SP1 pathway

SP1 pathway

Journal Pre-proof Via DNMT3b/SP1 pathway Wei Guo, Huiping Zhang, Anning Yang, Pengjun Ma, Lei Sun, Mei Deng, Caiyan Mao, Jiantuan Xiong, Jianmin Sun,...

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Journal Pre-proof Via DNMT3b/SP1 pathway

Wei Guo, Huiping Zhang, Anning Yang, Pengjun Ma, Lei Sun, Mei Deng, Caiyan Mao, Jiantuan Xiong, Jianmin Sun, Nan Wang, Shengchao Ma, Lihong Nie, Yideng Jiang PII:

S0022-2828(19)30370-0

DOI:

https://doi.org/10.1016/j.yjmcc.2019.11.145

Reference:

YJMCC 9082

To appear in:

Journal of Molecular and Cellular Cardiology

Received date:

11 October 2019

Accepted date:

4 November 2019

Please cite this article as: W. Guo, H. Zhang, A. Yang, et al., Via DNMT3b/SP1 pathway, Journal of Molecular and Cellular Cardiology(2019), https://doi.org/10.1016/ j.yjmcc.2019.11.145

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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.

© 2019 Published by Elsevier.

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Homocysteine accelerates atherosclerosis by inhibiting scavenger receptor class B member1 via DNMT3b/SP1 pathway Running Title: Hcy accelerates atherosclerosis through inhibition of SCARB1 Wei Guo1,6,7# , Huiping Zhang2# , Anning Yang1,6,7 , Pengjun Ma1,6,7 , Lei Sun1,6,7 , Mei Deng1,6,7 , Caiyan Mao1,6,7 , Jiantuan Xiong4 , Jianmin Sun3 , Nan Wang1,6 , Shengchao Ma1,6,7 , Lihong Nie 5 and Yideng

Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Ningxia Medical

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1

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Jiang1,6,7*

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University, Yinchuan, China.

Prenatal Diagnosis Center of Ningxia Medical University General Hospital, Yinchuan, China.

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Department of Pathogenic Biology and Immunology, School of Basic Medical Sciences, Ningxia

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Medical University, Yinchuan, China.

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College of Pharmacy, Ningxia Medical University, Yinchuan, China.

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Department of Physiology, School of Basic Medical Sciences, Ningxia Medical University, Yinchuan ,

China.

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4

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Ningxia Key Laboratory of Vascular Injury and Repair Research, Yinchuan, China.

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NHC Key Laboratory of Metabolic Cardiovascular Diseases Research (NingXia Medical University),

Yinchuan, China. #

These authors contributed equally to this work.

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*Corresponding author: Yideng Jiang, Ph.D, Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Ningxia Medical University, No.1160, Shengli Street, Yinchuan, Ningxia, 750004, China;

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Telephone number: +869516980056; Email: [email protected]

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Abstract Homocysteine (Hcy) is an independent risk factor for atherosclerosis, which is characterized by lipid accumulation in the atherosclerotic plaque. Increasing evidence supports that as the main receptor of high-density lipoprotein, scavenger receptor class B member 1 (SCARB1) is protective against atherosclerosis. However, the underlying mechanism regarding it in Hcy-mediated atherosclerosis

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remains unclear. Here, we found the remarkable inhibition of SCARB1 expression in atherosclerotic

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plaque and Hcy-treated foam cells, whereas overexpression of SCARB1 can suppress lipid accumulation

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in foam cells following Hcy treatment. Analysis of SCARB1 promoter showed that no significant change

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of methylation level was observed both in vivo and in vitro under Hcy treatment. Moreover, it was found

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that the negative regulation of DNMT3b on SCARB1 was due to the decreased recruitment of SP1 to

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SCARB1 promoter. Thus, we concluded that inhibition of SCARB1 expression induced by DNMT3b at

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least partly accelerated Hcy-mediated atherosclerosis through promoting lipid accumulation in foam cells ,

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which was attributed to the decreased binding of SP1 to SCARB1 promoter. In our point, these findings will provide novel insight into an epigenetic mechanism for atherosclerosis. Keywords: Atherosclerosis; Homocysteine; SCARB1; DNMT3b; SP1

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1. Introduction Atherosclerosis, a complex chronic disease characterized by lipid deposition in the arterial wall, is a major contributor to morbidity and mortality worldwide [1]. Emerging evidence demonstrated that homocysteine (Hcy), a key intermediate in the metabolism of sulfur-containing amino acid, has been

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recognized as an independent risk factor for atherosclerosis [2]. Although it was reported that

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Hcy-induced oxidative stress, endothelium dysfunction and vascular smooth muscle cells (VSMCs)

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proliferation also played important roles in the pathogenesis of atherosclerosis [3-5], the underlying

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mechanisms regarding Hcy-accelerated lipid deposition in atherosclerosis still need to be further

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elucidated.

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Scavenger receptor class B type 1 (SCARBI) is a cell-surface glycoprotein which belongs to the CD36

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superfamily together with scavenger receptor CD36 and lysosomal membrane protein 2 [6]. It was

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evidenced that SCARB1 is widely expressed in mammals including macrophages and endothelial cells. Additionally, SCARB1 was reported to play a vital role in cholesterol homeostasis [7, 8]. Ubiquitous deletion of SCARB1 can disturb HDL metabolism which reverses the transportation of cholesterol [6, 9]. On the other hand, overexpression of SCARB1 in SCARB1-deficient animals can reduce plasma HDL cholesterol levels [10]. Thus, it is reasonable to suspect that SCARB1 might be involved in atherosclerosis due to its regulating role in the metabolism of LDL and HDL which maintain cellular cholesterol balance.

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Gene transcriptional regulation involves epigenetics-transcriptional factor interaction, which plays a pivotal role in gene regulation processes and disease development at the molecular level [11]. DNA methylation is the most highly studied epigenetic marker in the modulation of gene transcription, which was regulated by DNA methyltransferases (DNMTs) and intermediate metabolites of the one-carbon pathway including Hcy. Meantime, several studies have suggested the tight correlation between plasma

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Hcy and DNA methylation [12]. In our previous study, it was found that Hcy-mediated FABP4 gene

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demethylation via DNMT1 can accelerate atherosclerosis in ApoE-/- mice [13]. However, a variety of

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DNA methylation aberrations at a genome-wide level and particular loci under similar pathological

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conditions was observed [14]. Besides, aberrant expression of DNMT3b, one kind of functional DNMTs

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in mammals, can inhibit gene expression and onset the bladder cancer by decreasing global DNA

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methylation [15]. More importantly, several investigations have documented that epigenetic modification

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including DNA methylation is initiated by the specific transcription factor that binds to the promoter of

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the gene [16]. As a member of the family of SP/KLF transcription factors, specificity protein 1 (SP1) can drive various cellular processes, such as development, proliferation, apoptosis, as well as lipid metabolism [17]. Meanwhile, it can trigger gene expression through binding to the GC-rich motifs present in the proximal promoter of a gene [18]. In breast cancer, Sp1-induced DNMT1 activity increase was responsible for p53-induced expression inhibition of p125 and the methylation of POLD1 gene promoter [19], while little is known about the association between SP1 and DNMT3b during the development of in atherosclerosis.

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Therefore, this study is aimed to elucidate the role of SCARB1 in Hcy-mediated atherosclerosis. The evidence indicated that the decreased recruitment of SP1 to SCARB1 promoter mediated by DNMT3b was responsible for the downregulation of SCARB1, which finally result in the lipid accumulation in foam cells. These findings provide new insight into Hcy-associated lipid accumulation and a new

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potential molecular target of SCARB1 for the therapy of Hcy-related cardiovascular diseases.

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2. Materials and methods

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2.1 Chemicals and Reagents

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Wizard® Genomic DNA Purification Kit was from Promega (Madison, USA), DL-Homocysteic Acid

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(DL-Hcy), PMA, ox-LDL,5-aza-2’-deoxycytidine (5-AZC), Nile Red, DC_05, TFD, and NanaomycinA, anti-SP1 antibodies were from Sigma-Aldrich (St. Louis, USA), anti-β-actin antibody was from Zsbio

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(Beijing, China), antibodies against SCARB1, DNMT3b, MOMA-2 and perilipin were from Abcam (MA,

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USA), HRP conjugated goat anti-rabbit IgG and goat anti-mouse-IgG were from Jackson

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ImmunoResearch (West Grove, USA), DNA methyltransferase activity assay kit was from Epigentek, (NY, USA),ChIP-IT kit was from Active Motif (CA,USA).

2.2 Animals

Six-week-old male Apolipoprotein E knockout (ApoE-/-) mice with C57BL/6J genetic background (Animal Center of Peking University Health Science Center, Beijing, China) were housed in a temperature-controlled (24 °C) facility with a 12-hour light/dark cycle and free access to food and water. After the acclimatization for one week, the ApoE-/- mice randomly divided into three groups were fed with

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the regular diet (control), regular diet plus 1.7% methionine (HMD) and regular diet plus 1.7% methionine, 0.006% folate, and 0.0004% vitamin B 12 (HMD+F+VB12 ) as previously described [13]. After 20 weeks, all of the mice were anesthetized by intraperitoneal (i.p.) pentobarbital (50 mg/kg body weight) or a bolus dose of 100 mg/kg if the mice moved or showed pain upon monitoring. Aortic tissues were frozen in the liquid nitrogen and storage at -80 °C for further analysis. This study followed the Guide for

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the Care and Use of Laboratory Animals published by the US National Institutes of Health. And the

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the Health Science Center of Ningxia Medical University.

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research protocol of this study was approved by the Committee on the Ethics of Animal Experiments of

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2.3 Tissue preparation and evaluation of atherosclerotic lesions in ApoE -/- mice After blood collection, murine aortas were removed when mice were anesthetized followed by

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snap-frozen in liquid nitrogen and embedded in optimum cutting temperature compound (OCT). Frozen

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sections from the region of the proximal aorta starting from the end of the aortic sinus and for 300 mm

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distally were taken according to Paigen et al [20]. Sections were stained with oil red O. Quantitative analysis of lipid-stained lesions was performed on sections starting just beyond the end of the aortic sinus. The lipid-stained lesions were measured by digitizing morphometry and reported as mm2 per lesion. Besides, aortic root cross-sections were stained with MOMA-2 (Abcam, MA, USA), a marker for macrophages (M) and perilipin (Abcam, MA, USA) [21].

2.4 Cell culture

Human monocytic leukemia cell line THP-1 cells purchased from the Chinese Academy of Life Sciences

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(Shanghai, China) were cultured in RPMI 1640 (GIBICO, USA) medium plus 10% heat-inactivated fetal bovine serum (HI-FBS; 56 °C, 45 min), 100 U/ml penicillin, and 100 mg/ml streptomycin at 37 °C in a 5% CO2 humidified atmosphere. Differentiation of THP-1 cells into macrophages was induced by 100 nmol/L PMA (Sigma-Aldrich, St Louis, USA) for 24 hours. Subsequently, the medium was replaced with fresh medium containing 100 μmol/L Hcy or supplemented with its antagonist (Folate+VB 12 ), and cells were

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incubated with 50 mg/L ox-LDL (Sigma-Aldrich, St Louis, USA) for 48 h to establish the model of

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THP-1 macrophage-derived foam cells.

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2.5 Construction of recombinant SCARB1, DNMT3b and SP1 adenoviral vectors

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Full length and fragments of human SCARB1, DNMT3b or SP1 gene were inserted into the replication-defective adenoviral shuttle vector pHBAd-CMV-IRES-GFP or adenoviral backbone plasmid

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pBHGlox (Delta) E1, 3Cre. They were linearized with PacI, and transfected into HEK293T cells.

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Subsequently, the virus was plaque purified, and a GFP-positive plaque was selected, grown on a large

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scale, and then infected macrophages cells before swallowing ox-LDL to form foam cells.

2.6 Knockdown of DNMT3b and SP1 expression by shRNA

The plasmid containing short hairpin RNAs (shRNAs) targeting DNMT3b and SP1 were purchased from GenePharma Corporation (Shanghai, China). The target sequences of DNMT3b shRNA-1, shRNA-2and shRNA-3 were 5′-GCUCGUCUCCUAUCGAAAATT-3′, 5′-UUUUCGAUAGGAGACGAGCTT-3′ and 5′-GCUACACACAGGACUUGAC-3′ respectively. The target sequences of SP1 shRNA-1, shRNA-2 and shRNA-3 were 5’-GGUAGCUCUAAGUUUUGAUTT-3’, 5′-AAAGCGCUUCAUGAGGAGUGA-3′

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and 5-AGCGCTTCATGAGGAG TGA-3′ respectively. A plasmid containing scrambled shRNA (LacZ) 5′-TGTTCGCATTATCCGAACCAT-3′ was used as a negative control. The macrophages cells were transfected with scramble shRNA, DNMT3b or SP1 shRNA according to the manufacturer’s instruction. Subsequently, ox-LDL was used to incubate cells to leads to the formation of foam cells [13]. The

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knockdown efficiency of which was analyzed by qRT-PCR and western blot.

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2.7 qRT-PCR

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Total RNA from aortic tissues in ApoE-/- mice and cultured cells were isolated by Trizol reagent

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(Invitrogen, Grand Island, USA) and reversely transcribed using the Revert Aid first strand cDNA

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synthesis kit (MBI, Vilnius, Lithuania). qRT-PCR was carried out on the FTC3000 qRT-PCR detection system under the following conditions: 45 cycles at 95 °C for 45 seconds, the annealing temperature for

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45 seconds and extension 60 °C for 60 seconds. Glyceraldehyde phosphate dehydrogenase (GAPDH) was

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used as the invariant control, mRNA abundance was determined by 2-△△ CT method. Primers used for

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qRT-PCR were listed in table 1.

Gene

Species

Table 1. Primers used for qRT-PCR Tm

Product

(°C)

(bp)

59.5

213

60.5

233

Primer sequence (5' to 3')

F: 5'-CGGATTTGGTCGTATTGGG-3' Human GAPDH

R: 5'-CGCTCCTGGAAGATGGTGAT-3' Mouse

F: 5'-GGTGAAGGTCGGTGTGAACG-3'

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R: 5'-CTCGCTCCTGGAAGATGGTG-3' F: 5'-CCTAACCAGGAGGCACACTC-3' Human

59.8

145

60.0

230

63.5

164

57.0

219

59.0

105

60.0

184

R: 5'-GGACCACAGGCTCAATCTTC-3' SCARB1 F: 5'-TTCGAACAGAGCGGAGCAAT-3' Mouse R: 5'-TCAGAGTAGGCCTGAATGGC-3'

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F: 5'-AGCTCTTACCTTACCATCGACCTCAC-3'

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Human

R: 5'-TACTCTGAACTGTCTCCATCTCCACTG-3' DNMT3b

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F: 5'- GAAGACGCACAACCAATG -3' Mouse

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R: 5'- AGAGCCCACCCTCAAAGA-3'

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F: 5'-TGATGTGTGGGCTTCTGAGT-3' Human SP1

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R: 5'-ATTCACTGGCTGATGCTCCT-3'

Mouse

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F: 5'-GGGTTCGCTTGCCTCGT-3'

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R: 5'-CATTGCCGCTACCCCCATTA-3'

F: forward primer and R: reverse primer.

2.8 Western blot

Western blot analysis was performed as previously described [13, 22]. Aortic tissues and foam cells were lysed in a lysis buffer (KeyGEN BioTECH, Nanjing, China) containing the protease inhibitor phenylmethanesulfonyl fluoride (PMSF) at 4°C for 30 min followed by centrifugation to remove cell debris. Protein concentration was measured using BCA protein assay kit (Keygen BioTECH, Nanjing,

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China). The protein was boiled and subjected to western blot with antibodies against SCARB1, DNMT3b, SP1 and β-actin (Abcam Inc., MA, USA) respectively. Optical densities of the bands were analyzed with Bio-Rad image analysis (Bio-Rad, CA, USA). 2.9 Nile red staining THP-1-derived foam cells seeding in glass coverslips were treated as above. Then cells were fixed with 4%

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formaldehyde for 20 minutes at room temperature and quenched with 0.1 M glycine. After washing three

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times for 5 min each with PBS, cells were incubated with 0.5 µg/ml of Nile Red (Sigma-Aldrich St. Louis,

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USA) for 30 minutes. Subsequently, cells were washed with PBS and the cover slips were mounted on

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glass slides with VectaShield (CA, USA) mounting media. Images were acquired using an Olympus

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confocal laser-scanning microscope at 450/500 nm excitation and 528 nm emission (Fluoview FV10i)

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with the Metamorph digital imaging system, and quantification was performed using Image J software.

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methylation

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2.10 Nested methylation-specific-polymerase chain reaction (nMS-PCR) for SCARB1 DNA

Genomic DNA was isolated from the aorta using the Wizard® Genomic DNA Purification Kit (Promega, Madison, USA). Nested methylation-specific-polymerase chain reaction (nMS-PCR) was used for the detection of methylation in the promoter of SCARB1, which consists of two-step PCR amplification after a standard sodium bisulfite DNA modification. The first step of nMS-PCR used an outer primer pair, and the second-step PCR was carried out with the following PCR primers: a methylation primer and an unmethylation primer. The primers for SCARB1 are listed in table 2. PCR products were purified with an

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agarose gel DNA fragment recovery kit according to the manufacturer's instructions and sequenced by Invitrogen (Carlsbad, USA). To reduce misprizing and increase efficiency, touchdown (TD) PCR was used in the amplification. Samples were subjected to 30 cycles in a TD program (94°C for 30 s; 66°C for 30 s and 72°C for 1 min), followed by a 1°C decrease of the annealing temperature in every cycle. After completion of the TD program, 20 cycles were run subsequently (94°C for 45 s, 51°C for 45 s and 72°C

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for 45 s), ending with a 5-min extension at 72°C. The PCR products were separated by 2% agarose gel

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containing ethidium bromide. DNA bands were visualized by ultraviolet light. Methylation was then

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calculated using the following formula: methylation% = methylation/ (methylation + unmethylation) ×

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100%.

Primer sequence (5' to 3')

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Gene

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Table 2. Primers used for nMS-PCR Product Tm (°C) (bp)

SCARB1-O

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F: 5'-AGAAAATGAGGGGATTGGTTAGTAT-3' 59.6

484

58.6

120

59.0

121

R: 5'-ATAATTACAAACAAATACTCCAAAAA-3' F: 5'-GTTATTTAGGTTGGAGTGCGG-3'

SCARB1-M R: 5'-CAATAACATACGCTTATCGCC-3' F: 5'-ATTTAGGTTGGAGTGTGGTG-3' SCARB1-U R: 5'-ACCACAATAACATACACTTATCACCC-3'

O: out primer; M: methylation primer; U: unmethylation primer; F: forward and R: reverse.

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2.11 Bisulfite Sequencing (BSP)

DNA was extracted from the foam cells using a DNA extraction kit (Axygen, Califonia, USA) followed by treatment with bisulphate (ZYMO RESEARCH, Germany). The PCR products were gel extracted (Axygen, Califonia, USA) and ligated into a plasmid vector using the pMD18-T Vector (Takara Bio,

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Califonia, USA). At least 10 separate clones were sequenced.

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USA). The plasmid was transformed into DH5a bacteria, and the plasmid DNA was isolated (Axygen,

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2.12 DNMT activity assay

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Nucleus was isolated from foam cells and aorta tissue of mice respectively. The enzyme activity was then

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assayed by using DNA methyltransferase activity assay kit (Epigentek, NY, USA) according to the

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manufacturer's instruction. Absorbance was read at 450 nm on a spectrophotometer.

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2.13 Quantitation of intracellular cholesterol

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Cell supernatant TC and TG were analyzed by Cholesterol Quantitation Kit (Biovision, Inc, CA, USA). Cells (1× 106 ) were treated with a 200 μl mixture of chloroform: isopropanol: NP-40 (7:11:0.1) in a 1.5 mL EP. The cell membrane was destructed by ultrasonic and the extract was spun at 15000×g for 10 minutes. The supernatant was air dried at 50 °C for 4 hours to remove chloroform. The samples were vacuumed at 50 °C for 30 minutes to remove trace organic solvent. The dried lipid was dissolved with 200 μl cholesterol assay buffer, and 20 μl was sued for cholesterol quantitation following the manufacturer’s protocol. The cholesterol content was measured by absorbance at 570 nm. Protein

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concentrations were determined by the BCA Protein Assay Kit according to the protocol provided by the manufacturer (Keygen BioTECH, Nanjing, China). The values of intracellular cholesterol were calculated as μg cholesterol/μg cellular protein.

2.14 Construction of deletion and site-directed mutagenesis of the SCARB1 promoter

by

PCR

with

the

same

reverse

primer

and

specific

forward

primer

sets:

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amplified

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Various length of SCARB1 promoter was amplified and inserted into a pGL3 vector. They were

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5’-CACGGGACGCGCTGCC-3’ for the SCARB1-p-(-1944/+321); 5’-CTCCGCAACTTCTCTCTGCT-3’

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for the SCARB1-p-(-834/+ 321); 5’-GTATAGGTTGGGGCGGAGTC-3’ for the SCARB1-p-(-166/+173),

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respectively. The PCR products were cloned into pUC57 cloning vectors. The mutant constructs of SCARB1 reporters were generated from pUC57-SCARB1-p-(-166/+32) plasmid using the Finnzymes

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Phusion® Hot Start High-Fidelity DNA Polymerase (Thermo Fisher Scientific Inc., MA, USA) and

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mutant primers. Mutagenesis primers were as follows: Mut-SP1-2F, forward, 5’-GTATAGGTTGTGTCT

ATACGA-3’; complement

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GAGTCGGATTC-3’ and complement strand Mut-SP1-2R, 5’-CCGAATCCGACTCAGACACAACCT Mut-SP1-1F,

forward

5’-ACGCAGTTGTGTGTCGTAGGCCTAAGTAC-3’

and

strand Mut-SP1-1R, 5’-GTACTTAGGCCTACGACACACAACTG CGTT-3’. The mutant

PUC57- SCARB1-p-(-166/+32) constructs were digested with KpnI and XhoI and then cloned into a pGL3 basic vector (Promega, Madison, USA) to establish the mutant SCARB1 reporter.

2.15 Luciferase reporter assay

To evaluate SCARB1 promoter activity, vectors containing wild-type or mutant SCARB1 promoter

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purchased from Yingbiotech Corporation (Shanghai, China) were sequenced before transfected into HEK293 or foam cells. Luc reporter assay was performed as described previously using a Promega Luc assay kit (Promega, Madison, USA) [23]. Promoter activity was expressed as relative light units (RLUs) of Luc activity and was normalized based on β–galactosidase activity.

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2.16 Chromatin immunoprecipitation (ChIP) and Re-ChIP

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ChIP assays were performed using ChIP-IT kit (Active Motif, Carlsbad, CA), and the detail procedures

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were according to the manufacturer's protocol. Antibodies against DNMT3b and SP1 were used for ChIP.

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Then, protein-G-Sepharose beads from the primary IP were washed and the complexes were eluted from

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the beads by incubation with 10 mM DTT at 37°C for 30 min. After a 50-fold dilution with 1× sonication buffer, the eluates were subjected to IP with the second antibody. After purification, Immunoprecipitated

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DNA and input were amplified by qRT-PCR with specific primers; SCARB1 promoter primers,

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SCARB1-1, forward 5’-CCAAAACGGAAGCGAGGC-3’ and reverse 5’-CAGCCTTGGGCTTCAGG

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ATT-3’; SCARB1-2, forward 5’-CATGGCGGGGCTTGTCTT-3’ and reverse 5’-GTGGTTTTAT GCCCCATCGC -3’; SCARB1-3, forward 5’-ACGAGGCCTCCTCAGCTC -3’ and reverse 5’-CAGC CTTGGGCTTCAGGATT-3’.

2.17 Immunoblotting and immunoprecipitation (IP) assays

Foam and HEK 293T cells were seeded in 10 cm dishes and treated with Hcy for 48 h before collection. Subsequently, they were washed once with ice-cold phosphate-buffered saline (PBS) and lysed in a lysis buffer containing 1% Triton X-100, 25 mM Tris( pH 7.5), 150 mM NaCl, 5 mM EDTA, 10% glycerol, 2

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mg/ml aprotinin and 1 mM PMSF, followed by centrifugation at 14000 g for 15 min at 4°C to remove cell debris. Supernatants were incubated with the indicated antibody for 1 h followed by incubation with protein A/G plus-agarose (Santa Cruz Technology) for 30 min at 4°C. The immunoprecipitate was washed three times in lysis buffer, boiled in SDS sample buffer for 5 min and separated by SDS–PAGE and transferred to PVDF membranes (Millipore, MA, USA). After being blocked with 5% nonfat milk,

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membranes were incubated with primary antibody for 1 h at room temperature or overnight at 4 °C and

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then horseradish peroxidase-conjugated secondary antibody was done for 1 h at room temperature.

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Results were visualized using ECL reagent (Millipore, MA, USA).

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2.18 Statistical analysis

Statistical analysis was performed using GraphPad Prism 6.0 Software (San Diego, USA). One-way

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ANOVA or unpaired Student's t-test was used to analyze the differences between the experimental groups.

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The correlation was analyzed using the Pearson correlation analysis. The representative histograms are

3. Results

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the mean±SD of three independent experiments. P< 0.05 was considered to be significant.

3.1 High methionine diet results in hyperhomocysteinemia (HHcy) and promotes atherosclerosis progression in ApoE-/- mice

To confirm the atherogenic effects of HHcy, we firstly administered the ApoE-/- mice with a high-methionine diet for 20 weeks to induce hyperhomocysteinemia (Fig. 1A). The results indicated that

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the atherosclerotic lesion area in the aortic root was significantly increased in ApoE-/- mice fed with a high-methionine diet as well as the lesion percentage of aortic sinus, while folate and vitamin B12 significantly attenuated the increase of atherosclerotic lesion (Fig. 1B, 1C). Pearson correlation analysis further showed a positive correlation between the atherosclerotic lesion area and serum levels of Hcy (r2 =0.7492, P<0.001) (Fig. 1D), suggested the successful establishment of HHcy model, which might

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promote atherosclerotic plaque formation. Considering MOMA-2 and perilipin are markers for

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monocyte/macrophage (MC and MФ) and lipid droplets, respectively. Double immunofluorescence

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staining against MOMA-2 and perilipin were then performed to examine the involvement of monocytes

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and lipid-related protein in HHcy-related atherosclerosis. As we expected that the atherosclerotic lesions

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were largely overlapped with macrophage marker (the green area) in high-methionine diet-treated mice,

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while folate and vitamin B12 inhibited the infiltration of macrophage in the lesions (Fig.1E, 1F). Moreover,

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MOMA-2 positive area was positively related to serum Hcy level (r2 =0.6984, P<0.001) (Fig. 1G). In

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addition, both of the perilipin positive area and the percentage of positive area in the lesion were markedly elevated in the high-methionine diet group in comparison with the regular diet group (Fig.1H). A positive correlation was found between perilipin positive area and serum Hcy levels (r2 =0.7028, P<0.001). From the well co-localization of perilipin and MOMA-2 in the lesion of ApoE-/- mice fed with high-methionine diet (Fig. 1I), it can be concluded that the lipid accumulation in monocyte/macrophage was significantly increased in this group as the perilipin and MOMA-2 double positive area represented the lipid accumulation in monocyte/macrophage. These results collectively suggested that elevated Hcy levels may aggravate the lipid accumulation and formation of the atherosclerotic plaque.

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Fig. 1. HHcy accelerates atherosclerotic plaque formation and lipid accumulation. 6-week-old ApoE-/mice were randomly divided into three groups, which were fed with a regular diet, Met (HMD), and Met plus folate plus vitamin B12 (HMD+F+VB12 ) respectively. After 20 weeks, serum and aorta were collected

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from these mice. (A) Serum Hcy levels in ApoE-/- mice were measured by the automatic biochemical analyzer. (B) Photomicrographs of mouse aortic sinus cross sections staining with oil red O. The blue arrow indicates the plaque region (Magnification:5×). (C, D) Quantitative analysis of lesions in the aortic sinuses. Atherosclerotic lesion area was defined as the neointimal region between the lumen and internal elastic lamina. The correlation of the atherosclerotic plaque area with Hcy levels was evaluated by

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Pearson correlation analysis. (E) Photomicrographs of mouse aortic sinus cross sections staining with

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MOMA-2 (MC/Mφ marker, green), perilipin (red), DAPI (blue). Merge image (yellow to orange) shows

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the accumulation of perilipin and MOMA-2 in the lesion. Scale bar, 200 μm. (F, G, H, I) Quantitative

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analysis of MC/Mφ area (F), perilipin positive area (H), and perilipin positive MC/Mφ area (I).

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Correlation analysis of Hcy level with MC/Mφ area, perilipin positive area, and perilipin positive

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MC/Mφ area were shown in scattered dot graphs. Each data point represents one mouse. MC, monocytes;

P<0.01.

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**

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Mφ, macrophages. Data are shown as means ± SD from three independent experiments. * P<0.05,

3.2 SCARB1 low expression was involved in Hcy-induced lipid accumulation in foam cells

SCARB1 , a key component of the reverse cholesterol transport pathway, plays an important role in the metabolism of HDL and LDL [24, 25]. To elucidate whether SCARB1 was involved in the lipid accumulation mediated by Hcy, double immunofluorescence staining assay was used to characterize the level of MOMA-2 and SCARB1. As shown in Fig 2A, the number of SCARB1 puncta in ApoE-/- mice with HHcy was significantly decreased, which was evidenced by the decreased co-localization with

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MOMA-2 in the atherosclerotic plaque. Consistent results were found for the mRNA and protein levels of SCARB1 in the aorta from ApoE-/- mice with the same treatment, which were also confirmed in foam cells cultured in vitro (Fig. 2B, 2C). These data suggested that Hcy may induce lipid accumulation in foam cells by inhibiting SCARB1 expression. To test this hypothesis, we establish a foam cell model with SCARB1 overexpression (Supplementary Fig. S1A). Fig. 2D showed that SCARB1 overexpression

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evidently decreased the content of total cholesterol (TC), cholesterol ester (CE) and TG (total triglyceride)

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but not FC in foam cells following with Hcy treatment. However, knockdown of SCARB1 obtained the

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contrary results (Fig.2E and Supplementary Fig. S1B). Nile red staining revealed that overexpression of

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SCARB1 lead to an overabundance of lipid bodies in foam cells in the presence of Hcy further supported

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our point that SCARB1 could attenuate Hcy-induced accumulation of lipid (Fig. 2F, 2G). Collectively,

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these data suggested that the enhanced lipid accumulation in Hcy-treated foam cells might be owing to the

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downregulation of SCARB1 during the formation of atheromatous plaque.

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Supplementary Fig. S1. SCARB1 expression in foam cells. (A) qRT-PCR and western blot were used to measure mRNA and protein levels of SCARB1 in foam cells infected with adenovirus express

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SCARB1 (Ad-SCARB1) and GFP (Ad-GFP) following Hcy. (B) mRNA and Protein detection of

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SCARB1 in foam cells infected with lentiviral shRNAs encoding SCARB1 (shSCARB1) or GFP

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(shRNA). Data are means ± SD from three independent experiments. * P< 0.05, ** P< 0.01.

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Fig. 2. Hcy promotes the accumulation of lipids in foam cells by the downregulation of SCARB1

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expression.

(A) Photomicrographs and quantitative analysis of double immunofluorescence staining for

MOMA-2 (green) and SCARB1 (red) in the aortic sinus from ApoE -/- mice. Nuclei were stained with DAPI. Scale bar, 200 μm. Yellow arrow indicated the double color staining. Quantification of the average SCARB1 intensity and co-localization with MOMA-2 was done, respectively (B, C). SCARB1 mRNA and protein expression levels in the aorta of ApoE-/- mice and foam cells under the treatment of Hcy or

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Hcy and folate plus vitamin B12 (D, E). Detection of free cholesterol (FC), total cholesterol (TC),

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cholesterol ester (CE) and triglyceride (TG) in foam cells after infection with Ad-GFP, Ad-SCARB1,

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shRNA or shSCARB1 following the treatment of Hcy. (F, G) Visualization of lipid droplets in foam cells

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using Nile Red staining (red). Nuclei were stained with DAPI (blue). Scale bar, 10 μm. Quantification of

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lipid droplets in the foam cells and approximately 5-10 cells per FOV from six different fields of view

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0.05, ** P< 0.01.

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were analyzed for each n value. Data are shown as means ± SD from three independent experiments. * P<

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3.3 SCARB1 downregulation mediated by Hcy is not in a DNA methylation-dependent manner DNA methylation is an important epigenetic regulation of gene expression, which often occurs at CpG islands that frequently coincide with the promoter region [26].To classify whether DNA methylation was involved in the transcriptional regulation of SCARB1, we analyzed DNA sequence at the promoter region of the SCARB1 gene using MethPrimer 2.0. Two CpG islands (CpG-1 island: a 201-bp long CpG island that starts from −1091 and extends up to −889, with a CG content of 50% and a CpG ratio of 0.6; and CpG-2 island: a 478-bp long CpG island that starts from −166 and extends up to +321, with a CG content

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of 50% and a CpG ratio of 0.6) were observed at the proximal promoter of this gene (Fig. 3A), indicating the potential regulation of DNA methylation on SCARB1 expression. To support our hypothesis, several fragments of SCARB 5’-flanking region (-1944/+321, −834/+321, −166/+321 and +321/+1) generated by PCR were inserted into the firefly luciferase vector pGL3. Luciferase reporter assay showed that the pGL3-SCARB1 construct (−1944/+1) which spans most of the CpG islands of SCARB1 promoter region

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possesses the highest promoter activity (Fig. 3B), indicating the presence of a regulatory element for

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SCARB1 in this region. However, further experiments performed by methylation-specific PCR (MSP)

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assay failed to show a significant change in the methylation of SCARB1 promoter from ApoE-/- mice fed

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with a high-methionine diet (Fig. 3C). Meantime, similar results were obtained from Hcy-treated foam

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cells as evidenced by bisulfite sequencing (BSP), showing the constitutive methylation at the CpG island

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from -1091 to -889, but not for the CpG island from -166 to 321 (Fig. 3D), implying that DNA

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methylation was not necessary for SCARB1 expression regulation. Of note, no significant change was

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observed in SCARB1 transcriptional activity in foam cells following treatment with DNA methyltransferases (DNMTs) inhibitor 5-azacytidine (AZC) in the presence of Hcy (Fig. 3E). These results suggested that DNA methylation may not be involved in the Hcy-mediated regulation of SCARB1 expression.

DNA methylation was not responsible for Hcy-mediated inhibition of SCARB1 expression. (A)

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A schematic diagram of putative CpG islands in the SCARB1 promoter identified by Meth Primer

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software. Criteria used for prediction were island size >100 bp, GC percentage >30% and

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observed/expected CpG ratio >0.6. Predicted CpG island was indicated by a blue color. (B) Promoter activity was analyzed by dual luciferase reporter assay. The full-length or deleted SCARB1 promoter luciferase vectors, as indicated on the left panel, were co-transfected with a Renilla reporter plasmid into HEK 293T cells. The luciferase activity was determined after 48 h transfection. Transcriptional activity was normalized by Renilla and expressed as fold activity of pGL3 promoter less vector. (C) The DNA methylation of SCARB1 promoter in the aortic tissue of ApoE -/- mice was detected by MSP. M indicates the methylated PCR band; U indicates the unmethylated PCR band. (D) The status methylation of SCARB1 promoter was analyzed by bisulfite sequencing (BSP) in foam cell exposed to Hcy or Hcy plus

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azacytidine (AZC) or Hcy plus folate and VB12 . Each circle denoted a CpG s ite; solid circles represent methylated sites and open circles represent unmethylated sites. (E) Foam cell co-transfected with the full-length SCARB1 promoter luciferase and Renilla reporter vectors were treated with Hcy or Hcy plus AZC for 48 h, followed with luciferase activity analysis. Results are expressed as the fold activity of pGL3 promoter less vector. Data are shown as mean ± SD from three independent experiments. * P< 0.05,

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P< 0.01, NS indicates no significance.

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3.4 Hcy inhibits SCARB1 expression by DNMT3b independent on its DNA methyltransferases activity

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DNMTs are the key enzyme for DNA methylation, which catalyzes the transfer of the methyl group from

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S-adenosyl-L-methionine (SAM or AdoMet) to the C5 position of cytosine [27]. Interestingly, Fig. 4A indicated that Hcy increased the activity of DNMTs in atherosclerotic plaques as well as foam cells

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although no significant alteration was found in SCARB1 promoter activity after AZC treatment, which

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indicated that there may exist other mechanisms behind DNMTs in Hcy-mediated SCARB1

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downregulation. Based on the above results, foam cells were then co-treated with Hcy and DC_05, Theaflavin-3,3’-digallate (TF3) or Nanaomycin A, the specific inhibitor of DNMT1, DNMT3a and DNMT3b, respectively. The result of Fig. 4B indicated that the expression of SCARB1 was evidently increased with the treatment of Nanaomycin A rather than DC_05 and TFD in the presence of Hcy. Meanwhile, the binding ability of DNMT3b to SCARB1 promoter was largely enhanced by Hcy in foam cells (Fig. 4C), implying the key regulatory role of DNMT3b on SCARB1 expression in response to Hcy. In addition, double immunofluorescence staining showed that the expression of DNMT3b was highly expressed in the atherosclerotic plaque from ApoE-/- mice with HHcy as evidenced by the well

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co-localization of DNMT3b and MOMA-2 (Fig. 4D, 4E), which further validate the importance of DNMT3b in the regulation of SCARB1. To further support our hypothesis, shDNMT3b was transfected into foam cells to construct a DNMT3b knock-down model (Supplementary Fig. S2A-C). From this model, it was found that DNA methylation levels at SCARB1 promoter did not show a significant change in the presence of Hcy (Fig. 4F), which was not in accordance with the significant increase in mRNA and

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protein expression of SCARB1 (Fig. 4G). In order to elucidate this phenomenon, a luciferase reporter

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assay was conducted using the vector containing SCARB1 promoter luciferase reporter constructs and

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DNMT3b wild-type or mutant (without the C terminus SAM-dependent Mtase C5-type) in foam cells,

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and the results demonstrated that both wild-type and mutant DNMT3b were able to significantly decrease

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the transcription activity of SCARB1 (Fig. 4H and Supplementary Fig. S2D), indicated that the negative

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regulation of DNMT3b on SCARB1 expression were not related with its DNA methyltransferases activity,

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which was further confirmed by western blot, as evidenced by no significant different in SCARB1

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expression was found between wild-type and mutant DNMT3b-infected foam cells (Fig. 4I). Taken together, these results suggested that the downregulation of SCARB1 transcription inhibited by DNMT3b following Hcy treatment was independent of its DNA methyltransferases activity.

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Supplementary Fig. S2. DNMT3b expression in foam cells. (A-C) Foam cells were infected with

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lentiviral shRNAs encoding DNMT3b (shDNMT3b) or GFP (shRNA), and the mRNA and protein expression of DNMT3b were determined by qRT-PCR and western blot, and shRNA3 was used for the

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further experiment. (D) Western blot was performed to detect DNMT3b protein expression in foam cells

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transfected with myc-tagged DNMT3b wild-type (WT-DNMT3b) and a mutant without the C terminus SAM-dependent Mtase C5-type (MUT-DNMT3b). Data are means ± SD from three independent experiments. * P< 0.05, ** P< 0.01, NS indicates no significance.

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Fig. 4. DNMT3b inhibited SCARB1 expression in foam cells in DNA methylation-independent manner. (A) DNMTs activity was measured in the aorta of ApoE-/- mice fed with high methionine diet and foam cells treated with Hcy plus AZC or Hcy plus folate and Vitamin B12 . (B) qRT-PCR and western blot were

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performed to detect the mRNA and protein expression of SCARB1 in foam cells co-treated with Hcy and DC_05, Theaflavin-3,3’-digallate (TF3), and Nanaomycin A, the inhibitor of DNMT1, DNMT3a and DNMT3b. (C) ChIP assay was performed to assess the binding of DNMT3b to SCARB1 promoter in foam cells treated with Hcy. The ChIP-enriched DNA fragments of SCARB1 promoter using IgG and DNMT3b antibody were amplified by PCR. Total input (5%) was used as a positive control. (D, E)

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Photomicrographs and quantitative analysis of double immunofluorescence for MOMA-2 (green) and

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DNMT3b (red) in the aortic sinus from ApoE-/- mice. Nuclei were stained with DAPI (blue). Scale bar,

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200 μm. Yellow arrow indicated the double color staining. (F) DNA methylation status of SCARB1

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promoter in foam cells infected with shDNMT3b or shRNA were detected by BSP. Each circle denoted a

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CpG site; solid circles represent methylated sites and open circles represent unmethylated sites. (G)

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qRT-PCR and western blot were used to detect the protein and mRNA expression of SCARB1 in foam

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cells treated as described above. (H) Foam cells were co-transfected with myc-tagged DNMT3b wild-type

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(WT-DNMT3b) and a mutant without the C terminus SAM-dependent Mtase C5-type (MUT-DNMT3b) plasmid and SCARB1 promoter luciferase reporter constructs. SCARB1 luciferase activities in extract were determined using luciferase reporter assay. Luciferase values were normalized to Renilla activities. (I) SCARB1 protein level in foam cells was detected by western blot after the infection of DNMT3b wild-type and a mutant without the C terminus SAM-dependent Mtase C5-type (MUT-DNMT3b) plasmid. Data are represented by means ± SD from three independent experiments , * P<0.05, indicates no significance.

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3.5 SP1 positively regulates the transcription of SCARB1 in foam cells SP1 is a well-investigated factor that regulates transcription through the binding to specific sequences 5’-(G/T)GGGCGG(G/A)(G/A)(C/T)-3’, and SCARB1 gene has been demonstrated to contain a G/C rich promoter [28], we therefore speculated that SP1 might play an important role in the regulation of SCARB1 expression. To this end, we first performed double immunofluorescence staining for MOMA-2

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and SP1. As shown in Fig.5A and 5B, the level of SP1 in atherosclerotic plaque from ApoE-/- mice with

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HHcy was lower than the mice with a regular diet, which was similarly attenuated by folate and vitamin

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B12 . Meanwhile, the in vitro and in vivo experiment showed that Hcy can inhibit the mRNA and protein

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expression of SP1 in foam cells (Fig. 5C-F). To know whether SP1 is involved in Hcy-mediated SCARB1

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downregulation, we established a foam cells model with SP1 overexpression (Supplemental Fig. S3A)

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and found that the significant elevation of SCARB1 promoter activity accompanied by the increase of

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mRNA and protein level of SCARB1 and the decrease content of TC and TG in foam cells (Fig. 5G, 5H

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and 5J). As expected, knockdown of SP1 obtained the contrary results (Fig. 5I, 5J, Supplementary Fig. S3B, S3C), and these observations were further confirmed by using mithramycin A (MTM), an inhibitor of SP1 (Supplementary Fig. S3D). Next, we analyzed SCARB1 promoter to get a well understanding of the regulation mechanisms of SCARB1 expression mediated by SP1, where three SP1 putative binding sites at -65/-55bp (SCARB1-1), -38/-28bp (SCARB1-2) and +7/+17bp (SCARB1-3) were found (Fig. 5K). ChIP assay using an anti-SP1 antibody was performed to validate the binding of SP1 to the predicted sites in vivo. The anti-SP1 antibody-enriched DNA sequences were amplified by PCR for identifying the three regions containing the putative SP1-binding sites, the results showed that SP1 clearly bound to the

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SCARB1 promoter at -65/-55, -38/-28 and +7/+17 sites (Fig. 5L). To further identify which binding site is functionally required for SP1-regulated SCARB1 promoter activation, we generated sequential deletions of these sites and performed luciferase reporter assay in the presence of Ad-SP1. pGL3-SCARB1-wt, which contains all three putative SP1-binding sites, showing the maximum promoter activity in cells following SP1 overexpression. Any of these sites mutations can reduce the transcription activity of

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SCARB1, indicating that all three sites are necessary for the transcription of SCARB1 (Fig. 5M). These

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results suggest that SP1 plays a positive regulatory role in SCARB1 expression to mediate lipid

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accumulation in foam cells.

Supplementary Fig. S3. SP1 expression and SCARB1 transcriptional activity in foam cells.

(A, B, C)

The mRNA and protein expression of SP1 was measured by qRT-PCR and western blot respectively in foam cells infected with Ad-SP1, Ad-GFP, shSP1 or shRNA in the presence of Hcy. (D) SCARB1 protein expression and promoter (-166/+32) transcriptional activity in foam cells were determined by western blot

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and luciferase reporter assay respectively in response to Hcy or Hcy plus mithramycin A (MTM). Promoter activity was normalized as the ratio between firefly luciferase and Renilla luciferase units.

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Results are represented by mean ± SD from three independent experiments. * P< 0.05,** P< 0.01.

Fig. 5. SP1 positively regulated SCARB1 expression in foam cells exposed to Hcy. (A,B) Photomicrographs and quantitative analysis of double immunofluorescence for MOMA-2 (green) and SP1 (red) in the aortic sinus from ApoE-/- mice. Nuclei were stained by DAPI (blue). Scale bar, 200 μm. Yellow arrow indicated the double color staining. (C to F) qRT-PCR and western blot were performed to measure SP1 mRNA and protein expressions in the aorta of ApoE-/- mice and foam cells following the

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treatment of Hcy or Hcy and folate plus vitamin B12 . (G, H, I) SCARB1 mRNA, protein expression and transcriptional activity in foam cells infected with Ad-SP1, Ad-GFP, shSP1 or shRNA in the presence of Hcy was detected by western blot, qRT-PCR and luciferase reporter assay respectively. Promoter activity was normalized as the ratio between firefly luciferase and Renilla luciferase units. (J) The contents of TC and TG were detected in foam cells treated as above. (K) A schematic of the SCARB1 promoter region

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shows the putative SP1-binding sites (TBS) in its proximal promoter region. SP1 binding sites are

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depicted by yellow boxes, and the transcriptional start site at G (+1) is marked in red. (L) ChIP assay

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demonstrated the direct binding of SP1 to the SCARB1 promoter in foam cells. The ChIP -enriched DNA

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fragments of the SCARB1 promoter using IgG and anti- SP1 and ant i-POL II antibody were amplified by

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PCR. Total input (5%) was used as a positive control. POL II: RNA polymerase II. (M) The foam cells

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were co-transfected with Ad-SP1 and the deletion constructs shown in the left panel and cloned into the

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pGL3 luciferase reporter plasmid, and transcriptional activity of SCARB1 proximal promoter was

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determined by luciferase reporter assay. Promoter activity was normalized as the ratio between firefly luciferase and Renilla luciferase units. Results are represented by mean ± SD from three independent experiments. * P< 0.05,** P< 0.01.

3.6 DNMT3b suppressed SCARB1 expression through the disruption of SP1 binding to SCARB1 promoter Given that Hcy-induced downregulation of SCARB1was DNA methylation-independent and the positive effect of SP1 on the transcription of SCARB1, we questioned whether there could be crosstalk between

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SCARB1 and SP1. To address this issue, foam cells were exposed to NanaomycinA, the specific inhibitor of DNMT3b. Fig. 6A showed that Nanaomycin A treatment did not affect the inhibition of SP1 mediated by Hcy, implying that SP1 was not the downstream target of DNMT3b. Surprisingly, although knockdown of DNMT3b could promote SCARB1 expression in foam cells treated with Hcy, no significant increase of SCARB1 transcription and expression were found as well as the content of TC and

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TG in foam cells following co-knockdown of DNMT3b and SP1 (Fig. 6B-D and Supplementary Fig.

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S4A), then we can speculate that DNMT3b might exert the inhibitory effect on SCARB1 through Sp1,

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which prompted us to examine the physical association between SP1 and DNMT3b. GST-SP1 and

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myc-DNMT3b were transiently transfected into the HEK293T cells , the result of Co-immunoprecipitation

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(Co-IP) experiment showed that exogenous SP1 could be co- precipitated with exogenous DNMT3b (Fig.

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6E). Conversely, exogenous DNMT3b was also co-precipitated with exogenous SP1 (Supplementary Fig.

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S4B). The interaction between endogenous SP1 and endogenous DNMT3b was also readily detected in

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foam cells by Co-IP assay (Fig. 6F), which means the interaction between DNMT3b and SP1 both in vitro and in vivo. Besides, co-localization experiments showed that both SP1 and DNMT3b were distributed throughout both the nucleus and the cytoplasm (Fig. 6G), as reported previously [29, 30].To further clarify the association between SCARB1 and SP1, we performed chromatin ChIP and Re-ChIP assays experiments. The results exhibited that DNMT3b was recruited to the SP1 binding region of SCARB1 promoter in foam cells with or without the treatment of Hcy (Fig. 6H and Supplementary Fig. S4C), suggesting a competitive binding between SP1 and DNMT3b on SCARB1 promoter in foam cells. Meanwhile, enhanced binding of SP1 to SCARB1 promoter was observed in foam cells treated with Hcy

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following DNMT3b knockdown, while DNMT3b overexpression obtained the contrary result (Supplementary Fig. S4D), indicating the key role of SP1 for DNMT3b in foam cells lipid accumulation. To determine the domain responsibility of the interaction between DNMT3b and SP1, we generated a series of deletion mutants for DNMT3b (Fig. 6I) and co-transfected them with GST-SP1 in HEK 293T cells. Pull-down assay showed that the deletion of 580 residues of DNMT3b, including the PWWP

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domain at the N terminus (1-298), or the SAM-dependent Mtase C5-type domain at the C terminus

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(572-853) abolished the interaction with SP1 (Fig. 6J,lanes 2 and 4). Conversely, deletion of 273 residues

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of DNMT3b, including the AAD domain at the central amino acid residues (299-571), retained the

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interaction with SP1 (Fig. 3C, lanes 3 and 5), suggesting that the amino acid residues from 1-298 and

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572-853 are the core domain required for its interaction with SP1.

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We next identified the region in SPl that associates with DNMT3b and constructed four deletion mutants

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of SP1 as described in Fig. 6K. After coexpressing these four deletion mutants of SPl with myc-DNMT3b

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in HEK 293T cells (Fig. 6K), GST pull-down assay was then performed. We found that the 610 residues deletion at the N terminus (1-610) of SP1, including the Repressor domain and transaction domain disrupted the interaction between SP1 and DNMT3b, indicating that the N-terminal region of SPl was responsible for its interaction with DNMT3b (Fig. 6L). Taken together, our results suggested that DNMT3b mediate the lipid accumulation in foam cells was due to the competitively binding to SCARB1 promoter with SP1, at least partly through the interaction with SP1.

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Supplementary Fig. S4. DNMT3b represses SCARB1 transcription via interaction with SP1. (A) Foam

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cells infected with shDNMT3b or Ad-DNMT3b were transfected with SCARB1 promoter luciferase reporter constructs, and the transcriptional activity of SCARB1 proximal promoter was determined by

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luciferase reporter assay. Promoter activity was normalized as the ratio between firefly luciferase and

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Renilla luciferase units. (B) HEK 293T cells were transfected with myc-DNMT3b and GST-SP1 solely or

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jointly, and immunoprecipitations were performed with anti-GST-conjugated beads. The presence of SP1 and DNMT3b was detected in the immunoprecipitates (top) and the input (bottom) using anti-GST or anti-myc antibody respectively. (C) Chromatin immunoprecipitated with the anti-SP1 antibody in foam cells following the treatment of Hcy was eluted and subjected to a Re-ChIP using either the anti-SP1 or the anti-DNMT3b. PCR was performed with primers flanking the SP1 binding regions of SCARB1. Representative gels are shown. (D) ChIP assay demonstrated the binding of SP1 on SCARB1 promoter in foam cells infected with shDNMT3b and Ad-DNMT3b solely or jointly following Hcy treatment. The

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ChIP-enriched DNA fragments of the SCARB1 promoter using IgG and an anti-SP1 antibody were amplified by PCR. Total input (5%) was used as a positive control. Data are means ± SD from three

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independent experiments. * P< 0.05 ** P< 0.01,NS indicates no significance.

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Fig. 6. SP1 was required for DNMT3b to inhibit the expression of SCARB1. (A) The protein expression

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detection of SP1 in foam cells with Hcy treatment or Hcy plus Nanaomycin A using western blot. (B) shDNMT3b and shSP1 were transfected into foam cells solely or jointly following the treatment of Hcy, and the SCARB1 protein and mRNA expression were detected by western blot and qRT-PCR. (C, D) Contents of TC and TG in foam cells after infection with the indicated adenovirus in the absence or presence of Hcy. (E) HEK 293T cells were transfected with myc-DNMT3b and GST-SP1 solely or jointly,

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and immunoprecipitations were performed using anti-myc-conjugated beads. The presence of DNMT3b

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and SP1was detected in the immunoprecipitates (top) and the input (bottom) using anti-myc or anti-GST

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antibody respectively. (F) Co-immunoprecipitation (Co-IP) assay was performed to determine the

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endogenous interaction between SP1 and DNMT3b in foam cells using the antibodies against SP1 and

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DNMT3b. IgG was used as a negative control. (G) Confocal immunofluorescence of foam cells

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expressing DNMT3b (green) together with SP1 (red). Representative photograph showed the

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co-localization of DNMT3b-SP1 (indicated with yellow signals) in merged images, nuclei were stained

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with DAPI (blue). Scale bar, 10 µm. (H) Chromatin immunoprecipitated with the anti-SP1 antibody in foam cells was eluted and subjected to a Re-ChIP using either the anti-SP1 or the anti-DNMT3b. PCR was performed with primers flanking the SP1 binding regions of SCARB1. Representative gels are shown. (I) Schematic diagram showing the structure of DNMT3b and the different deletion constructs. (J) HEK293T cells were transfected with myc or myc-tagged DNMT3b domain constructs together with GST-tagged SP1, Extracts were immunoprecipitated with anti-myc antibodies, and bound SP1 was examined by western blot. The stars indicate the location of the fragments in the gels. (K) Schematic diagram showing the structure of SP1 and the different deletion mutants. (L) HEK293 cells were

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transiently co-transfected with plasmids encoding GST or GST-tagged deletion mutants of SP1, and then cell lysates were used for GST pull-down assay, followed by western blot with an anti-GST antibody. The stars indicate the location of the fragments in the gels. Results are represented by mean ± SD from three

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independent experiments. * P< 0.05 ** P< 0.01,NS indicates no significance.

Fig 7. A proposed model for the regulation of SCARB1 expression in Hcy-mediated lipid

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accumulation in foam cells. DNMT3b-mediated the downregulation of SCARB1 promotes foam cells

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lipid accumulation in response to Hcy, which was attributed to its increased recruitment ability to the SP1 binding region of SCARB1 promoter, but not dependent on its DNA methyltransferases activity.

4. Discussion

Atherosclerosis is a chronic lipid-driven disease in large and medium arterial walls [31]. As the major component of atherosclerotic lesions, macrophages played a critical role in the development of atherosclerosis by uptaking ox-LDL into foam cells [32]. Recently, various risk factors including Hcy in

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atherosclerosis have been identified [33]. In this study, we found that DNMT3b-mediated inhibition of SCARB1 facilitated lipid accumulation in foam cells in the presence of Hcy, which did not depend on its DNA methylation activity. According to our point, these results will provide a new perspective for a comprehensive understanding of Hcy-related disease.

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The intracellular lipid homeostasis of macrophages is dynamically regulated by low-density lipoprotein

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(ox-LDL) uptake and cholesterol (CE) efflux [34]. We previously reported that HHcy can accelerate

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atherosclerosis and lipid accumulation in ApoE-/- mice with a high methionine diet [35]. The current study,

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which is in agreement with our previous findings , further demonstrated that HHcy can increase lipid

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accumulation in MOMA-2 positive monocyte/macrophage. It was reported that Hcy could stimulate macrophage CD36 expression, which contributes to the uptake of ox-LDL and foam cell formation [36].

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On contrary, both ABCA1 and ABCG1 can promote excessive cholesterol efflux from macrophages. Jin

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et al demonstrated that ABCA1 and ABCG1 were decreased under Hcy treatment [37]. These findings

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demonstrated that Hcy can cause accumulative CE stores as cytoplasmic lipid droplets and subsequently trigger the formation of foam cells. These findings led to the hypothesis that HHcy contributes to atherosclerosis via promoting lipid accumulation. However, evidence from cell lines is limited. Meanwhile, the potential mechanism for Hcy-mediated abnormal lipid metabolism in macrophage needs to be further addressed.

SCARB1 is a glycoprotein with two transmembrane domains, the primary role of which is to facilitate uptake of cholesteryl esters (CE) from HDL in the liver [38]. Disruption of SCARB1 in mice resulted in

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elevated HDL cholesterol levels due to the defects in selective HDL-cholesterol clearance [10]. SCARB1 was also involved in the bidirectional flux of free cholesterol between peripheral cells and HDL [39]. Outside of these reports, SCARB1 has been found to be implicated in cardiovascular diseases in recent years. Our study revealed that SCARB1 expression was decreased in both atherosclerotic plaques from ApoE-/- mice with HHcy and Hcy-treated foam cells, and overexpression of SCARB1 could abrogate

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Hcy-induced foam cells lipid accumulation. This observation is consistent with previous reports that

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deletion of ABCA1 and SCARB1 in bone marrow-derived cells enhances macrophage foam cell

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formation in vivo and atherosclerotic lesion development in LDL receptor knockout mice on a Western

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diet [40]. Although the present work supports the importance of SCARB1 in the homeostasis of

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cholesterol in Hcy-treated foam cells, it does not exclude the involvement of other members in the

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scavenger receptors family. Whether SCARB1 is involved in athermanous plaque formation in ApoE-/-

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mice fed with a high methionine diet is an interesting question for future investigation.

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Hcy is an intermediate product released during the methionine cycle, which provides methyl groups for DNA and protein in the presence of S-adenosylmethionine (SAM) to alter the methylation status [41]. As the most widely investigated epigenetic modification, DNA methylation usually occurs at CpG islands or GC-rich regions and is mostly associated with transcriptional repression [42]. Here, no significant DNA methylation alteration was observed in both aortas from ApoE-/- mice with HHcy and Hcy-treated foam cells although bioinformatics identified two islands in SCARB1 promoter. Even more intriguingly, the present study exhibited that DNMT3b, one of the DNMTs in mammal negatively regulated SCARB1

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expression in Hcy-treated foam cells, which is independent of its DNA methyltransferases activity. These results were inconsistent with the previous study that the DNMT3b upregulation induced the hypermethylation of the p53 promoter and finally resulted in a decrease of p53 expression [5]. Obviously, it is not plausible to clarify the down-regulation of SCARB1 expression from the perspective of DNA methylation due to this phenomenon was not abided by the DNA methylation pattern. Since gene

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expression regulation is a complex process involving many mechanisms, the possible explanation is that

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DNMT3b alters SCARB1 expression through other pathways or interactions with other proteins. Tang X

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et al reported that autocrine TGF-β1/miR-200s/miR-221/DNMT3B regulatory loop is responsible for the

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maintenance of cancer-associated fibroblasts (CAF) status to drive breast cancer cell proliferation [43].

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Additionally, there is also evidence to support the point that Zinc-fingers and homeoboxes 1 (ZHX1)

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interact with DNMT3b in the nucleus and enhance DNMT3b-mediated transcriptional repression [44].

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Apart from the epigenetic modification such as DNA methylation, the role of transcription factors (TFs)

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in regulating gene expression has also been extensively elucidated [45]. Some evidence demonstrated that TFs can regulate the expression of multiple downstream genes by binding DNA directly or indirectly through the formation of protein complexes. As a member of Sp/Kruppel transcription factor family, SP1 is implicated in gene trans-activation in response to a variety of cellular signals [17]. In this study, we found that SP1 positively regulates SCARB1 expression in foam cells under Hcy treatment. One reason is that SP1 can initiate gene transcription through binding to the GC box in the gene promoter, and SCARB1 promoter region is rich in GC dinucleotide, which is similar to those previous reports [46, 47]. Another reason is that SP1 might exert its effect on SCARB1 in an indirect manner. It was reported that wild-type

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p53 negatively regulated DNMT1 expression by forming a complex with SP1protein and chromatin modifiers on the DNMT1 promoter [48]. Furthermore, DNMT3b was found to co-exist with SP1 in a complex formation to decrease the SP1 binding to SCARB1 promoter in Hcy-treated foam cells. The new regulation mechanism of DNMT3b wholly agrees with the previous report that DNMT3b can perform its biological functions in a DNA methyl-transferase activity-independent manner [49, 50]. Our studies

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strongly suggest that SP1 is required for DNMT3b to inhibit SCARB1 expression in foam cells in

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response to Hcy. Covalent-bond-forming domains of proteins are specific structures to exercise certain

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biological functions in biological macromolecules and the key for protein-protein interactions. DNMT3b

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protein consists of PWWP, AAD and SAM-dependent Mtase C5-type domains, while the protein of SP1

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consists of repressor domain, transaction domain and VZV IE62-binding domain. Our results indicated

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that the amino acids 1-298 and 572-853 of DNMT3b and 1-610 at the N terminus of SP1 are required for

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the interaction between SP1 and DNMT3b. Considering the redundant sequences found in the domains of

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both SP1 and DNMT3b protein, it would be necessary for us to excavate the key domain of DNMT3b to determine whether it was responsible for the corresponding phenotypes and gain further insight into the DNMT3b regulation mechanism in the development of atherosclerosis. In conclusion, our study reveals that the inhibition of SCARB1 expression regulated by DNMT3b facilitated Hcy-mediated lipid accumulation in foam cells, which is necessary for the development of atherosclerosis. Furthermore, we also revealed that the competitively binding to SCARB1 promoter with SP1 is responsible for DNMT3b to downregulate SCARB1 expression in foam cells under Hcy treatment (Fig. 7). These findings may deepen our understanding of atherosclerosis.

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Footnotes

Abbreviations:

Hcy homocysteine

HHcy hyperhomocysteinemia

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ApoE-/- Apolipoprotein E knockout

high methionine diet

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HMD

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DNMTs DNA methyltransferases

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TC total cholesterol

FC

AZC

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CE cholesterol ester

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TG triglyceride

free cholesterol

5-azacytidine

HDL high-density lipoprotein

MTM mithramycin A

TF3 Theaflavin-3,3’-digallate

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SCARB1 scavenger receptor class B member1

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (81560288, 81560084, 81700404, 81570452, 81560086). West China first-Class Disciplines Basic Medical Sciences at Ningxia Medical

University

(NXYLXK2017B07).

Ningxia

Natural

Science

Foundation

of

China

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(2019AAC03075).

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The authors declare that they have no conflict of interest.

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Conflict of Interest

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Highlights 

Downregulation of SCARB1 facilitated Hcy-mediated lipid accumulation in foam cells.



DNA methylation was not responsible for Hcy-mediated downregulation of SCARB1 expression.



Inhibition of SCARB1 expression mediated by DNMT3b is independent on its DNA methyltransferases activity.

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DNMT3b interferes SP1 binding to SCARB1 promoter leading to SCARB1 downregulation.

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