Supplemental Information. Integrative Transcriptome Analyses. of Metabolic Responses in Mice Define. Pivotal LncRNA Metabolic Regulators

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1 Cell Metabolism, Volume 24 Supplemental Information Integrative Transcriptome Analyses of Metabolic Responses in Mice Define Pivotal LncRNA Metabolic Regulators Ling Yang, Ping Li, Wenjing Yang, Xiangbo Ruan, Kurtis Kiesewetter, Jun Zhu, and Haiming Cao

2 A. B. Mouse feeding Liver RNA extracaon Ad Libitum Fast GCK AK35241 AK3369 AK539 AK79937 Gm16551 AK Ad libitum Fast Expression Profiling 18,431 non-redundant lncrnas 25,376 coding transcripts Fast Fast (285) (237) lncrnas AK45415 AK7778 AK1936 AK18645 AK85787 AK47141 AK16437 Ad libitum Fast C. RPKM Correla,on between mircoarray and RNA-seq >.5 r =.8 >.1 r =.75 > (all) r =.71 Figure S1, related to Figure 1. Genome-wide analyses of liver lncrnas and in mice during fasting and refeeding. (A) Experimental outline. MRNAs and lncrnas in the livers of mice fed ad libitum (Ad Libitum), subject to a 24-hour fast (Fast), or a 24-hour fast followed by a 4-hour refeeding () were analyzed by Mouse LncRNA Array V2. (8 x 6K, ArrayStar). P value <.5 and fold change >= 2 were used to identify differentially expressed genes (Fast vs Ad libitum or vs Fast, n=4). The numbers of genes regulated by Fast and are indicated, and the numbers of genes regulated by Fast and reversed by are indicated in brackets. (B) Validation of microarray results by quantitative real-time PCR analysis. Expression levels of one mrna, glucose kinase (GCK) and 13 lncrnas were quantified. Error bars are SEM, n=4. P<.5 (Fast vs Ad libitum), p<.5 ( vs Fast). (C) Pearson correlation of log2-transformd fold changes between microarray and RNA-seq for differentially expressed lncrnas identified by microarray.

3 A. B. Liver Adipose Muscle Group Condi+on Sample ID Group Condi+on Sample ID Group Condi+on Sample ID CH-1 FR-A1 FR-M1 Ad CH-2 Ad FR-A2 Ad FR-M2 Libitum CH-3 Libitum FR-A3 Libitum FR-M3 CH-4 FR-A4 FR-M4 CH-9 FR-A9 FR-M9 Group1 Fast CH-1 FR-A1 FR-M1 Group1 Fast Group1 Fast CH-11 FR-A11 FR-M11 CH-12 FR-A12 FR-M12 CH-17 FR-A17 FR-M17 CH-18 FR-A18 FR-M18 CH-19 FR-A19 FR-M19 CH-2 FR-A2 FR-M2 HD-L6 HD-F6 Normal HD-L9 Normal HD-F9 Normal HD-M9 diet HD-L15 diet HD-F15 diet HD-M15 control HD-L26 control HD-F26 control HD-M26 HD-L32 HD-F32 HD-M32 HD-L7 HD-F7 HD-M7 HD-L13 HD-F13 HD-M13 Group2 HFD 48h HD-L14 Group2 H48h HD-F14 Group2 H48h HD-M14 HD-L16 HD-F16 HD-M16 HD-L17 HD-F17 HD-L19 HD-F19 HD-L25 HD-F25 HD-M25 HFD 12w HD-L3 H12w HD-F3 H12w HD-M3 HD-L31 HD-F31 HD-M31 HD-L33 HD-F33 HD-M33 OB-L2 OB-F2 OB-M2 OB-L6 OB-F6 WT OB-L14 WT OB-F14 WT OB-M14 OB-L18 OB-F18 OB-M18 Group3 OB-L2 Group3 OB-F2 Group3 OB-M2 OB-L3 OB-F3 OB-L7 OB-F7 OB-M7 ob/ob OB-L9 ob/ob OB-F9 ob/ob OB-M9 OB-L13 OB-F13 OB-M13 OB-L15 OB-F15 OB-M15 C. lncrna proportion % 13.99% Exon number Relaonship of lncrnas to 3.92% 5.49% 8.95% 54.19% >14 Bidirec1onal Intergenic Intronic an1sense An1sense Sense intronic Sense overlapping D. E. F. Fast Liver Fast Adipose Fast Muscle oxidation reduction steroid biosynthetic process sterol biosynthetic process cholesterol biosynthetic process steroid metabolic process detection of abiotic stimulus response to abiotic stimulus regulation of cyclic nucleotide metabolic process regulation of nucleotide metabolic process energy reserve metabolic process..1.2 regulation of apoptosis regulation of programmed cell death regulation of cell death negative regulation of apoptosis negative regulation of programmed cell death..5.1 steroid biosynthetic process cofactor metabolic process oxidation reduction hexose metabolic process coenzyme metabolic process Liver..2.4 defense response intracellular signaling cascade positive regulation of biosynthetic process positive regulation of nitrogen compound metabolic process positive regulation of cellular biosynthetic process Adipose..2.4 positive regulation of cellular biosynthetic process positive regulation of biosynthetic process positive regulation of macromolecule metabolic process positive regulation of gene expression positive regulation of nitrogen compound metabolic process Muscle h HFD Liver oxidation reduction proteolysis involved in cellular protein catabolic process cellular protein catabolic process modification-dependent protein catabolic process modification-dependent macromolecule catabolic process..1.2 lipid biosynthetic process sterol biosynthetic process sterol metabolic process cholesterol biosynthetic process M phase of mitotic cell cycle 48h HFD Adipose fat cell differentiation acute-phase response acute inflammatory response white fat cell differentiation response to wounding 48h HFD Muscle w HFD Liver 12w HFD Adipose 12w HFD Muscle drug metabolic process positive regulation of macromolecule metabolic process regulation of multicellular organism growth positive regulation of gene expression cell surface receptor linked signal transduction..5.1 response to wounding inflammatory response cell activation chemotaxis taxis muscle cell differentiation RNA splicing mrna metabolic process mrna processing RNA splicing..5.1 ob/ob Liver ob/ob Adipose ob/ob Muscle oxidation reduction fatty acid metabolic process response to wounding lipid biosynthetic process steroid metabolic process..1.2 inflammatory response response to wounding immune response defense response phagocytosis in utero embryonic development chordate embryonic development embryonic development ending in birth or egg hatching regulation of growth cellular component morphogenesis..5.1

4 Figure S2, related to Figure 1. Sample information and biological processes regulated by various metabolic conditions. (A) Sample information. (B) Distribution of exon numbers of the lncrna transcripts covered in this study. This exon information is obtained from the NONCODE 216 mouse database. (C) Distribution of different types of lncrnas covered in this study based on their relative position to nearby coding genes. (D-F) Top 5 gene ontology (GO) biological process terms for differentially expressed in each metabolic condition in liver (D), adipose (E), and muscle tissue (F).

5 A. B. C. Liver Adipose Muscle steroid biosynthetic process oxidation reduction acyl-coa metabolic process steroid metabolic process lipid biosynthetic process cholesterol biosynthetic process cholesterol metabolic process sterol metabolic process fatty acid metabolic process sterol biosynthetic process cofactor metabolic process coenzyme metabolic process isoprenoid biosynthetic process isoprenoid metabolic process acute inflammatory response M phase of mitotic cell cycle mitosis nuclear division mitotic cell cycle organelle fission M phase cell division cell cycle phase cell cycle cell cycle process lipid biosynthetic process steroid metabolic process hexose metabolic process cholesterol metabolic process sterol metabolic process acute-phase response fatty acid metabolic process fatty acid biosynthetic process acute inflammatory response osteoblast development negative regulation of molecular function organic acid biosynthetic process carboxylic acid biosynthetic process response to cytokine stimulus negative regulation of cell differentiation D. E. F. Liver Adipose Muscle lncrnas lncrnas lncrnas G. H. I. Liver Adipose Muscle lncrnas 29 lncrnas lncrnas Figure S3, related to Figure 2. GO biological process terms for highly regulated and correlation analyses of metabolically sensitive lncrnas with coding genes. (A-C) Top GO terms for that were regulated at least three times in liver (A), adipose (B) and muscle (C). (D-F) Heat map with hierarchical clustering of the positive correlations between protein coding and the metabolically sensitive lncrnas in liver (D), adipose (E), and muscle (F) by Pearson s correlation method. (G-I) Heat map with hierarchical clustering of the negative correlations between protein coding and the metabolically sensitive lncrna genes in liver (G), adipose (H), and muscle (I) by Pearson s correlation method.

6 A. Exisng annotaon from Ensemble B. C YFP SREBP1c lacz sh Gm16551 sh2 D. mg/dl Plasma TG lacz sh Gm16551 sh2 Iden%fied mouse liver transcript by RACE E. mg/g liver lacz sh Liver TG Gm16551 sh1 Gm16551 sh2 I. J. IdenEEes: 67% E value: 4e Gm16551 sequence (Ensemble) Human homolog of Gm16551 (RP11-29K1.2, Ensemble) F. Relative gene expression lacz sh+yfp Gm16551 sh1+yfp Gm lacz sh+dnsrebp1c Gm16551 sh1+dnsrebp1c PCK Fetal Heart Fetal Kidney Fetal Liver FAS G. Gm16551 human homolog Fetal Muscle Heart Gm16551 RNA level Kidney YFP+Vector SREBP1c+Vector SREBP1c+Gm16551 Liver Muscle. Gm16551 K. H. Onecut1 mg/g liver Human Liver Gm16551 human homolog ACLY Normal liver FAS Liver TG YFP+Vector SREBP1c+Vector SREBP1c+Gm16551 Fatty liver SCD1 Figure S4, related to Figure 4. Analysis of an SREBP1c induced lncrna, Gm (A) Browser shot of Gm16551 region from Ensembl and the Gm16551 transcript is defined by RACE using mouse liver samples. (B) Expression levels of fatty acid synthase (FAS) gene in mouse liver transduced with YFP or constitutively active mouse SREBP1c adenoviruses. Error bars are SEM, n=5. P<.5. (C) Expression levels of Gm16551, Onecut1, ACLY, FAS and SCD1 in the livers of control (lacz sh) and Gm16551 knockdown (Gm16551 sh2) mice. Error bars are SEM, n=7. P<.5. (D) Plasma TG levels in control (lacz sh) and Gm16551 knockdown (Gm16551 sh2) mice. Error bars are SEM, n=7. P<.5. (E) Liver TG contents in control (lacz sh) and Gm16551 knockdown (Gm16551 sh1/sh2) mice. Error bars are SEM, n=6. P<.5. (F) Expression levels of Gm16551 and PCK1 in the livers of mice receiving lacz sh plus YFP, lacz plus dominant negative SREBP1c, Gm16551 knockdown (Gm16551 sh1) plus YFP, and dominant negative SREBP1c plus Gm16551 knockdown adenoviruses. Error bars are SEM, n=5. P<.5 (compared to control). (G) Expression levels of Gm16551 in the livers of mice receiving YFP plus vector, active SREBP1c plus vector, and active SREBP1c plus

7 Gm16551 adenoviruses. Error bars are SEM, n=7. P<.5. (H) Liver TG contents in mice receiving YFP plus vector, active SREBP1c plus vector, and active SREBP1c plus Gm16551 adenoviruses. Error bars are SEM, n=7. P<.5. (I) The alignment of mouse Gm16551 with its human homolog. (J) Quantitative real-time PCR analysis of human homolog of Gm16551 in human tissues of fetal heart, adult heart, kidney, liver and muscle. (K) Expression levels of human homolog of Gm16551 in the liver of patients with fatty liver disease. Error bars are SEM, n=5. P<.5. SUPPLEMENTAL TABLES AND LEGENDS Table S1, related to Figure 1. RNA-seq results of differentially expressed lncrnas identified by microarray. One sheet in this table, for those differentially expressed lncrnas (fasting vs ad libitum) identified by microarray, NONCODE IDs and their log2 transformed fold changes in both microarray and RNA-seq are listed. Table S2, related to Figure 2. Metabolically sensitive and lncrnas in liver, adipose and muscle tissue. Four sheets in this table, seqnames of metabolically sensitive from liver, adipose and muscle are listed in one sheet. Metabolically sensitive lncrnas for each tissue are listed in individual sheet with their chromosome, transcript start and end sites, strand, exons number, transcript length information. Table S3, related to Figure 2. Correlation values of liver, adipose, and muscle mrna and metabolically sensitive lncrna gene pairs. Three sheets in this table, the correlation coefficient of each pair of mrna and lncrna in liver, adipose or muscle was calculated by Pearson s correlation method. The first row is seqnames of lncrnas and the first column is the seqnames of coding genes. Table S4, related to Figure 2. GO terms of correlated with representative metabolically sensitive lncrnas in liver, adipose and muscle tissue. Twenty-four sheets are in this table, 8 metabolically sensitive lncrnas were selected for each tissue, and gene ontology

8 (GO) biological process terms for positively and negatively correlated with these lncrnas are listed. Table S5, related to Figure 4. Human homologs of metabolically sensitive lncrnas. One sheet in this table, including the mouse transcript information, human transcript information, and blastn results. Table S6, related to Experimental Procedures. A list of qrt-pcr primers. Mouse genes Sense primer An/-sense primer AK85787 TCTGGCTGCCTGTGGAAGA AGTGCTAGAGAGCCAAGAGTTTTACA uc9kuu.1 CCGAAAGCTGCTTAACTCCTAAGA GAGATCCAGCAATGCACTTCAC AK16911 AAGTAGGGAGCCGAGGGTAACT AACGCCAGCTCCCATACCT AK253 TCTCCAGGATCATGTGCTCC GGCTGGCTGGTTTTGTTGTT ENSMUST1617 (Gm16551) AAAATCAACGCAAAGGAAAGGA CGGCAGGTTGCTTCTGACA uc8txr.1 AGCAGCAACATTATCAGCATCTTC TGGCAGCGGCAGTATCTG AK3369 TGAAGCAGGTCTAAATGGGACAA TGCTCATGGTGACTGGTATGG AK9289 GATCCGGCCCTTACTGTGAG CCCACAGACAACTTTCCACA FGF21 CTGCTGGGGGTCTACCAAG CTGCGCCTACCACTGTTCC FAS GGAGGTGGTGATAGCCGGTAT TGGGTAATCCATAGAGCCCAG SREBP1c GGAGCCATGGATTGCACATT GGCCCGGGAAGTCACTGT G6P CGACTCGCTATCTCCAAGTGA GTTGAACCAGTCTCCGACCA PCK1 CCCAAGGCAACTTAAGGGCTAT CTGAGGTGCCAGGAGCAACT PK TCAAGGCAGGGATGAACATTG CACGGGTCTGTAGCTGAGTG RPIA AAGGCCGAGGAGGCTAAGAA CTTTCAGCTATTCGCTGCACA GLUT2 TCAGAAGACAAGATCACCGGA GTCATAGCCGAACTGGAAGGA GCK CCGTGATCCGGGAAGAGAA GGGAAACCTGACAGGGATGAG apoa4 ATCAAGAAGGAGCTGGAGGA GGTGCTCCTGCAACTTCTGC apoc2 ATGGGGTCTCGGTTCTTCCT GTCTTCTGGTACAGGTCTTTGG IGFBP1 CTGCCAAACTGCAACAAGAATG GGTCCCCTCTAGTCTCCAGA ACC1 CTTCCTGACAAACGAGTCTGG CTGCCGAAACATCTCTGGGA HNF4a AAATGTGCAGGTGTTGACCA CACGCTCCTCCTGAAGAATC FOXO1 GGGTCCCACAGCAACGATG CGAGGACGAAATGTACTCCAGTT chrebp CTGGGGACCTAAACAGGAGC GAAGCCACCCTATAGCTCCC GPAM CCTCTTTTGCCACAACATCA CGAGCCTCCGTCTTATGAAA ACLY CAGCCAAGGCAATTTCAGAGC CTCGACGTTTGATTAACTGGTCT SCD1 TTC TTG CGA TAC ACT CTG GTG C CGG GAT TGA ATG TTC TTG TCG T Ac/n GGCTGTATTCCCCTCCATCG CCAGTTGGTAACAATGCCATGT 18s AGTCCCTGCCCTTTGTACACA CGATCCGAGGGCCTCACTA Human gene Sense primer An/-sense primer H-lncLMS5 ATGGCTGCAAATGTGTGTGT CACAAGCCCAAATTCCCTCC

9 SUPPLEMENTAL EXPERIMENTAL PROCEDURES RNA extraction and quantitative real-time PCR analysis Total RNA was isolated from tissue samples or cells using Trizol reagent (Invitrogen). After Turbo DNA-free DNase treatment (Ambion), reverse transcription was carried out with the SuperScript III First-Strand Synthesis system (Invitrogen) using 1 µg of RNAs. Quantitative real-time RT-PCR was performed on a ViiA 7 Real-Time PCR System (Applied Biosystems Inc.). The PCR program was: 2 min 3 s at 95 C for enzyme activation, 4 cycles of 15 s at 95 C, and 1 min at 6 C. Melting curve analysis was performed to confirm the specific real-time PCR products. Primer sequences used are provided in Table S6. RNA-seq We selected ad Libitum and fasting liver samples to run RNA-seq on an Illumina HiSeq3 sequencer and obtained ~1 million strand-specific paired 5-mer reads per sample. The raw reads were mapped to the same NONCODE 216 mouse database using BWA (bwa aln; bwa sampe with the default parameters). Proper-paired reads were then counted on each noncoding transcript to calculate the RPKM by normalizing the read count on each transcript with transcript length and total library size. For those differentially expressed lncrnas identified by microarray (fasting vs ad libtum), the correlation between Microarray and RNA-seq was calculated by Pearson Correlation using log2-transformed fold changes. Pseudogenes were excluded from this correlation analysis because differentiating them from their corresponding coding genes using an RNA-seq mapping process is challenging. Isolation and culture of mouse primary hepatocytes Primary hepatocytes were isolated from male C57BL/6 mice fed with a normal chow diet. Mice were anesthetized with Ketamine (1mg/kg) and Xylazine (1mg/kg), and the livers were perfused with Krebs Ringer buffer with glucose at a rate of 5ml/min for 8 minutes, followed by continuous perfusion with the same buffer containing collagenase (Liberase TM Research Grade, Roche) for 1 minutes. Hepatocytes were harvested and purified with Percoll. The viability of hepatocytes was examined by trypan blue exclusion. Only cell isolates with viability over 9% were used. Hepatocytes were plated onto collagen-coated plates in DMEM (no glucose)

10 supplemented with 5.5mM glucose, 2mM GlutaMAX, 1nM Dexamethasone, 1nM insulin and 1% Cosmic Calf Serum (CCS). 5-6hrs after plating, cells were switched to maintenance medium as indicated in each experiment. Immunoblotting For immunoblotting analyses, livers were lysed in RIPA buffer (Cell Signaling Technology) containing phosphatase inhibitors (Sigma) and a protease inhibitor cocktail (Roche). The lysates were subjected to SDS PAGE, transferred to polyvinylidene fluoride (PVDF) membranes, and incubated with the primary antibody followed by the fluorescence conjugated secondary antibody (LI-COR). The bound antibody was visualized using a quantitative fluorescence imaging system (LI-COR). The primary antibodies used were an ACLY antibody (4332s, Cell Signaling), FAS antibody (sc-214, Santa Cruz), SCD1 antibody (2794s, Cell Signaling), and Tubulin antibody (2146s, Cell Signaling). Triglyceride (TG) Measurement Liver and plasma TG levels were measured using a kit from Sigma following the manufacturer s protocols.