Hepatic Elovl6 gene expression is regulated by the synergistic action of ChREBP and SREBP-1c

Accepted Manuscript Hepatic Elovl6 gene expression is regulated by the synergistic action of ChREBP and SREBP-1c Jin-Sik Bae, Ah-Reum Oh, Ho-Jae Lee, Yong-ho Ahn, Ji-Young Cha PII:

S0006-291X(16)31317-1

DOI:

10.1016/j.bbrc.2016.08.061

Reference:

YBBRC 36262

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 2 August 2016 Accepted Date: 9 August 2016

Please cite this article as: J.-S. Bae, A.-R. Oh, H.-J. Lee, Y.-h. Ahn, J.-Y. Cha, Hepatic Elovl6 gene expression is regulated by the synergistic action of ChREBP and SREBP-1c, Biochemical and Biophysical Research Communications (2016), doi: 10.1016/j.bbrc.2016.08.061. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Hepatic Elovl6 gene expression is regulated by the synergistic action of ChREBP and SREBP-1c

a

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Jin-Sik Baea, Ah-Reum Oha, Ho-Jae Leea, Yong-ho Ahnb, Ji-Young Chaa,c,*

Department of Biochemistry, Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Incheon

21999, Republic of Korea

Department of Biochemistry and Molecular Biology, Yonsei University College of Medicine, Seoul

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b

120-752, Republic of Korea

Gachon Medical Research Institute, Gil Medical Center, Incheon 21565, Republic of Korea

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c

*

Corresponding author: Ji-Young Cha, PhD, Department of Biochemistry, Lee Gil Ya Cancer and

Diabetes Institute, Gachon University, 155 Gaetbeol-ro, Yeonsu-gu, Incheon 21999, Republic of Korea.

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Tel. +82 32 899 6070, Fax: +82 32 899 6032, E-mail: [email protected].

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Competing interests: The authors have no competing interests to declare.

Abbreviations: ELOVL6, Elongation of very long chain fatty acids protein 6; ChREBP, Carbohydrate response element binding protein; Mlx, Max-like protein X; ChoRE, Carbohydrate response element; SREBP-1c, sterol regulatory element-binding protein-1c; KO, knockout; WT, wild type; qPCR, quantitative real-time polymerase chain reaction; ChIP, chromatin immunoprecipitation; Lpk, Lpyruvate kinase; Dgat2, Diacylglycerol O-Acyltransferase 2

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ACCEPTED MANUSCRIPT Abstract Elongation of very long chain fatty acids protein 6 (ELOVL6), a rate-limiting enzyme for the elongation of saturated and monounsaturated fatty acids with 12, 14, and 16 carbons, plays a key role in energy metabolism and insulin sensitivity. Hepatic Elovl6 expression is upregulated in the fasting-

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refeeding response and in leptin-deficient ob/ob mice. Mouse Elovl6 has been shown to be a direct target of sterol regulatory element binding protein-1 (SREBP-1) in response to insulin. In the present study, we demonstrated that mouse and human Elovl6 expression is under the direct transcriptional

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control of carbohydrate response element binding protein (ChREBP), a mediator of glucose-induced gene expression. Serial deletion and site-directed mutagenesis studies revealed functional

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carbohydrate response elements (ChoREs) in the mouse and human Elovl6 promoters and gel shift assays and chromatin immunoprecipitation assays confirmed the binding of ChREBP to the Elovl6ChoRE sites. In addition, the ectopic co-expression of ChREBP and SREBP-1c in HepG2 cells synergistically stimulated Elovl6 promoter activity and this synergistic activation was abolished by mutating the Elovl6 promoter ChoREs. Taken together, these results suggest that the synergistic action

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of ChREBP and SREBP-1c is necessary for the maximal induction of Elovl6 expression in the liver.

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Keywords: Elovl6; carbohydrate; ChREBP; SREBP-1c; transcriptional regulation

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ACCEPTED MANUSCRIPT 1. Introduction In mammals, fatty acids are predominantly synthesized in the liver by de novo lipogenesis and incorporated into triglycerides, which ultimately serve as an important source of energy during fasting [1]. The end product of de novo lipogenesis is usually oleic acid (C18:1n-9) or vaccenic acid (C18:1n-

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7), whereas the primary fatty acid synthesized by fatty acid synthase, a multifunction enzyme complex, is palmitic acid (C16:0). Therefore, the fatty acids either synthesized by fatty acid synthase or derived from the diet can be further desaturated and/or elongated into long chain and very long

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chain fatty acids. Recently, several studies have shown that proper desaturation and elongation are essential to the maintenance of lipid homeostasis [2-5]. Further, dysregulation of these processes is

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closely related to the development of metabolic syndrome, a condition characterized by central obesity, dyslipidemia, elevated blood glucose, and hypertension [6].

Elongation of very long chain fatty acids protein 6 (ELOVL6) is a microsomal enzyme involved in the elongation of saturated fatty acids and monounsaturated fatty acids with 12, 14, and 16 carbons to

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18-carbon fatty acids (C18) [7]. ELOVL6 is ubiquitously expressed, especially in tissues with high lipid content such as the liver, brown and white adipose tissues, and the brain [4, 7]. Further, the deletion of ELOVL6 in a mouse model prevents the development of diet-induced insulin resistance

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and hyperglycemia, which suggests that altered fatty acid composition [decreased stearate (C18:0) and oleate (C18:1n-9) levels and increased palmitate (C16:0) and palmitoleate (C16:1n-7) levels] is a new

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determinant for insulin sensitivity [2, 8]. The regulation of ELOVL6 expression plays an important role in the management of hepatic lipid composition in response to changes in dietary and hormonal status [9, 10]. Elovl6 mRNA levels are markedly increased in the liver and adipose tissue during the fasting-refeeding response and sterol regulatory element-binding protein-1c (SREBP-1c) is reportedly a transcriptional activator responsible for its nutritional regulation [4, 11]. However, this refeeding response is not completely blunted in SREBP-1 deficient mice, suggesting that SREBP-1-independent expression is also involved in Elovl6 expression [4]. 3

ACCEPTED MANUSCRIPT Carbohydrate response element-binding protein (ChREBP), which contains a basic helix-loophelix/leucine zipper (bHLH/LZ) motif, has been identified as a major glucose-responsive transcription factor [12]. In response to a carbohydrate rich diet, ChREBP cooperates with SREBP to stimulate glycolytic and lipogenic gene expression [13, 14]. As well, glucose increases ChREBP gene

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expression and nuclear translocation. In the refeeding condition, cytosolic ChREBP is rapidly translocated to the nucleus where it forms a heterodimer with Max-like protein X (Mlx) and binds to the carbohydrate response element (ChoRE) for transcriptional regulation of its target genes. Although several studies have indicated that ChREBP is involved in the control of Elovl6 expression in the

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refeeding condition, the direct interaction of ChREBP with regulatory elements in the Elovl6 promoter has not yet been reported [15, 16]. In this study, we identified the ChoREs in the Elovl6 promoter

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region that are involved in transcriptional induction upon refeeding. We also demonstrated that ChREBP and SREBP-1c synergistically regulate hepatic Elovl6 expression.

2.1. Animals

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2. Materials and Methods

ChREBP knockout (KO) mice were purchased from Jackson Laboratory (Sacramento, CA). Age-

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matched male wild type (WT; C57Bl/6J) and ChREBP KO mice were maintained in a temperatureand light-controlled room under a 12 h light/dark cycle. For fasting-refeeding studies, mice were

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either fasted for 24 h or fasted for 24 h and refed a high carbohydrate diet (60% sucrose, Research Diets, New Brunswick, NJ) for 12 h before they were euthanized and their livers were removed and snap-frozen in liquid nitrogen. The livers were stored at –80°C until RNA isolation. Animal studies were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of the Lee Gil Ya Cancer and Diabetes Institute, Gachon University.

2.2. RNA isolation and quantitative real-time polymerase chain reaction (qPCR) 4

ACCEPTED MANUSCRIPT Total RNA was isolated from mouse livers using RNAiso Plus (Takara, Shiga, Japan). Purified total RNA was treated with RNase-free DNase (Roche, Penzberg, Germany) and reverse-transcribed using a QuantiTect® Reverse Transcription Kit (Qiagen, Hilden, Germany). Quantitative gene expression analyses were performed on a 7900HT Fast Real-Time PCR System (Life Technologies,

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Carlsbad, CA) using SYBR® Premix Ex Taq™ II, ROX Plus (Takara, Shiga, Japan). The expression levels were calculated using the 2-∆∆CT method [17] with ribosomal protein, large, P0 (Rplp0) serving as the invariant control.

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2.3. Cell culture and luciferase assay

Human hepatocellular carcinoma HepG2 cells were obtained from the American Type Culture

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Collection (ATCC, Manassas, VA) and maintained in 25 mM glucose Dulbecco’s Modified Eagle’s Medium (DMEM, Welgene, Gyeongsan, Korea) supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin. For the luciferase assay, the cells were transfected with the indicated plasmids using polyethylenimine (Sigma, St. Louis, MO). Luciferase activities were

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measured using the Luciferase Assay System (Promega, Madison, WI) according to the manufacturer’s instructions. The results were shown as arbitrary units normalized to β-galactosidase

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activity. All of the experiments were performed in triplicate and were repeated at least three times.

2.4. Plasmid construction

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The mouse Elovl6 promoter (-2035/+241 bp region, translation start as +1) were cloned into the Sac I and Xho I sites of the pGL4 vector (Promega, Madison, WI). Serial deletion constructs from the mouse Elovl6 promoter construct were prepared by amplifying the indicated regions and subcloned into the pGL4b vector. Mutant clones M1, M2, and M3 were generated by introducing substitution mutations into the mouse Elovl6 promoter construct (-2035/+241). The human ELOVL6 promoter reporter construct was generated by PCR amplifying the proximal promoter of ELOVL6 (-1107/+246) from HepG2 cell genomic DNA and inserting the products into the Xho I and Hind III sites of the 5

ACCEPTED MANUSCRIPT pGL4b vector. The expression construct encoding the nuclear form of human SREBP-1c was generated by PCR amplification and insertion into the pcDNA3-2×FLAG vector. Mouse ChREBP and Mlx expression vectors were kind gifts from Dr. Howard C. Towle [18]. The integrity of each plasmid

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was verified by DNA sequencing.

2.5. In vitro translation and gel shift assay

In vitro-translated FLAG-tagged ChREBP and Myc-tagged Mlx proteins were prepared using a coupled transcription/translation kit (Promega, Madison, WI). Gel shift assays were performed as

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previously described [15]. For the supershift assay, anti-FLAG (Sigma, St. Louis, MO) or anti-IgG

were visualized by autoradiography.

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(Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies were added to the reaction. The results

2.6. Chromatin immunoprecipitation (ChIP) assay

ChIP assays were performed as previously described [15] and the resulting purified ChIP DNA

2.7. Statistical analysis

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samples were analyzed by qPCR.

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The statistical analysis was carried out using SPSS (Version 17.0; SPSS Inc., Chicago, IL). Data were analyzed using the Mann-Whitney U test. A p value less than 0.05 was considered statistically

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significant.

3. Results and Discussion

3.1. Elovl6 expression in response to refeeding is abolished in ChREBP KO mice To evaluate the effect of ChREBP deletion on Elovl6 expression, we determined the expression of Elovl6 in the livers of WT (C57BL/6J) and ChREBP KO mice under different nutritional conditions. Elovl6 mRNA levels were low during fasting but were strongly induced upon feeding with a high 6

ACCEPTED MANUSCRIPT carbohydrate diet (Fig. 1A), which was in agreement with the results of a previous study [4]. However, this induction pattern was completely blunted in ChREBP KO mice, indicating that ChREBP could be a major transcription factor responsible for the expression of Elovl6 in response to refeeding. The mRNA levels of a representative ChREBP target, L-pyruvate kinase (Lpk ) were markedly suppressed

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in ChREBP KO mice compared to WT mice, particularly in the refed state. The effect of refeeding on Diacylglycerol O-Acyltransferase 2 (Dgat2) mRNA levels, a recently reported direct target of ChREBP, were decreased in ChREBP KO mice [19]. As expected, Chrebp mRNA levels were increased in refed WT mice but its expression was not detected in ChREBP KO mice. ChREBP has

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two isoforms, ChREBP-α, a canonical ChREBP isoform, and ChREBP-β, a recently identified short isoform, whose levels are strongly correlated with ChREBP activity [20]. Chrebp-β mRNA levels and

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the fold change in Elovl6 mRNA levels were significantly increased after refeeding. The mRNA levels of Mlx, a heterodimer partner of ChREBP, were slightly induced by refeeding in WT mice but were not altered by refeeding in ChREBP KO mice.

Next, we determined the mRNA levels of the Srebp genes, which are known transcription factors for

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hepatic Elovl6 expression after refeeding [4, 11]. The mRNA levels of Srebp-1a, -1c, and -2 were significantly increased by refeeding in WT mice. Unexpectedly, the observed induction of Srebp-1a and -1c in the fasting-refeeding response was significantly suppressed in ChREBP KO mice after

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refeeding. These two transcription factors are expressed by the same gene, but they possess different promoters and transcription start sites, which results in different forms of exon1. These results suggest

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the possibility that SREBP-1c is a direct or indirect target of ChREBP. In fact, genome-wide analysis of ChREBP binding sites revealed that human SREBP-1 was a direct target of ChREBP in HepG2 cells and its promoter region is conserved in the mouse [15]. Collectively, the Elovl6 transcriptional induction pattern upon refeeding correlates better with the patterns observed in ChREBP than with those of SREBP-1c.

3.2. The Elovl6 promoter contains functional ChoRE elements 7

ACCEPTED MANUSCRIPT According to previously reported results [15] and in silico analysis, we identified two putative ChREBP binding sites (ChoREs, -567/-551 bp region and -334/-318 bp region) in the mouse Elovl6 promoter region (Supplementary Fig. 1). ChoRE1 is well conserved between species, but ChoRE2 is quite variable between species and is not conserved in humans (Supplementary Fig. 1). To investigate

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whether ChREBP influences the transcriptional activity of Elovl6, we generated promoter-luciferase plasmid constructs. A nearly 2-kb fragment in the 5’-flanking region of the mouse gene was cloned and ligated into a luciferase reporter plasmid (-2035/+241, Fig. 2A) and transfected into HepG2 cells along with expression vectors for ChREBP and Mlx (ChREBP/Mlx). ChREBP was cotransfected with

target

genes

[18].

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constructs

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Mlx because ChREBP works with its heterodimer partner Mlx to bind and activate glucose-responsive containing

putative

Elovl6

promoter

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ChoREs -2035/+241, -1550/+241, -914/+241, and -600/+241) exhibited increased transcriptional activity in cells overexpressing ChREBP/Mlx (~2.5–3-fold). Deletion constructs (-400/+241 and -200/+241) were not activated by ChREBP/Mlx, suggesting the majority of ChREBP/Mlxmediated transcription requires the mouse Elovl6 promoter regions containing putative ChoREs. Next,

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we investigated the effect of ChREBP/Mlx on the human ELOVL6 promoter (Fig. 2B) and determined that the human ELOVL6 promoter-luciferase construct (-1107/+246) exhibited higher luciferase activity (6-fold) than the mouse Elovl6 promoter-luciferase constructs (-2035/+241, -1550/+241, -

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914/+241, and -600/+241), indicating that human ChoRE1 might be a stronger functional site for

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ChREBP than mouse ChoRE1 and/or ChoRE2.

3.3. ChREBP directly binds to ChoRE on the Elovl6 promoter The direct interaction between ChREBP and ChoRE on the Elovl6 promoter was confirmed by a gel shift assay, an in vitro biochemical method (Fig. 3A-D). HEK293 cells were transfected with ChREBP expression vectors and its heterodimer partner Mlx and nuclear extracts were prepared. The nuclear extracts were incubated with a radiolabeled oligonucleotide (probe) containing each of the mouse or human ChoRE sequences (Fig. 3A), and the assay results indicated the strong binding of the 8

ACCEPTED MANUSCRIPT ChREBP/Mlx complex to each of the mouse and human ChoRE-containing probes. Conversely, no shifted bands were observed with the mutated ChoRE probe, which failed to compete out the ChREBP/Mlx band. The ChREBP/Mlx band was supershifted by antibodies against FLAG and Myc that were tagged with the ChREBP and Mlx transcription factors, which indicated the specific binding

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of ChREBP/Mlx to the ChoRE on the mouse and human Elovl6 promoters. In vivo association of ChREBP/Mlx to the Elovl6 promoter in the mouse liver was assessed by a ChIP assay (Fig. 3E). The enrichment of ChREBP to the mouse Elovl6 promoter ChoREs was significantly

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enhanced under the refeeding condition compared with the fasted condition, indicating that refeeding affects the binding of ChREBP to DNA. The direct binding of ChREBP to the human ELOVL6

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promoter was previously reported [15]. Collectively, these findings indicate that ChREBP/Mlx directly binds to the Elovl6 promoter ChoRE and its interaction is dependent on the feeding condition.

3.4. ChREBP and SREBP-1c synergistically activate Elovl6 promoter activity

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To test the functional role of each ChoRE in the Elovl6 promoter, the ChoRE sites were mutated and the transcriptional activities were determined. The mutation of ChoRE1 and/or ChoRE2 (M1, M2, or M3) completely abolished the transcriptional activation by ChREBP/Mlx that was predicted by the

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deletion studies and the gel shift assay (Fig. 4A). To delineate the relationship between ChREBP and SREBP-1c upon Elovl6 expression, SREBP-1c and/or ChREBP/Mlx were cotransfected with

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the -2035/+241 construct or a mutant (M1, M2, or M3). The Elovl6 promoter was activated by SREBP-1c and was synergistically activated by both ChREBP/Mlx and SREBP-1c. The activation of the Elovl6 promoter by SREBP-1c was not changed by the mutation of ChoRE1 or ChoRE2. However, the synergistic activation by ChREBP/Mlx and SREBP-1c was affected by the mutation of ChoRE and was completely abolished by the mutation of both ChoRE1 and ChoRE2. A similar regulation pattern by ChREBP/Mlx and SREBP-1c was observed with the human ELOVL6 promoter construct

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ACCEPTED MANUSCRIPT (Fig. 4B), which suggests that the synergistic action of ChREBP/Mlx and SREBP-1c is necessary for the maximal induction of Elovl6 gene expression in the liver. In this study, we clearly show that mouse and human Elovl6 are direct targets of ChREBP. The mouse Elovl6 promoter contains two ChoREs, whereas the human Elovl6 promoter contains only one ChoRE,

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but its binding affinity and functional activity are slightly stronger than the mouse ChoREs. Upon refeeding, the binding of ChREBP to the Elovl6 promoter was increased, which resulted in the induction of Elovl6. The results of the mutation study showed that a mutation in either ChoRE1 or

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ChoRE2 abolishes the activation of the Elovl6 promoter activity by ChREBP and that both of the ChoREs are involved in the synergistic activation of Elovl6 by SREBP-1c and ChREBP.

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Unexpectedly, the expression of Elovl6 induced by refeeding was completely blunted in ChREBP KO mice, whereas the Elovl6 refeeding response in SREBP-1c KO mice remained comparable to WT mice [4]. These results indicate that ChREBP directly and primarily regulates Elovl6 induction upon

Acknowledgments

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refeeding.

We thank Yun-Seung Jeong for assistance with the ChIP assay. We also thank Seung-ho Cho for help

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with plasmid construction. This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and and

by the

Korea

Mouse

Phenotyping Project

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Technology (NRF-2012R1A1A3018738)

(2013M3A9D5072550) of the Ministry of Science, ICT, and Future Planning through the National Research Foundation awarded to J-Y Cha and (NRF-2012R1A1A2044473) awarded to J-S Bae.

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Figure legends

Fig. 1. Hepatic Elovl6 mRNA expression in fasted or refed WT (C57BL/6J) and ChREBP KO mice. WT (n=6) and ChREBP KO (n=6) mice were either fasted for 24 h or fasted for 24 h and refed a high

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carbohydrate diet for 12 h. Hepatic mRNA levels of Elovl6, Dgat2, Lpk, Chrebp-α, Chrebp-β, Mlx, Srebp-1a, Srebp-1c, and Srebp-2 were measured by qPCR. Expression levels were normalized to the

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expression of Rplp0. Each value represents the mRNA level relative to that of the fasted group, which was arbitrarily defined as 1. Values represent the mean ± S.E.M. of six independent samples. * = p<0.05 vs. the fasted group.

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Fig. 2. Identification of the functional ChoRE in the Elovl6 promoter. The effect of deletion on the ChREBP-mediated mouse (A) and human (B) Elovl6 promoter activity. The promoter activities were measured by cotransfecting 50 ng of ChREBP and 50 ng of Mlx expression or empty vectors into

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HepG2 cell lines. A schematic diagram of the serial deletion constructs of the Elovl6 promoter

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reporter (left) and ChREBP-mediated Elovl6 promoter activity (right). Results are expressed as the mean ± standard deviation of three independent experiments. Data are expressed as fold increases relative to the basal activity. * = p<0.05 vs. basal activity.

Fig. 3. ChREBP and Mlx bind to the Elovl6 ChoREs as a heteromeric complex. (A) Nucleotide sequences of the oligonucleotides used as probes and competitors. (B-D) Electrophoretic mobility shift assays were performed using the indicated oligonucleotide probes. All lanes contain the indicated 13

ACCEPTED MANUSCRIPT labeled-probe and lanes 3–13 contain 5 µg of HEK293 nuclear extract. Lanes 3 and 4 contain HEK293 mock-transfected nuclear extracts. The remaining lanes contain extract from HEK293 cells transfected with FLAG-tagged ChREBP and Myc-tagged Mlx. The black arrow indicates the position of the ChREBP-Mlx complex. The white arrow indicates the position of the antibody-supershifted

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complexes. WT, wild type; M, Mutant. (E) ChIP assay for ChREBP binding to the Elovl6 gene ChoREs. Schematics of the amplification of the Elovl6 promoter in the ChIP assay are presented. Chromatin was extracted from the livers of mice fasted for 24 h or refed for 12 h after 24 h fasting (n=3 per group). The chromatin was immunoprecipitated with antibody against control IgG or anti-

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ChREBP antibody. The data are presented as fold increases determined by the qPCR signal from anti-

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ChREBP relative to control IgG. Values represent the mean ± standard deviation of three independent samples. * = p<0.05 vs. IgG and # = p<0.05 vs. fasted with anti-ChREBP.

Fig. 4. The synergistic effect of ChREBP and SREBP-1c and the effect of a putative ChoRE mutation

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on the Elovl6 promoter activity. Mouse (A) and human (B) Elovl6 promoter activities were measured by cotransfecting 50 ng of ChREBP and 50 ng of Mlx and/or 10 ng of nSREBP-1c expression vectors into HepG2 cell lines. A schematic diagram of the mutation constructs of the Elovl6 promoter reporter

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(left) and ChREBP- and/or SREBP-1c-mediated Elovl6 promoter activity (right). Normalized luciferase activities are shown as the means ± standard deviation of three independent experiments

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performed in triplicate. * = p<0.05 vs. basal activity.

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ACCEPTED MANUSCRIPT Hepatic Elovl6 gene expression is regulated by the synergistic action of ChREBP and SREBP-1c Jin-Sik Baea, Ah-Reum Oha, Ho-Jae Leea, Yong-ho Ahnb, Ji-Young Chaa,c,*

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Supplementary Figure1. Multiple sequence alignment of the -642/+244 of the mouse Elovl6 promoter fragment and its corresponding rat and human sequences. The red boxes indicate putative ChREBP and known SREBP responsive elements on the mouse Elovl6 promoter and its corresponding rat and

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human sequences. The black box indicates the translation start codon.

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ACCEPTED MANUSCRIPT Highlights Elovl6 expression in response to refeeding is totally blunted in ChREBP-/- mice

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ChREBP/Mlx overexpression increases mouse and human Elovl6 promoter activities

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Direct binding of ChREBP/Mlx to ChoRE is dependent on feeding conditions

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ChREBP and SREBP-1c synergistically activate Elovl6 promoter activity

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