MCB Accepted Manuscript Posted Online 26 January 2015 Mol. Cell. Biol. doi:10.1128/MCB.01404-14 Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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(Article, Molecular and Cellular Biology)
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Lysine-specific demethylase LSD2 suppresses lipid influx and
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metabolism in hepatic cells
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Katsuya Nagaoka1,2, Shinjiro Hino1*, Akihisa Sakamoto1, Kotaro Anan1, Ryuta Takase1,
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Takashi Umehara3, Shigeyuki Yokoyama3, Yutaka Sasaki2 & Mitsuyoshi Nakao1,4*
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Department of Medical Cell Biology, Institute of Molecular Embryology and Genetics, 2 Department of Gastroenterology and Hepatology, Graduate School of Medical
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Sciences, Kumamoto University, Kumamoto, 860-0811, Japan, RIKEN Systems and Structural Biology Center, Yokohama, Japan, 4 Core Research for Evolutional Science and Technology (CREST), Japan Science and
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Technology Agency, Tokyo, Japan.
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*Address correspondence to:
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Mitsuyoshi Nakao M.D., Ph.D. Shinjiro Hino Ph.D.
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Department of Medical Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University 2-2-1 Honjo, chuo-ku, Kumamoto 860-0811, Japan. Phone: +81-96-373-6800; Fax: +81-96-373-6804 E-mail:
[email protected] or
[email protected] 25
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ABSTRACT
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Cells link environmental fluctuations such as nutrition to metabolic remodeling.
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Epigenetic factors are thought to be involved in such cellular process, but the molecular
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basis remains unclear. Here we report that the lysine-specific demethylase LSD2
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suppresses the flux and metabolism of lipids to maintain the energy balance in hepatic
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cells. Using transcriptome and chromatin immunoprecipitation-sequencing analyses, we
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reveal that LSD2 represses the genes involved in lipid influx and metabolism, through
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demethylation of histone H3K4. Selective recruitment of LSD2 at lipid metabolism
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gene loci was mediated in part by a stress-responsive transcription factor c-Jun.
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Intriguingly, LSD2 depletion increased the intracellular levels of many lipid metabolites,
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which was accompanied by an increased susceptibility to toxic cell damage in response
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to fatty acid exposure. Our data demonstrate that LSD2 maintains metabolic plasticity
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under fluctuating environment in hepatocytes, by mediating the crosstalk between the
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epigenome and metabolism.
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INTRODUCTION
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Organisms and cells must adjust their energy strategy to fluctuating nutrient
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availability and other environmental conditions. Epigenetic mechanisms have been
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implicated in the phenotypic plasticity in response to environmental changes, as well as
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in consistent execution of the developmental program (1). It has been shown that
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nutrients and dietary composition potently influence epigenetic marks including DNA
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methylation and histone methylation/acetylation in both humans and animal models (2).
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Because chromatin-modifying enzymes utilize nutrient-derived metabolites as
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substrates and coenzymes, epigenome formation is, by nature, influenced by nutritional
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and metabolic conditions (3-6). Lysine-specific demethylase 1 and 2 (LSD1 and LSD2),
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also respectively known as KDM1A and KDM1B, comprise the flavin-dependent amine
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oxidase family of histone demethylases (7). These enzymes require flavin adenine
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dinucleotide (FAD) as a coenzyme for the removal of methyl groups from the lysine
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residue of histone H3 and other proteins (8, 9). FAD is a vitamin B2-derived metabolite
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that serves as a redox cofactor in key metabolic processes such as fatty acid oxidation
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and succinate dehydrogenation in the tricarboxylic acid cycle (10). Thus, the cellular
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metabolic state may influence the demethylase activity of these proteins. Indeed, we and
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others have previously demonstrated that LSD1 controls energy metabolism genes in
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response to extracellular conditions (11, 12), suggesting that FAD-dependent epigenetic
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factors may link environmental information to metabolic programming. LSD2 was
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identified as a second flavin-dependent histone demethylase that targets methylated
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lysines 4 and 9 of histone H3 (H3K4 and H3K9, respectively) (8, 13-15). Although
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LSD2 has been implicated in the establishment of maternal genomic imprinting in
3
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oocytes (16), little is known about its biological functions, particularly in relation to
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metabolic control.
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In the liver, hepatocytes play a crucial role in the homeostatic control of lipid
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metabolism (17). Hepatocytes incorporate adipose- and diet-derived fatty acids, which
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are either stored by themselves as neutral lipids or redistributed to other tissues in the
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form of very low-density lipoproteins (18). When hepatocytes are exposed to an
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intolerably high amount of fatty acids, for example due to over-feeding, excessive fatty
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acids and their toxic metabolites accumulate in the cells, often leading to the lipotoxic
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liver injury known as nonalcoholic fatty liver disease (NAFLD) (19, 20). Epigenetic
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alterations in the liver have been linked to insulin resistance and NAFLD in humans
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(21), and diet-induced steatosis in mice (22). A recent report by Ahrens et al. examined
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the DNA methylation profiles of liver biopsies from patients with NAFLD and
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non-alcoholic steatohepatitis (NASH), an advanced form of NAFLD (23). Of particular
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note, some disease-state dependent methylation patterns could be reversed after
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improvement of the disease condition by bariatric surgery (23), suggesting that hepatic
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lipid homeostasis is associated with epigenetic plasticity. However, we still lack
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knowledge of whether a specific epigenetic factor could be involved in the homeostatic
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control of hepatic lipid metabolism.
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In the present study, we provide direct evidence that LSD2 plays an essential
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role in the homeostatic control of lipid metabolism in hepatocytes. Our integrative
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investigations
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immunoprecipitation-sequencing (ChIP-seq) analyses reveal that LSD2 suppresses lipid
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transport and metabolism by repressing key metabolic genes through the regulation of
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methylated H3K4 (H3K4me). We further show that LSD2 depletion leads to enhanced
using
the
transcriptome,
4
metabolome
and
chromatin
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lipotoxic cell damage under fatty acid exposure. We propose an epigenetic mechanism
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for ensuring metabolic plasticity in response to lipid overload, in which LSD2 maintains
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the proper expression of lipid metabolism genes in hepatocytes.
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MATERIALS and METHODS
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Cell culture
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HepG2 cells were cultured in high glucose (25 mM D-glucose) Dulbecco’s
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modified Eagle’s medium (Sigma) supplemented with 10% (v/v) heat-inactivated fetal
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bovine serum and penicillin/streptomycin. For the knockdown experiments, specific
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siRNAs were introduced to the cells using RNAiMAX reagent (Invitrogen) when they
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were approximately 50% confluent. After being cultured for 3 to 4 days, semiconfluent
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cells were harvested for subsequent analyses. Target sequences used for siRNA design
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are listed in Table S1. siRNA against firefly luciferase gene was used as a control. For
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the knockdown of c-Jun gene, siGENOME Human JUN siRNA SMARTpool
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(Dharmacon) was used, while siGENOME Non-Targeting siRNA SMARTpool #1
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(Dharmacon) was used as a control.
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Antibodies
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Anti-LSD2 polyclonal antibody was raised in rabbits by administering
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recombinant human LSD2 protein (region 26 – 822), which was prepared using a
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baculovirus expression system. Anti-LSD2 antisera were purified by affinity
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chromatography of the IgG fraction, and were titrated prior to use. Anti-histone H3 5
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(ab1791), anti-H3K4me1 (ab8895) and anti-H3K27ac (ab4729) antibodies were
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purchased from Abcam. The following antibodies were also used: anti-histone
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H3K4me2 (07-030, Millipore), anti-normal rabbit IgG (sc-2027, Santa Cruz), anti-Flag
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M2 (F1804, Sigma-Aldrich).
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Plasmid construction
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To construct pcDNA3-Flag-hLSD2, an LSD2 expression vector, a fragment
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(from +1 to +2,469) of the human LSD2 gene was PCR-amplified using cDNAs from
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HepG2 cells, and cloned into EcoRV and XbaI sites of the pcDNA3-Flag-mock vector.
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Gene expression analysis
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Total RNA from tissues and cells was extracted using Trizol reagent
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(Invitrogen). cDNAs were produced using a ReverTra Ace qPCR RT-Kit (Toyobo).
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Quantitative RT-PCR was performed by the SYBR green method using Thunderbird
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reagents (Toyobo) and an ABI 7300 Sequence Detector (Applied Biosciences). Data are
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presented as mean ± SD. Statistical analyses were performed using two-tailed Student’s
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t-test. Primers used in this study are listed in Table S1.
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Microarray analysis
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Genome-wide expression analysis was performed using a GeneChip Human
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Genome Array U133 Plus 2.0 in combination with a GeneChip Hybridization, Wash and
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Stain Kit (Affymetrix). We prepared three HepG2 samples, by introducing each of three
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different siRNAs against LSD2. We also prepared two control-KD samples. Total RNA
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from cells was extracted, and the sample integrity was confirmed using a Bioanalyzer 6
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RNA 6000 Nano Assay (Agilent). Data annotation analysis was performed using
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GeneSpring GX software (Agilent). GSEA was done using GSEA ver. 2.0 software
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provided
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(http://www.broadinstitute.org/gsea/).
by
the
Broad
Institute
of
MIT
and
Harvard
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Chromatin immunoprecipitation
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In the ChIP experiments for detecting modified histones, cells were
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cross-linked with 1% formaldehyde. Following cell lysis, isolated nuclei were subjected
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to sonication for chromatin fragmentation. Chromatin fragments were incubated at 4 °C
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overnight with appropriate antibodies, followed by a pull-down assay using protein
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A/G-conjugated agarose beads. Purified DNAs were subjected to quantitative PCR
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(qPCR) using the primer sets listed in Table S1. To detect LSD2 enrichment on
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genomic DNA, we employed a protocol for detecting indirect associations between
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protein and DNA (24). Briefly, enhanced cross-linking of chromatin using
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formaldehyde
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3′-dithiobispropionimidate 2HCl (Sigma) was performed to increase the stability of
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protein-DNA complexes. Chromatin fragmentation was done by sonication in regular
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RIPA buffer containing 0.1% SDS, followed by immunoprecipitation, as described
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above.
and
the
protein-protein
chemical
cross-linker
dimethyl
3,
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ChIP-seq analysis
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For ChIP-seq analysis to detect LSD2 or H3K4me1/DNA interactions, 1×107
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HepG2 cells were collected for a ChIP experiment. After crosslinking, chromatin DNA
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was fragmented using a Covaris S220 sonicator (Covaris Inc.), followed by 7
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immunoprecipitation with either an anti-LSD2 or an anti-H3K4me1 polyclonal antibody.
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The protein-bound chromatin fraction was collected using Dynabeads Protein A/G (Life
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Technologies), and the DNA was purified. A DNA fragment library for sequencing was
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constructed using an Ion Fragment Library Kit (Life Technologies). Adopter-ligated
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DNA fragments were purified using Agencourt AMPure XP (Beckman Coulter Inc.).
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High throughput sequencing was performed using Ion PGM and Ion Proton
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semiconductor sequencers (Life Technologies), according to the manufacturer’s
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instruction. All sequence data obtained from Ion PGM and Proton were merged before
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alignment onto the human reference genome hg19 using the BWA algorithm (CLC
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Genomics Workbench Software). With the same software, the duplicate reads and low
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read/mapping quality reads were trimmed out. For LSD2 ChIP-seq, final numbers of
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mapped reads were 36,409,704 reads for LSD2, and 27,141,872 reads for Input. For
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H3K4me1 ChIP-seq, final numbers of mapped reads were 33,087,078 reads for the
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control siRNA sample, and 53,725,212 reads for input of the control siRNA sample;
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49,410,448 reads for the LSD2-KD sample, and 58,829,288 reads for input of the
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LSD2-KD sample.
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Peak detection was done using the MACS algorithm in Avadis NGS software.
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LSD2 binding sites were detected based on the LSD2 peaks significantly enriched over
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input peaks at a cutoff value of p=10-5. H3K4me1 peaks enhanced by LSD2-KD were
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detected based on the H3K4me1 peaks in LSD2-KD samples significantly enriched over
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H3K4me1 peaks in control siRNA sample peaks at a cutoff value of p=10-5.
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For identification of the direct regulatory target genes of LSD2, we first
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detected 414,095 LSD2 peaks using the MACS algorithm (25) and selected 15,532
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robust peaks that met the criteria of >25 reads and >5-fold enrichment. Based on this 8
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peak detection, 6,079 neighboring genes were identified within 5,000 bases of the LSD2
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peaks using Avadis NGS software with the gene annotation provided by Ensembl.
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Among these genes, we identified 226 genes with >1.5-fold expression change in
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response to LSD2-KD.
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For identifying genes with both increased H3K4me1 and expression change in
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response to LSD2-KD, we first detected 180,505 exclusive H3K4me1 peaks under
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LSD2-KD using the MACS algorithm, and selected 12,735 robust peaks that met the
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criterion of >30 reads and >5-fold enrichment. Based on this peak detection, 5,768
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neighboring genes were identified within 5,000 bases of the H3K4me1 peaks using
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Avadis NGS with the gene annotation provided by Ensembl. Of these genes, we
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identified 207 with >1.5-fold expression change in response to LSD2-KD.
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Visualization of ChIP-Seq data with smoothing (smoothing window size = 2
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bp) was done using Avadis NGS. ChIP-seq data for chromatin modifications in HepG2
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cells were obtained from the ENCODE/Broad Institute via the UCSC Genome Browser
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website (http://genome.ucsc.edu/). The accession numbers of the files are as follows:
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wgEncodeEH000082
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(GSM733743) for H3K27ac. ChIP-seq data for p300 enrichment in HepG2 cells was
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from ENCODE/Stanford/Yale/USC/Harvard (wgEncodeEH001862), FAIRE data was
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from ENCODE/OpenChrom (UNC Chapel Hill) (wgEncodeEH000546 (GSM864354)),
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c-Jun data was from ENCODE/Stanford/Yale/USC/Harvard (wgEncodeEH001794) and
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c-Myc data was from ENCODE/Open Chrom (UT Austin) (wgEncodeEH000542
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(GSM822286)).
(GSM733693)
for
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Correlation analyses of ChIP-seq peaks 9
H3K4me2
and
wgEncodeEH000094
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Correlations of LSD2 peaks with histone modifications were analyzed using
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GenomeInspector
(Genomatix)
according
to
the
manufacturer’s
instructions
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(https://www.genomatix.de/online_help/help_regionminer/GenomeInspector.html).
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reference data sets, histone modification data of HepG2 cells were obtained from the
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ENCODE/Broad Institute via the UCSC Genome Browser website. The accession
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numbers of the files used in the analyses are as follows: wgEncodeEH001749
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(GSM798321) for H3K4me1, wgEncodeEH001023 (GSM733754) for H3K27me3.
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LSD2 peaks were selected using the criteria of >28 reads and >5-fold enrichment from
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the peaks detected using the MACS algorithm. Peaks for H3K4me1 and H3K27me3
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were detected using NGS analyzer (Genomatix) or the MACS algorithm in Avadis
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NGS.
As
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Metabolomic analysis
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Metabolomic analyses were performed using CE-TOFMS and LC-TOFMS at
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Human Metabolome Technologies (HMT, Japan, http://humanmetabolome.com).
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Triplicate samples of control and LSD2-KD2 cells were subjected to both CE-TOFMS
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and LC-TOFMS, while LSD2-KD1 was analyzed by LC-TOFMS. Cells were washed
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with PBS and collected by trypsinization, then washed twice with 5% (w/w) mannitol
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solution at room temperature. Cells were re-dispersed in LC/MS grade methanol
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(Wako) for CE-TOFMS or LC/MS grade ethanol (Wako) for LC-TOFMS, both
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containing HMT’s Internal Standard Solution 1. Peaks were extracted as previously
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described (26). After quantification, metabolite concentrations were normalized by cell
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number. Of ~900 (for CE-TOFMS) and ~500 (for LC-MS) metabolites from the HMT
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databases, 275 metabolites were detected above the signal-to noise threshold (listed in 10
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Table S4). The databases include glycolysis and TCA cycle intermediates, amino acids,
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nucleic acids, nucleotides, nucleosides, Coenzyme A, organic acids, nicotinamide
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coenzymes, fatty acids, bile acids, lipids, steroid derivatives and polyphenols.
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Clustering of metabolites according to changes in concentration was
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performed using Cluster 3.0, which was obtained from the Laboratory of DNA
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Information Analysis of the Human Genome Center in the Institute of Medical Science,
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The
240
(http://bonsai.hgc.jp/~mdehoon/software/cluster/software.htm#ctv/).
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clustering was calculated using Pearson correlation (centered correlation) and complete
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Linkage. Clustering results were visualized using Java TreeView software (27).
University
of
Tokyo Hierarchical
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Fatty acid uptake assay
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Cellular fatty acid uptake was tested using QBT Fatty Acid Uptake Assay Kit
246
(Molecular Devices), which uses dodecanoic acid conjugated with BODIPY, a
247
fluorescent dye (BODIPY-DA). Fatty acid uptake assay stock solutions were dissolved
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completely by adding 10 ml of 1× HBSS buffer (1× Hank’s balanced salt solution with
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20 mM HEPES and 0.2% fatty acid-free bovine serum albumin). HepG2 cells were
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cultured in the medium containing the assay reagent for 10 min at 37 °C. Cells were
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either subjected to microscopic analysis, or trypsinized for fluorescence-activated cell
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sorting analysis, using a FACS Canto cytometer (Becton Dickinson).
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Lipotoxicity analysis
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To analyze cellular lipotoxicity, siRNA-introduced HepG2 cells were treated
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with bovine serum albumin (BSA)-conjugated oleic acid for 48 hours. Following 11
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trypsinization, cells were counted using an automatic cell imaging counter, Cytorecon
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(GE Healthcare). For oleic acid conjugation to BSA, a sodium oleate (≥99% (capillary
259
GC), Sigma) solution in 150 mM NaCl was added to a fatty acid-free BSA solution
260
(Wako). The final molar ratio of oleic acid to BSA was 6:1.
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Animal studies
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Animal experiments were conducted in accordance with the guidelines of the
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Animal Care and Use Committee of Kumamoto University. For NAFLD induction,
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7-week-old male C57BL/6J mice were fed a high fat diet (HFD) containing 21% kcal
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fat (A02082003BP: Research Diets Inc.) or a methionine/choline-deficient diet (MCD,
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A02082002BG: Research Diets Inc.), which also contains 21% kcal fat for four weeks.
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Body weight was monitored weekly throughout the test period. After 16-hour fasting,
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liver tissues were dissected, and the sections were either snap-frozen in liquid nitrogen
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for RNA analyses or fixed with formalin for histological analyses.
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Statistical analyses All statistical analyses between two groups were done by Student’s t-test unless otherwise stated. Equality of variance was examined using F-test.
275 276
Accession Number
277
The accession number of microarray and ChIP-seq data in GEO is GSE59695.
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RESULTS 12
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LSD2 selectively represses lipid metabolism genes in hepatocytes
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Because LSD1 regulates the metabolic genes under diverse cellular contexts
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(11, 12, 28), we tested, in this study, whether another FAD-dependent demethylase
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LSD2 could be involved in the metabolic programming. During our initial examinations
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in mouse tissues, we found that LSD2 was expressed more highly in the liver than in
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other metabolic tissues (Fig. 1A). To gain insight into the role of LSD2 function in
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hepatocytes, we depleted LSD2 in HepG2 human hepatic cells using three different
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siRNAs (Fig. 1B), then carried out an expression microarray experiment. We detected
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1,362 probe sets with more than 1.5-fold difference between the control and
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LSD2-knockdown (KD) cells (Fig. 1C). Of these, 906 probe sets were up-regulated,
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while 456 were down-regulated. Using gene set enrichment analysis (GSEA) (29, 30),
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we established that genes associated with “metabolism of lipids and lipoproteins” were
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significantly enriched in the probe sets that were up-regulated by LSD2-KD (statistical
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threshold, p-values