Fine-tuning of gene expression through the Mettl3-Mettl14-Dnmt1 axis controls ESC differentiation

. Author manuscript; available in PMC: 2025 Nov 20.

Published in final edited form as: Cell. 2025 Jan 17;188(4):998–1018.e26. doi: 10.1016/j.cell.2024.12.009

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER

Antibodies

Mouse monoclonal anti-β-Actin	Sigma Aldrich	Cat#A5316; RRID:AB_476743
Rabbit polyclonal anti-Dnmt1	Abcam	Cat#ab19905; RRID:AB_731983
Rabbit monoclonal anti-Dnmt1	Cell Signaling Technology	Cat#5032; RRID:AB_10548197
Mouse monoclonal anti-DNMT1	Invitrogen	Cat# 60B1220.1; RRID:AB_838131
Rabbit polyclonal anti-METTL3	Proteintech	Cat#15073–1-AP; RRID:AB_2142033
Rabbit polyclonal anti-METTL14	Sigma Aldrich	Cat#HPA038002; RRID:AB_10672401
Mouse monoclonal anti-FLAG^® M2	Sigma Aldrich	Cat#F3165; RRID:AB_259529
Mouse monoclonal anti-6X His tag^®	Abcam	Cat#ab18184; RRID:AB_444306
Mouse monoclonal anti-Myc-tag	Cell Signaling Technology	Cat#2276; RRID:AB_331783
Mouse monoclonal anti-GAL4 (DBD) (RK5C1)	Santa Cruz Biotechnology	Cat#sc-510; RRID:AB_627655
Mouse monoclonal anti-GST tag	Cell Signaling Technology	Cat# 2624; RRID:AB_2189875
Mouse monoclonal anti-TBP	Abcam	Cat#ab818;RRID: AB_306337
Rabbit polyclonal anti-H3	Abcam	Cat#ab1791;RRID:AB_302613
Rabbit monoclonal anti-H3	Cell Signaling Technology	Cat#4499;RRID: AB_10544537
Rabbit monoclonal anti-SETD2	Cell Signaling Technology	Cat# 80290; RRID:AB_3105876
Rabbit monoclonal anti-Dnmt3a	Abcam	Cat#ab307503; RRID:AB_3105875
Rabbit monoclonal anti-DNMT3B	Cell Signaling Technology	Cat# 57868;RRID: AB_2799534
Rabbit monoclonal anti-H3K36me3	Cell Signaling Technology	Cat# 4909; RRID:AB_1950412
Rabbit monoclonal anti-FOXA2	Cell Signaling Technology	Cat# 8186; RRID:AB_10891055
Rabbit monoclonal anti-GATA4	Abcam	Cat# ab307823; RRID:AB_3105880
Rabbit monoclonal anti-GATA6	Cell Signaling Technology	Cat# 5851; RRID:AB_10705521
Rabbit monoclonal anti-SOX17	Abcam	Cat# ab224637; RRID:AB_2801385
Mouse monoclonal anti-GAPDH	Proteintech	Cat#60004–1-Ig; RRID:AB_2107436
Goat polyclonal secondary antibody anti-Mouse Alexa Fluor 594	ThermoFisher Scientific	Cat# A-11005; RRID: AB_2534073
Rabbit monoclonal anti-m⁶A	Synaptic Systems	Cat#202003; RRID:AB_2279214
Mouse monoclonal anti-5mC	Diagenode	Cat# C15200003; RRID:AB_3105883

Bacterial and virus strains

BL21 DE3	NEB	C2527H

Chemicals, peptides, and recombinant proteins

STM-2457 METTL3 inhibitor	MedChemExpress	HY-134836; CAS: 2499663-01-1
FB23–2 FTO demethylase inhibitor	MedChemExpress	HY-127103; CAS: 2243736-45-8
MEK inhibitor PD0325901	Axon MedChem	Cat#1408; CAS: 391210-10-9
GSK3 inhibitor CHIR99021	Axon MedChem	Cat#1386; CAS: 252917-06-9
Actinomycin D	Sigma-Aldrich	Cat#SBR00013; CAS: 50-76-0
Cycloheximide	Sigma-Aldrich	Cat#C4859; CAS: 66-81-9
Lipofectamine 2000	Invitrogen	Cat# 11668019
Lipofectamine 3000	Invitrogen	Cat# L3000–008
FuGENE Transfection Reagent	Promega	Cat# E2311
TRIzol LS	Invitrogen	Cat#10296028
Isopropylthiogalactosidase (IPTG)	Sigma	Cat#3I5502–1G
cOmplete^™ Mini Protease Inhibitor Cocktail	Roche	Cat#11836153001
L-Glutathione reduced	Sigma-Aldrich	Cat# G4251–5G
RNase-Free DNase Set	Qiagen	Cat#79254
NanoBRET 618 fluorescent ligand	Promega	Cat#G9801
NanoBRET furimazine substrate	Promega	Cat#N1571
Human Recombinant DNMT1	Active Motif	Cat#31404; GenPept:P26358
Human Recombinant METTL3	Active Motif	Cat#31567; GenPept:Q86U44
Human Recombinant METTL14	Active Motif	Cat#31568; GenPept:Q9HCE5
Human Recombinant Histone H2A	This paper	N/A
Human Recombinant Histone H2B	This paper	N/A
Human Recombinant Histone H3.1	This paper	N/A
Human Recombinant Histone H4	This paper	N/A
Human Recombinant Biotinylated Nucleosome	This paper	N/A
Human Recombinant Biotinylated Nucleosome-H3K36me3	EpiCypher	Cat#16–0320; GenPept: P04908, O60814, P68431, P62805
Human Recombinant GST- DNMT3B-PWWP	Active Motif	Cat#31542; GenPept: Q9UBC3
Human Recombinant GST-METTL3	This paper	N/A
Human Recombinant GST-METTL14	This paper	N/A
Human Recombinant GST-METTL14-NLS	This paper	N/A
Human Recombinant GST-METTL14-MTD	This paper	N/A
Human Recombinant GST-METTL14-RGG	This paper	N/A
Human Recombinant GST-METTL14-RGG1	This paper	N/A
Human Recombinant GST-METTL14-RGG2	This paper	N/A
ESGRO^® Recombinant Mouse LIF Protein	Merck Millipore	Cat#ESG1107

Critical commercial assays

RNeasy Kit	Qiagen	Cat#74004
mRNA Miniprep Kit	Sigma-Aldrich	Cat#MRN10
QIAamp DNA Mini Kit	Qiagen	Cat#51306
EZ DNA 462 Methylation Kit	Zymo Research	Cat#D5002
HotStarTaq DNA Polymerase	Qiagen	Cat#203205
DC^™ Protein Assay Kit	Bio-Rad	Cat#5000112

Deposited data

All raw gel data are deposited at Mendeley Data	This paper	https://doi.org/10.17632/k9w73kftmt.1
Raw and analyzed data	This paper	GEO: GSE184757
Human and mouse rRNA and tRNA sequences for sequencing data filtering	The National Center for Biotechnology Information (NCBI)	https://www.ncbi.nlm.nih.gov/nuccore
Human reference genome build 37 (GRCh37), transcriptome and annotation version 85 for sequencing data analyses	Ensembl	https://ftp.ensembl.org/pub/grch37/release-85/
Human reference annotation version 28 for Infinium array analyses	Gencode	https://www.gencodegenes.org/human/release_28.html
Mouse reference annotation version M25 for Infinium array analyses	Gencode	https://www.gencodegenes.org/mouse/release_M25.html
Human long noncoding transcriptome and annotation version 5.2	LNCipedia	https://lncipedia.org/
Mouse reference genome, build 38 (GRCm38), transcriptome and annotation version 99	Ensembl	https://ftp.ensembl.org/pub/release-99/
Illumina Manifests for infinium probe target chromosomic positions	Illumina	https://support.illumina.com/

Experimental models: Cell lines

HeLa	ATCC	N/A
HeLa METTL3 knockout	This study	N/A
COS-7	Laboratory of Sriharsa Pradhan	N/A
HEK293	Promega Corporation	N/A
HEK293^5XUAS	Laboratory of Bastian Stielow	N/A
mouse embryonic stem cells J1 WT/Mettl3 knockout	Laboratory of Howard Y. Chang	N/A
mouse embryonic stem cells J1 WT/Dnmt1 knockout	Laboratory of Fabio Spada	N/A
mouse embryonic stem cells E14TG2a WT/Mettl14 knockout	Laboratory of Laixin Xia	N/A
mouse embryonic stem cells E14TG2a Mettl14 knockout + Mettl14^WT or Mettl14^ΔRGG	This study	N/A

Oligonucleotides

DNA oligos	This study	See Table S7

Recombinant DNA

Plasmid: pcDNA3.1-Myc/His-METTL3	This study	N/A
Plasmid: pcDNA3.1-Myc/His-METTL14	This study	N/A
Plasmid: RFP-DNMT1	This study	N/A
Plasmid: NanoLuc-METTL3	This study	N/A
Plasmid: NanoLuc-METTL14	This study	N/A
Plasmid: HaloTag-DNMT1	This study	N/A
Plasmid: pGex-4T1-GST-METTL14 (full-length or partial)	This study	N/A
Plasmid: pFlag-CMV2-METTL14 (full-length or partial)	This study	N/A
Plasmid: pcDNA3-Myc-DNMT1	Addgene	Cat#36939
Plasmid: pcDNA3-GAL4-METTL14 (full-length or partial)	This study	N/A
Plasmid: pFlag-CMV2-DNMT1	This study	N/A
Plasmid: pLV-EF1a-IRES-Mettl14 (full-length or partial)	This study	N/A

Software and algorithms

Graphpad Prism 7	GraphPad	https://www.graphpad.com/
Biorender	Biorender	https://www.biorender.com
R version 4.2.2	The R Project	https://cran.r-project.org/
Heatmapper webtool	Meissner et al.⁷³	http://www.heatmapper.ca/
Zen version 2.1	Zeiss	https://www.zeiss.com/
ImageJ	Xia et al.⁴⁶	https://imagej.nih.gov/ij/
LiftOver webtool	Bisia et al.⁷⁰	https://genome.ucsc.edu/cgi-bin/hgLiftOver
FastQC version 0.11.5	Yang et al.⁵⁰	https://github.com/s-andrews/FastQC
AfterQC version 0.9.6	Su et al.⁵¹	https://github.com/OpenGene/AfterQC
Bowtie2 version 2.3.4.1	Collignon et al.⁵²	https://bowtie-bio.sourceforge.net/bowtie2
Trimmomatic version 0.33	Kalkan et al.⁵³	http://www.usadellab.org/cms/?page=trimmomatic
STAR version 2.6.0c	Zhang et al.⁵⁴	https://github.com/alexdobin/STAR
RSEM version 1.3.1	Ke et al.⁶¹	https://github.com/deweylab/RSEM
samtools version 1.6.2	Hayashi et al.⁶²	http://www.htslib.org/
HTseq counts version 0.9.1	Herzog et al.⁵⁷	https://pypi.python.org/pypi/HTSeq
tximport version 1.10.1	Krishnakumar et al.⁶³	https://bioconductor.org/packages/release/bioc/html/tximport.html
DESeq2 version 1.22.2	Yang et al.⁶⁴	https://bioconductor.org/packages/release/bioc/html/DESeq2.html
m6aViewer version 1.6.1	Neumann et al.⁵⁸	http://dna2.leeds.ac.uk/m6a/
Bedtools version 2.27.1	Wang et al.⁷¹	https://github.com/arq5x/bedtools2
bamTobw	Kim et al.⁵⁹	https://github.com/YangLab/bamTobw
IGV tool version 2.9.4	Varley et al.⁶⁰	https://software.broadinstitute.org/software/igv/