KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| anti-MITF monoclonal antibody C5 | Made in the lab of Dr. David E. Fisher | (King et al., 1999) C5 |
| Mouse monoclonal anti-tyrosinase antibody, clone T311 | Sigma-Adlrich | Cat# 05-647: RRID:AB_309873 |
| Donkey anti-rabbit IgG-HRP | ThermoFisher Scientific | Cat# 45-000-683: RRID: AB_2721111 |
| Amersham ECL mouse IgG, HRP | ThermoFisher Scientific | Cat#45000680;RRID: AB_2721110 |
| Monoclonal anti-β-actin-peroxidase | Sigma Aldrich | Cat# A3854;RRID:AB_262011 |
| Alexa Fluor 555 goat anti-rabbit IgG (H+L), secondary antibody | ThermoFisher Scientific | Cat# A-21428;RRID: AB_2535849 |
| Alexa Fluor 647 goat anti mouse IgG (G+L); fluorescence conjugated secondary antibody | ThermoFisher Scientific | Cat# A-21236;RRID: AB_2535805 |
| Alexa Fluor 594 F(ab)2 fragment of goat anti-rabbit IgG (G+L); fluorescence conjugated secondary antibody | ThermoFisher Scientific | Cat# A-11072;RRID: AB_2534116 |
| Alexa Fluor 488-conjugated donkey anti-mouse secondary antibodies | ThermoFisher Scientific | Cat# A-21202;RRID: AB_141607 |
| Anti-Cyclobutane Pyrimidine Dimers (CPDs) mAb antibody (Clone TDM-2) | Cosmo Bio USA | Cat# CAC-NM-DND-001; RRID: AB_1962813 |
| Rabbit anti-γ-H2AX (P-ser139) polyclonal antibody | NOVUS Biologicals | Cat# NB100-384;RRID:AB_10002815 |
| Mouse monoclonal anti-Mitofusin 2 antibody [6A8] | Abcam | Cat# ab56889;RRID: AB_2142629 |
| Rabbit polyclonal anti TRP2/DCT antibody | Abcam | Cat# ab74073;RRID:AB_1524517 |
| Mouse monoclonal anti-NNT antibody [8B4BB10] | Abcam | Cat# ab110352;RRID:AB_10887748 |
| Rabbit anti-NNT (C-terminal) polyclonal antibody | Abcam | Cat# ab214212; RRID:AB_2889980 |
| Mouse anti-8-oxo-dG monoclonal antibody | Trevigen | Cat# 4354-MC-050; RRID:AB_1857195 |
| IDH1 (D2H1) Rabbit mAb | Cell Signaling Technology | Cat# 8137;RRID: AB_10950504 |
| Mouse monoclonal p53 antibody [PAb 240] | Abcam | Cat# ab26;RRID:AB_303198 |
| Rabbit monoclonal TRP1 antibody [EPR21960] | Abcam | Cat# ab235447; RRID:AB_2889980 |
| Mouse monoclonal antibody Pmel17 (E-7) | Santa Cruz Biotechnology | Cat# sc-377325; RRID:AB_2889982 |
| LC3B (D11) rabbit monoclonal antibody | Cell Signaling Technology | Cat#38668S RRID:AB_2137707 |
| Biological Samples | ||
| Full thickness human breast and abdominal skin explants | Massachusetts General Hospital | IRB# 2013P000093 |
| Paraffin-embedded formalin fixed slides, prepared from breast and abdominal biopsy samples | Massachusetts General Hospital | IRB# 2013P000093 |
| Human skin samples for genome wide association study (GWAS) | Massachusetts General Hospital or the Cooperative Human Tissue Network | IRB# 2013P000093 |
| Chemicals, Peptides, and Recombinant Proteins | ||
| 3-isobutyl-1-methylxanthine (IBMX) | Sigma-Aldrich | Cat# I5879 |
| 12-O-tetradecanoylphorbol-13-acetate (TPA) | Sigma-Aldrich | Cat# 16561-29-8 |
| Ham's F10 | Thermo Fisher Scientific | Cat# MT10070CV |
| N6,2′-O-Dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt | Sigma-Aldrich | Cat# D0627 |
| Penicillin-Streptomycin | Thermo Fisher Scientific | Cat# 15140163 |
| Na3VO4 | Sigma-Aldrich | Cat# 450243 |
| Medium 254 | Life Technologies | Cat# M254500 |
| 0.05% Trypsin-EDTA w/ phenol red | Life Technologies | Cat# 25300120 |
| Human Melanocyte Growth Supplement (HMGS) | Life Technologies | Cat# S0025 |
| Bovine Serum Albumin | Sigma | Cat#A7030 |
| Goat serum | Sigma-Aldrich | Cat# G9023 |
| RPMI (Roswell Park Memorial Institute 1640 Medium) | Life Technologies | Cat# 11875119 |
| RIPA lysis buffer | Sigma-Aldrich | Cat# R0278 |
| FetalPlex Animal Serum Complex | Gemini Bio-Products | Cat# 100-602 |
| Western Lightning Plus-ECL | PerkinElmer | Cat # NEL105001EA |
| Non-fat milk powder | Boston BioProducts | Cat# P-1400 |
| Protein Block | Agilent | Cat# X090930-2 |
| Antibody Diluent | DAKO | Cat# S3022 |
| VECTASHIELD® HardSet™ Antifade Mounting Medium with DAPI | Vector Laboratories | Cat# H-1500 |
| synthetic melanin | Sigma Aldrich | Cat# M8631 |
| N,N-Dicyclohexylcarbodiimide [DCC] | Sigma Aldrich | Cat# D80002 |
| 2,3-Butanedione [2,3BD] | Sigma Aldrich | Cat# B85307 |
| Palmitoyl coenzyme A lithium salt | Sigma Aldrich | Cat# #P9716 |
| cycloheximide (CHX) | Sigma Aldrich | Cat# C7698 |
| NADPH | Sigma Aldrich | Cat# N7505 |
| N-Acetyl-L-cysteine (NAC) | Sigma Aldrich | Cat# A7250 |
| MitoTEMPO | ThermoFisher Scientific | Cat# 501872447 |
| Hydrogen peroxide solution | Sigma Aldrich | Cat# 216763 |
| SYBR FAST qPCR master mix | Kapa Biosystems | Cat# KK4600; |
| Protease and Phosphatase Inhibitor | ThermoFisher Scientific | Cat# PI78445 |
| Western Lightning Plus-ECL, Enhanced Chemiluminescence Substrate | Perkin Elmer | Cat# NEL105001EA |
| MitoSOX Red | ThermoFisher Scientific | Cat# M36008 |
| CM-H2DCFDA | ThermoFisher Scientific | Cat# C6827 |
| NucBlue | ThermoFisher Scientific | Cat# R37605 |
| Polybrene | Sigma-Aldrich | Cat# TR-1003 |
| Paraformaldehyde 16% | ThermoFisher Scientific | Cat# 50980487 |
| Ethanol | Thermo Fisher Scientific | Cat# 04355226 |
| Triton X-100 | Sigma Aldrich | Cat# T8787 |
| TWEEN® 20 | Sigma Aldrich | Cat# P7949 |
| Forskolin from Coleus forskohlii, ≥98% | Sigma Aldrich | Cat# F6886 |
| Lipofectamine RNAiMAX Transfection Reagent | Life Technologies | Cat# 13778150 |
| IQ5 High-fidelity DNA Polymerase | New England Biolabs | Cat# M0491S |
| Critical Commercial Assays | ||
| Direct cAMP ELISA Kit | Enzo Life Sciences | Cat# ADI-901-066 |
| GSH/GSSG-Glo Assay | Promega | Cat# V6611 |
| CellTiter-Glo Luminescent Cell Viability Assay | Promega | Cat# G7570 |
| Pierce BCA protein assay | ThermoFisher Scientific | Cat# 23225 |
| KAPA Library Quantification Kits | Roche | Cat# 7960140001 |
| MiSeq Reagent Kits v2 (300 cycles) | Illumina | Cat# MS-102-2002 |
| MaxBlock Autofluorescence Reducing Reagent Kit | MaxVision Biosciences | Cat# MB-L |
| Fontana-Masson Stain Kit (Melanin Stain) | Abcam | Cat# ab150669 |
| Dual Reporter System | GeneCopoeia | Cat# HPRM39435-LvPM02 |
| Secrete-Pair Gaussia Luciferase Assay Kit | GeneCopoeia | Cat# LF062 |
| NADP/NADPH-Glo Assay | Promega | Cat# G9082 |
| QUANTI-Blue™ Solution | InvivoGen | Cat# rep-qbs |
| RNeasy Plus Mini Kit | Qiagen | Cat# 74136 |
| Deposited Data | ||
| Raw data supporting the human genetics association analyses | https://www.dropbox.com/scl/fi/ahdfnjo4puwzdz8ayw2ix/supporting-data-human-genetic-associations-2.xlsx?dl=0&rlkey=gvbpat4tjb6bnekgnxuu1a6yh | |
| Publicly Available Data | ||
| Ensembl database information for promoter ENSR00000180214 of the NNT gene | Ensembl | http://www.ensembl.org/Homo_sapiens/Regulation/Summary?fdb=funcgen;r=5:43600000- |
| GTEx expression database | GTEx | www.gtexportal.org |
| GWAS summary statistics from the CANDELA cohort | GWAS Central | http://www.gwascentral.org/study/HGVST3308 |
| GWAS summary statistics from the UK Biobank cohort | https://cnsgenomics.com/software/gcta/#DataResource | |
| Experimental Models: Cell Lines | ||
| Primary human melanocytes (isolated from neonatal foreskins) | Massachusetts General Hospital | IRB# 2013P000093 |
| Human melanoma cell line UACC257 | National Cancer Institute Division of Cancer Treatment and Diagnosis (DCTD) Tumor Repository | |
| Human melanoma cell line SK-MEL-30 | Memorial Sloan Kettering Cancer Center | https://www.mskcc.org/ |
| Mouse Melan-A cell line | Wellcome Trust Functional Genomics Cell Bank | Bennett et al., 1987 |
| Primary human keratinocytes isolated from discarded surgical human skin tissue (e.g., foreskins) | Massachusetts General Hospital | IRB# 2013P000093 |
| Primary human fibroblasts isolated from discarded surgical human skin tissue (e.g., foreskins) | Massachusetts General Hospital | IRB# 2013P000093 |
| Lenti-X™ 293T cells | Clontech | Cat# 632180 |
| Experimental Models: Organisms/Strains | ||
| C57BL/6J mice | Jackson Laboratory | Stock No: 000664 |
| C57BL/6NJ mice | Jackson Laboratory | Stock No: 005304 |
| Casper zebrafish (mitfa−/−;roy−/−) embryos | Laboratory of Dr. Leonard I. Zon | |
| Oligonucleotides | ||
| nheIkozakHAhNNT_f1: forward, 5’-ctagctagcCCGCCACCATGTACCCATACGATGTTCCAGATTACGCTGCAAACCTATTGAAAACAGTGGTGACTG-3’ | eurofins Genomics | For PLMJ1- HA-NNT |
| hNNTnheI_r1: reverse, 5’-ctagctagcTTACTTCTGATAGGATTCTCTAACTTTCGC-3’ | eurofins Genomics | For PLMJ1- HA-NNT |
| nheIkozakhMFN2_f1: forward, 5’-ctagctagcGCCACCATGTCCCTGCTCTTCTCTCGATGC-3’ | eurofins Genomics | For PLMJ1- HA MFN2 |
| hMFN2(HA)nheI_r1: reverse, 5’-ctagctagcTTAGGATCCAGCAGCGTAATCTGGAAC-3’ | eurofins Genomics | For PLMJ1- HA MFN2 |
| RT-Primers for NNT, IDH1, MFN2, TYRP1, DCT/TRP2, MITF, POMC, PPARGC1A, Tyrosinase: See Table in STAR Methods | ||
| Additional oligonucleotides that were used for nnt knockout in zebrafish are in Supplementary Table 3 | ||
| Recombinant DNA | ||
| CRISPR MiniCoopR-U6:gRNA-mitfa:Cas9 plasmid | Addgene | Cloned from Addgene plasmid ID 118840 |
| pMiniCoopR-mitfa:NNT expression plasmid | Addgene | Cloned from Addgene plasmid ID 118850 |
| pLMJ1-NNT-HA | This manuscript | Based on Addgene plasmid, # 19319 |
| pLMJ1-EGFP plasmid | Laboratory of Dr. David Sabatini | Addgene plasmid, # 19319; http://n2t.net/addgene:19319; RRID:Addgene_19319, (Sancak et al., 2008) |
| pcDNA3.1 Mfn2HA | Laboratory of Dr. Allan Weissman | Addgene plasmid, # 139192; http://n2t.net/addgene:139192; RRID:Addgene_139192 ((Leboucher et al., 2012) |
| PLMJ1-MFN2-HA | This manuscript | Based on Addgene plasmid, # 139192 and Addgene plasmid, # 19319 |
| Software and Algorithms | ||
| GraphPad Prism 8.4.3 (471) | GraphPad | https://www.graphpad.com/scientific-software/prism/ |
| ImageJ (v1.8.0) | National Institutes of Health | https://imagej.nih.gov/ij/ |
| FIJI software for pixel-based color quantification | FIJI | https://imagej.net/Fiji |
| Off-target prediction software (for design of guide RNAs) | (Bae et al., 2014) | http://www.rgenome.net/cas-offinder/ |
| On-target prediction software (for design of guide RNAs); CRISPRscan and CHOPCHOP | (Moreno-Mateoset al., 2015) (Labun et al., 2019) |
https://www.crisprscan.org/ https://chopchop.cbu.uib.no/ |
| Axiovision REL 4.7 software | Zeiss | https://carl-zeiss-axiovision-rel.software.informer.com/4.7/ |
| CRISPResso2 software (for genome editing) | Kendell Clement et al | Nat Biotechnol. 2019 March; 37(3):224–226. |
| MACH software | http://csg.sph.umich.edu/abecasis/mach/index.html | |
| GCTA program | Yang et al., 2011 | https://cnsgenomics.com/software/gcta/ |
| PLINK program | https://www.cog-genomics.org/plink/1.9/ | |
| BioRender | BioRender.com | |
| Fiji | Saalfeld, S., Schmid, B., et al. (2012) | https://imagej.net/Fiji |
| NDP.view2 Viewing software | HAMAMATSU | https://www.hamamatsu.com/us/en/product/type/U12388-01/index.html |
| Other | ||
| siGENOME Human MITF siRNA SMARTpool | Dharmacon | Cat# M-008674-00-0005 |
| ON-TARGETplus Human IDH1 siRNA SMARTpool | Horizon Inspired Cell Solutions | Cat# L-008294-01-0005 |
| ON-TARGETplus Human MFN2 siRNA SMARTpool | Horizon Inspired Cell Solutions | Cat# L-012961-00-0005 |
| ON-TARGETplus Human NNT siRNA SMARTpool | Horizon Inspired Cell Solutions | Cat# L-009809-00-0005 |
| ON-TARGETplus Human PPRGC1A siRNA SMARTpool | Horizon Inspired Cell Solutions | Cat# L-005111-00-0005 |
| ON-TARGETplus Human Tyrosinase siRNA SMARTpool | Horizon Inspired Cell Solutions | Cat# L-012555-00-0005 |
| ON-TARGETplus non-targeting siRNA control pool | Horizon Inspired Cell Solutions | Cat# D-001810-10-05 |
| 4-15% Criterion TGX Precast Midi Protein gels | Bio-Rad Laboratories | Cat# 5671084 |
| Chamber slides | ThermoFisher Scientific | Cat# 125657 |