Myt Transcription Factors prevent stress-response gene overactivation to enable postnatal pancreatic β-cell proliferation, function, and survival

. Author manuscript; available in PMC: 2021 May 18.

Published in final edited form as: Dev Cell. 2020 Apr 30;53(4):390–405.e10. doi: 10.1016/j.devcel.2020.04.003

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies (with fold of dilution indicted)
488-donkey anti Guinea Pig (1:1000)	Jackson Immunoresearch	706-545-148 RRID:AB_2340472
488-donkey anti goat igG (1:1000)	Jackson Immunoresearch	705-545-147 RRID:AB_2336933
Alexa Fluor® 647 AffiniPure Donkey Anti-Guinea Pig IgG (H+L) (1:1000)	Jackson Immunoresearch	706-605-148 RRID:AB_2340476
Alexa Fluor® 488 AffiniPure Donkey Anti-Rabbit IgG (H+L) (1:1000)	Jackson Immunoresearch	711-545-152 RRID:AB_2313584
Alexa Fluor® 647 AffiniPure Donkey Anti-Rabbit (1:1000)	Jackson Immunoresearch	711-605-152 RRID:AB_2492288
Alexa Fluor® 647 AffiniPure Donkey Anti-Mouse IgG (H+L) (1:1000)	Jackson Immunoresearch	715-605-150 RRID:AB_2340862
Alexa Fluor® 594 AffiniPure Donkey Anti-Mouse (1:1000)	Jackson Immunoresearch	715-585-150 RRID:AB_2340854
Alexa Fluor® 594 AffiniPure Donkey Anti-Goat IgG (H+L) (1:1000)	Jackson Immunoresearch	705-585-003 RRID:AB_2340432
Alexa 647 AffiniPure Rabbit Anti-Syrian Hamster IgG (H+L) (1:1000)	Jackson Immunoresearch	307-605-003 RRID:AB_2339601
Rat anti-BrdU (1:500)	ABcam	Ab-6326 RRID:AB_305426
biotin anti Rat (1:1000)	Jackson Immunoresearch	712-065-151 (RRID: N/A)
Cy3-Donkey anti-mouse (1:1000)	Jackson Immunoreserach	715-165-150 RRID:AB_2340813
Cy3-streptavidin (1:1000)	Vector laboroatories	SA-1300 (RRID: N/A)
Cy5- streptavidin (1:1000)	Vector laboroatories	SA-1500 (RRID: N/A)
FITC anti Rabbit (1:1000)	Jackson Immunoresearch	711-095-152 RRID:AB_2315776
Goat anti-Pdx1 (1:2000)	ABcam	AB47383 RRID:AB_2162359
Goat anti-insulin (1:500)	Santa Cruz	sc-7839 RRID:AB_2296108
Guinea pig anti insulin (1:1000)	Dako	A0564 RRID: NA
Rabbit anti-Myt1 (1:1000)	This lab. Mech. Dev. 2007. 124, 698–910.	NA
Mouse anti-glucagon (1:5000)	Millipore	MabN238 RRID:NA
Rabbit anti-Pdx1 (1:5000)	ABcam	AB47267 RRID:AB_777179
Rabbit anti-Nkx6.1 (1:5000)	Gift of P. Serup, Copenhagen, Denmark	NA
Rabbit anti-MafB (1:1000)	Bethyl	A700–046 RRID: NA
Rabbit anti-Cleaved Caspase 3 (1:500)	SYSY	#105173 RRID:AB_887838
Rabbit anti-glucagon (1:100)	ABcam	AB92517 RRID:AB_10561971
Rabbit anti-Ki67(1:500)	Abcam	Ab 15580 RRID: AB_443209
Rabbit anti-HA (1:500)	Cell Signaling Technology	#3724 RRID: AB_1549585
Guinea pig anti-Myt2 (Myt1L) (1:1000)	This lab. See *M of this paper.	NA
Rat anti-Myt3 (1:500)	This lab. See *M of this paper.	NA
Normal IgG (1 μg/IP)	Cell Signaling	#2729S RRID: AB_1031062
Rabbit anti-H3K27Ac (1 μg/IP)	Millipore	#07–360 RRID: AB_310550
Bacterial and Virus Strains
Bl-21	Vanderbilt MPB Core	NA
DH5-alpha	Vanderbilt MPB Core	NA
Xl-1	Vanderbilt MPB Core	NA
Biological Samples
Bovine serum albumin	Sigma	A9418
Donkey Serum	Jackson Immunoresearch	017-000-121 RRID:AB_2337258
Fetal Bovine Serum	Atlanta Biologicals	S10250
Human islet (normal)	IIDP	NA
Human islet (normal)	Clinical Islet Transplantation Laboratory, University of Louisville, Louisville, KY	NA
Human islet (diabetic)	Clinical Islet Transplantation Laboratory, University of Louisville, Louisville, KY	NA
Chemicals, Peptides, and other key reagents
Collagenase from Clostridium histolyticum	Sigma-Aldrich	C5138
DMSO	Sigma-Aldrich	D2650
Doxycycline	Sigma-Aldrich	D9891
DAPI	Sigma-Aldrich	D9542
paraformaldehyde	Sigma-Aldrich	P6148
Trypsin	Sigma-Aldrich	C5138–5G
SuperScript III Reverse Transcriptase	ThermoFisher	18080085
RNaseOUT Recombinant Ribonuclease Inhibitor	ThermoFisher	10777019
T4 DNA Polymerase	NEB	M0203S
paraformaldehyde	Sigma	P6148
HBSS	ThermoFisher	14025092
High capacity cDNA synthesis	Applied BIosystems	4368814
RPMI1066	Sigma-Aldrich	R8758
Fetal Bovine Serum	Corning	35010-cv
BSA	Sigma-Aldrich	9048468
Nano-Glo® Dual-Luciferase® Reporter Assay System	Promega	N1610
CRML-1066	ThermoFisher	11530037
Cyclophilin B Control siRNA	Dharmacon	D-001820-01-05
Nontargeting Control siRNA	Dharmacon	D-001810-01-05
Human MYT1 targeting siRNA	Dharmacon	L-005271-00-0005
Human MYT2 (MYT1L) targeting siRNA	Dharmacon	L-022771-00-0005
Human MYT3 (ST18) targeting siRNA	Dharmacon	L-008451-02-0005
SYBER Green qPCR mix	Biorad	1705060
Critical Commercial Assays
Serum Glucagon assay	The Vanderbilt Hormone Assays & Analytical Cores	NA
Magna ChIP™ HiSens Chromatin Immunoprecipitation Kit	Millipore	17–10460
Experimental Models: Cell lines
HEK293	ATCC	CRL-11268
EndoC-βH1	UniverCell-Biosolutions	EndoC-βH1
Experimental Models: Organisms
Myt1F/F	This lab	NA
Myt2F/F	This lab	NA
Myt3F/F	This lab	NA
Pdx1Cre	Jackson laboratories	Tg(Pdx1-Cre)89.1
InsCre	Jackson laboratories	B6(Cg)-Ins1^{tm1.1(cre)Thor}/J
Rosa26-ATF4^LoxTG	Jackson laboratories	B6;129X1-Gt(ROSA)26Sor^tm2(ATF4)Myz/J
Db/+	Jackson laboratories	B6.BKS(D)-Lepr^db/J
Pdx1CreER	This lab (Gu et al., 2002)	NA
Ai9	Jackson laboratories	Gt(ROSA)26Sor^{tm9(CAG-tdTomato)Hze}
HspOE	This lab/this work	N/A
ICR	Charles Rivers	CD-1
DNA oligos for Genotyping
TTGAAACAAGTGCAGGTGTTCG	IDT	Pdx1Cre
ATGTGGCGTCCACGGGGAGT	IDT	Pdx1Cre
CCCTAGGCTCTCTGTGGTTGCAC	IDT	TetO-Hsp
GGTCGCTGAACTCATTGGCGAT	IDT	TetO-Hsp
CCAGAAGCTTGGTGTAGAGCAG	IDT	rTTA
GGCTGGCTCTGCACCTTGGTGATC	IDT	rTTA
TCCACCTGTCCGTTCAGTG	IDT	Myt1F
AGATCCTTCCAGGGTGGAGA	IDT	Myt1F
GTATGGGGAAACTGCTGAATGAA	IDT	Myt1LF
GCATCCAGACAGACTGCGGTGA	IDT	Myt1LF
TGAGACTGAGACTACTTGTTAGC	IDT	Myt3F
GCTTTCTGGGTTCATTTCTG	IDT	Myt3F
GGTGTAGCTGATGATCCGAATA	IDT	Ins1Cre
CATGATCTCCGGTATTGAAAC	IDT	Ins1Cre
GGCTCCTCTCTTACATGGATCT	IDT	ATF4OE allele
CTTCGACCAGTCGGGTTTG	IDT	ATF4OE allele
DNA oligos for Real-time RT-PCR or mouse tissues
CCAGTAGCCTGGGAAGACAT	IDT	hspa1b
CAGTGCCAAGACGTTTGTTT	IDT
TGAAACCTCTGCAAATTCAAA	IDT	dnajb9
GCTTCAGACATTTAGTAACCCAAG	IDT
CAGAGAGCTGTCTTGGAGCA	IDT	hspb8
TCAAACACACACACAATGCC	IDT
TGACATGGATCAGGTGGAGT	IDT	DNAja4
GGCTCTTCGTCATCTTCCTC	IDT
CAGGGCCAACAGAGGTCACA	IDT	Ddit3
TCCTCTTCCTCCTGGGCCAT	IDT
AGAGTTATTGGATATGGGCTGCCA	IDT	Dnajb4
GGCAGGGAGATGCTTCCTCA	IDT
TCATCGGCGACAAGTCGGAA	IDT	Hspa2
AAGGTCTGCGTCTGCTTGGT	IDT
ACAGCCTCCCTAAGCCCTCT	IDT	Hspb6
GAGGCCTGCGTGGTGATAGA	IDT
AGGAGTGCTGCCGAAACTGT	IDT	Hspe1
ACTCTTTCCTTTCCCTCCTGACC	IDT
ATAAGGCCCGAAGCCAGAGG	IDT	Atf5
TGTGACGCTGGAGACAGACG	IDT
CCCACCAGACAATCTGCCTTCT	IDT	atf4
CCAGGTGGGTCATAAGGTTTGGG	IDT
TGTGTTTAATCCCGAGAGGA	IDT	Hsph1
GGTCAGGTAGCCAAATGCTT	IDT
ACTGGACTCTTGCCCTCTGT	IDT	DNAjb1
CCCACACAGTGGAGACTGAC	IDT
AGTGGGCAGCCATGAGGTTT	IDT	Myt1
GGACATAAAGAAGGAACTTC	IDT
GCTGGTAAGGAACTAGGTCCT	IDT	XBP1
CTCACGGCCTTGTGGTTGAGA	IDT
GGATGCAGGGATGATGTTCT	IDT	GAPDH
AACTTTGGCATTGTGGAAGG	IDT
CCGGGTTATGTGAGCCCAA	IDT	Nkx6.1
CTGGACAGCAAATCTTCGCC	IDT
CACCAGTTCAAGCTCAACAAG	IDT	Mnx1
GGTTCTGGAACCAAATCTTCAC	IDT
GTTCTGGGGCTCAAGATCAA	IDT	Insm1
GTCAGCGTACTCCTCCTTGC	IDT
GATCTGAATCCTACAAGCTCCTG	IDT	Hnf4a
CCCTCGTGTCACATCTTCTTT	IDT
TGAAGCCTATGCTGCACTTG	IDT	Isl1
ACGTGCTTTGTTAGGGATGG	IDT
CAGACCCATAACACTCAAGAGG	IDT	NeuroD1
GGAGTGTGTGTTGGCATTTATT	IDT
GGTAGTTGCTCGCCATCCA	IDT	Mafb
CCCAGCTTCAGTCCGACTGA	IDT
CCAGGTTGTCTAAATTGG	IDT	Pdx1
GTTGGGTATAGCCGGAGAGA	IDT
AAGAACACGTAAGGCCCAAG	IDT	Glut2
AGCAACTGGGTCTGCAATTT	IDT
ACCACGTGCGCTTGGAGGAG	IDT	MafA
ACCTCCTCCTTGCTGAA	IDT
GCAGATGCAAAAGTCCAGGTG	IDT	Pax6
CAGGTTGCGAAGAACTCTGTTT	IDT
DNA oligos for ChIP-PCR in regulatory regions
CAGTTGGGATTAAAGGCTTGAG	IDT	Hspa1a-a1
GAACAGGGCCGAAGATGAGA	IDT	Hspa1a-a1
ATCTCCCTGGGTCTCCTAAA	IDT	Hspa1a-a2
GTTGTGTACAGTTTGTTGTGATTTG	IDT	Hspa1a-a2
AGCCTAATTCCCGAATGAAA	IDT	Hspa1b-a1
TCCCAGTACCAGTCCTCTCA	IDT	Hspa1b-a1
GTGTCAATAGCAGCACCAGCA	IDT	Hspa1b-a2
GGTTCGCTAGAGAGTACGGAT	IDT	Hspa1b-a2
TCCTCCGGCTCGCTGATTG	IDT	Hspa1b-a3
GCGCCTTTAAGGAGTCTTCA	IDT	Hspa1b-a3
GGAGAGATGGCTCAGTGGTT	IDT	HspH1-a1
TGTTGGGATTTGAACTGAGG	IDT	HspH1-a1
CTGGAGTCAACTGGAACGAA	IDT	HspH1-a2
GCTAAAACTGACCGAGCTTGC	IDT	HspH1-a2
AATCATCTCTTTCAAGAACACC	IDT	Dnajb1-a1
TGGTGAAGTTGACATTATTCTG	IDT	Dnajb1-a1
CCCAAGGTCACACAGCCAGA	IDT	Dnajb1-a2
CAGGGAATGGGACTGCCTGA	IDT	Dnajb1-a2
CAAAGAGAGGACATAGTTACCCT	IDT	Atf4-a1
CCCAAAGAAGTCACAAGTGC	IDT	Atf4-a1
CCAGGTGCAGAGCCAATAG	IDT	Atf4-a2
GTGGGACACCTAGCCAAAGCT	IDT	Atf4-a2
TGCTAGGCAGAATGACTCAAA	IDT	Albumin
GAGACCGTAATAAATTCAACTGTATCC	IDT	Albumin
Oligos for MytZf-Vp16 fusion/reporter construction
CGAGCTCGGTACCCGGGTCGAGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCCCGAATTCGAGCTCGGTACCCGGGGATCCTCTAGTCAGCTGACGC	IDT	CMV minimal promoter
GAATTCCACCATGGCGTACCCATACGATGTTCCTGACTATGCTGGCTCTTATCCGTATGACGTCCCTGACTATGCAACTGGAGGGGAAGTCACCCTAACCAACT	IDT	5’ fusion
TCTGGCTGTCCCAGAGCAACCTTTGCTGGAAGTGGGTCCGCGTACAGCCGCGCGCGTACGA	IDT	junction
GCGGCCGCTACCCACCGTACTCGTCAATTCCAAGGGCA	IDT	3’ fusion
Oligos for Real-time RT-PCR of human tissues
GGCCACATCACCGGGAACTA	IDT	MYT1
AGTGGGCAGCCATGAGGTTT	IDT	MYT1
CTCACACACCGCAGCTTGTC	IDT	MYT2
GCCCGCTGTTTGATGGTCAG	IDT	MYT2
CTTTGGTATCGTGGAAGGACTC	IDT	GAPDH
AGTAGAGGCAGGGATGATGT	IDT	GAPDH
CAGCTGCTGCCATCCTGAAC	IDT	MYT3
GGCATGCAGACTCTGTGGCT	IDT	MYT3
CATCCACAGCCAGCCATTCG	IDT	ATF4
TTACCTGGAGGTGGCCAAGC	IDT	ATF4
TGCAGAGATGGCAGCTGAGT	IDT	DDIT3
GCAGGTCCTCATACCAGGCT	IDT	DDIT3
GGGAGGCCTTCTCCAGGAAC	IDT	DNAJB1
GTGGCTGCACAGTGAACGTC	IDT	DNAJB1
GGTCAGCACCATGGACGAGA	IDT	HSPA1
AAGCACTGGCCTTTCCAGGT	IDT	HSPA1
GGCTGAGGAAGTGGGACCTC	IDT	HSPH1
GTTGCCTGCCTCACTCTGC	IDT	HSPH1
CTGGAGAACTACTGCAACTAGAC	IDT	INS
TGCTGGTTCAAGGGCTTTAT	IDT	INS
GGCTCCTCTCTTACATGGATCT	IDT	ATF4
CTTCGACCAGTCGGGTTTG	IDT	ATF4
DNA oligos for H3K27Ac ChIP-PCR near promoter regions
GGCGTCTCGCGTGATAACCT	IDT	Atf4-RTO21
GTGGGACACCTAGCCAAAGCT	IDT	Atf4-RTO21
GCTGATTGGCCCAGCGGAGA	IDT	Hspa1a-RTO24
CTGCGCCTTTAAGGAGTCTT	IDT	Hspa1a-RTO24
TCCTCCGGCTCGCTGATTG	IDT	Hspa1b-RTO33
GCGCCTTTAAGGAGTCTTCA	IDT	Hspa1b-RTO33
AGGAAGCGGAAGTGGCACGTG	IDT	Hsph1-RTO49
CTACTGAGGAGAACTTTCCAGA	IDT	Hsph1-RTO49
GGCGTTGACCAGAGTAGAGA	IDT	Dnajb1-RTO41
CTCCGTGACGCAGCACAT	IDT	Dnajb1-RTO41
GGTCCAAAGAGAGGGAGGAG	IDT	Gapdh
GCTACGTGCACCCGTAAAGC	IDT	Gapdh
Software and Algorisms
ImageJ	Schneider et al., 2012 PMCID: PMC5554542	https://imagej.nih.gov/ij/
TopHat and Cufflink	Trapnell et al, 2012 PMCID: PMC3334321	https://ccb.jhu.edu/software/tophat/index.shtml
Webgestalt	Wang et al., 2017 PMCID: PMC5570149	http://www.webgestalt.org/
bumphunter	Jaffe et al., 2012 PMCID: PMC3304533	http://bioconductor.org/packages/release/bioc/html/bumphunter.html
Aperio ImageScope – Pathology slide viewing Software	NA	https://www.leicabiosystems.com/digital-pathology/manage/aperio-imagescope/
IBM SPSS Statistics software	IBM SPSS Statistics for Windows, Version 25.0. Armonk, NY: IBM Corp.	https://www.ibm.com/analytics/spss-statistics-software
Excel	Microsoft Office	https://office.microsoft.com/excel
Deposited data used
E-MTAB-2266	ArrayExpress	https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-2266/
E-MTAB-6615	ArrayExpress	https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-6615/
Db islet expression	GEO	https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE107489
Plasmids used
Firefly luciferase-eGFP fusion	Addgene	#119816
Renila luciferase	Addgene	#118016
A1a-3kb -firefly luciferase	This study	NA
A1a-2kb -firefly luciferase	This study	NA
A1a-1kb -firefly luciferase	This study	NA
A1b-3kb -firefly luciferase	This study	NA
A1b-2kb -firefly luciferase	This study	NA
A1b-1kb -firefly luciferase	This study	NA
H1–3kb -firefly luciferase	This study	NA
H1–2kb -firefly luciferase	This study	NA
H1–1kb -firefly luciferase	This study	NA
Jb-5kb -firefly luciferase	This study	NA
Jb-3.5kb -firefly luciferase	This study	NA
Jb-2kb-firefly luciferase	This study	NA
A-1.6kB-firefly luciferase	This study	NA
A-1kB-firefly luciferase	This study	NA
WT-Mini-Hspa1a-luc	This study	NA
Mut-Mini-Hspa1a-luc	This study	NA
CMV-Myt1ZF-Vp16	This study	NA