

m^6 A peak center m^6 A peak center

−1.0

−0.5

0.0

0.5

1.0

1.5

2.0

−1

0

1

2

3

Control ASO

LINE1 ASO

Mettl3-/- mESC

WT mESC

m^6 A Level

High Medium Low

PCC = −0.79 PCC = −0.61

Active

TSS

Less active

Decay

TSS

carRNAs

carRNAs

Fig. 4. The m^6 A level of carRNAs affects local chromatin state and
downstream transcription.(A) Profiles of H3K27ac (top) and H3K4me3
(bottom) level changes on gene body together with 2.5 kb upstream of the
TSS (transcription start site) and 2.5 kb downstream of the TTS (transcription
termination site) in WT andMettl3KO mESCs. Genes were categorized into
two groups according to whether they harbor upstream m^6 A-marked carRNAs
(m^6 A) or not (non-m^6 A). (BandC)ProfilesofEP300(B)orYY1(C)DNA
binding at their peak center and flanking 2.5 kb regions in WT andMettl3KO
mESCs. (DandE) Profiles of EP300 (D) and YY1 (E) DNA binding at the
center of m^6 A peaks overlapped with carRNAs and its flanking 2.5 kb regions
in WT mESCs. m^6 A peaks were categorized into highly (high), moderately
(medium), or lowly (low) methylated groups, according to their m^6 A levels in
WT mESCs. (FandG) The correlation between changes in m^6 A level of the
carRNAs and changes in EP300 (F) or YY1 (G) DNA binding at genomic
regions that show m^6 A differences withMettl3KO. The genomic regions were

categorized into 100 bins on thebasis of fold change rank of m^6 A level

uponMettl3KO. (H) Barplots showing H3K27ac level changes at genomic

regions that are m^6 Amethylated(m^6 A only, without EP300 and YY1 binding),

bound by EP300 or YY1 (EP300/YY1 only, without m^6 AcarRNA),andm^6 A

methylated with EP300 and YY1 binding (m^6 A + EP300/YY1). The last group

showed the highest increase uponMettl3KO. (I) Analysis of chromatin

accessibility inMettl3KO mESCs treated with control or LINE1 antisense

oligos (ASOs). DNase I–treated TUNEL assay was performed. (J) A dCas13b-

FTO (WT or inactive mutant) construct with gRNA targeting the seRNA of

Arntlwas used to reduce the m^6 A level ofArntlseRNA. After treatment,

increased half lifetime of the target seRNA, elevated local H3K27ac and

H3K4me3 levels, and increasedArntltranscription rate were observed,

accompanied by the decreased seRNA m^6 A level. (K)Aschematicmodel

showing how m^6 A affects transcription by regulating the decay of upstream

carRNAs stability and chromatin state.

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