CTCF调控小鼠AML12肝细胞系脂质代谢功能与基因表达

doi:10.3969/j.issn.1674-8115.2024.09.002

上海交通大学学报（医学版） ›› 2024, Vol. 44 ›› Issue (9): 1069-1082.doi: 10.3969/j.issn.1674-8115.2024.09.002

• 论著 · 基础研究 • 上一篇

CTCF调控小鼠AML12肝细胞系脂质代谢功能与基因表达

陈怀煌(), 左武(), 卞迁()

上海交通大学医学院附属第九人民医院上海精准医学研究院，上海 200011

收稿日期:2024-05-03 接受日期:2024-05-22 出版日期:2024-09-28 发布日期:2024-10-09
通讯作者: 卞迁 E-mail:chh1142268531@163.com;wuzuo@sjtu.edu.cn;qianbian@shsmu.edu.cn
作者简介:陈怀煌（1999—），男，硕士生；电子信箱：chh1142268531@163.com
左　武（1994—），男，博士；电子信箱：wuzuo@sjtu.edu.cn。第一联系人：（陈怀煌、左　武并列第一作者）
基金资助:
国家自然科学基金(32200432);上海市自然科学基金(23ZR1435700);上海高水平地方高校创新研究团队(SHSMUZLCX20211700);上海交通大学医学院“双百人”项目(20171920)

CTCF regulates lipid metabolism and gene expression in mouse AML12 liver cell line

CHEN Huaihuang(), ZUO Wu(), BIAN Qian()

Shanghai Institute of Precision Medicine, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China

Received:2024-05-03 Accepted:2024-05-22 Online:2024-09-28 Published:2024-10-09
Contact: BIAN Qian E-mail:chh1142268531@163.com;wuzuo@sjtu.edu.cn;qianbian@shsmu.edu.cn
Supported by:
National Natural Science Foundation of China(32200432);Natural Science Foundation of Shanghai(23ZR1435700);Innovative Research Team of High-Level Local Universities in Shanghai(SHSMUZLCX20211700);“Two-hundred Talents” Program of Shanghai Jiao Tong University School of Medicine(20171920)

摘要/Abstract

摘要：

目的·明确CCCTC结合因子（CCCTC-binding factor，CTCF）对肝细胞脂质代谢的调控作用，并探究CTCF调控肝细胞基因表达的作用机制。方法·通过慢病毒将稳定表达Ctcf shRNA的DNA序列整合到小鼠永生化的AML12肝细胞系中，实现对Ctcf的稳定敲低。通过反转录实时荧光定量聚合酶链反应（RT-qPCR）及蛋白质印迹法（Western blotting）验证Ctcf的敲低效率。碘化丙啶（propidium iodide，PI）染色测定细胞周期，使用CCK-8法绘制细胞生长曲线，检测Ctcf敲低对细胞生长的影响。油红O染色标记细胞内脂质，检测CTCF对AML12细胞脂质代谢和脂滴累积的影响。使用CUT & Tag测序技术分析Ctcf敲低后CTCF在全基因组的结合变化。结合RNA测序（RNA-seq）的转录组数据分析CTCF结合变化后的转录组变化，使用基因本体论（GO）、京都基因和基因组数据库（KEGG）富集分析及基因集富集分析（GSEA）揭示Ctcf敲低对AML12肝细胞基因的表达影响，并探究差异基因与CTCF结合变化间的关联。结果·RT-qPCR结果表明Ctcf在 mRNA水平敲低了63.4%，Western blotting验证了CTCF在蛋白表达层面降低了57.7%（均P<0.05）。生长曲线及周期实验确定了Ctcf敲低后细胞增殖阻滞于G₁/G₀期。并且AML12细胞在Ctcf敲低后自发出现细胞内脂质蓄积（P<0.05）。CTCF在全基因组的结合呈现出显著变化，大多数CTCF结合差异区域表现出CTCF结合减少，但仍有部分区域CTCF结合增加。转录组数据显示Ctcf敲低导致1 344个基因出现显著的表达变化，在上调基因中富集出与脂滴堆积相关的脂质代谢通路。CTCF结合上调峰关联的差异基因富集在脂质转运与脂质定位相关通路，而CTCF结合下调峰关联的差异基因主要富集在DNA损伤修复、细胞凋亡、细胞周期等生物学过程，但CTCF在基因组的结合变化并不足以导致邻近基因的表达上调或下调。结论·CTCF通过调控脂质代谢相关基因的表达影响肝细胞的代谢功能，然而CTCF在基因组上的结合变化与邻近基因的表达缺乏显著的相关性，可能主要通过远端调控的方式对基因表达产生影响。

关键词: CCCTC结合因子（CTCF）, AML12细胞系, 脂质代谢, 转录调控

Abstract:

Objective ·To clarify the regulatory role of CCCTC-binding factor (CTCF) in lipid metabolism in liver cells, and explore the mechanisms by which CTCF regulates liver cell gene expression. Methods ·Immortalized AML12 liver cell line was used as a model to investigate the functions of CTCF in liver cells. To stably knock down Ctcf, DNA sequences stably expressing Ctcf shRNA were integrated into AML12 cells through lentivirus. The knockdown efficiency of Ctcf was verified by RT-qPCR and Western blotting. The effects of Ctcf knockdown on cell growth and cell cycle were assessed by performing CCK-8 assay and propidium iodide (PI) staining. Intracellular lipids, labeled with Oil Red O staining, were analyzed and quantified to detect the effect of CTCF on lipid metabolism and lipid droplet accumulation in AML12 cells. Changes in CTCF genome distribution after Ctcf knockdown were analyzed using the Cleavage Under Targets and Tagmentation (CUT&Tag) method. Transcriptome changes in AML12 cells after Ctcf knockdown were quantified by RNA sequencing (RNA-seq). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, and gene set enrichment analysis (GSEA) were employed to evaluate the functions of differentially expressed genes. The correlation between gene expression changes and CTCF binding changes was further assessed by performing statistical analyses. Results ·The result of RT-qPCR showed that Ctcf is downregulated 63.4% in mRNA level and 57.7% in protein level (both P<0.05). Assay of the growth curve and cycle phase confirmed that cell proliferation was inhibited in the G₁/G₀ phase after Ctcf knockdown. After Ctcf knockdown, AML12 cells exhibited spontaneous accumulation of intracellular lipids, indicating dysregulation of lipid metabolism (P<0.05). Genome-wide CTCF binding analysis revealed significant changes, with most differential CTCF peaks showing decreased binding, although a subset of regions exhibited increased CTCF binding. Transcriptome analyses revealed that knocking down Ctcf resulted in significant expression changes in 1 344 genes. These differentially expressed genes were enriched in lipid metabolism pathways. Further analysis showed that genes associated with regions of increased CTCF binding were enriched in pathways related to lipid transport and localization, whereas genes associated with regions of decreased CTCF binding were mainly enriched in processes such as DNA damage repair, apoptosis, and cell cycle regulation. However, the binding changes of CTCF in the genome were not sufficient to lead to the expression changes of their neighboring genes. Conclusion ·CTCF affects the metabolic function of liver cells by regulating the expression of lipid metabolism-related genes. However, the binding changes of CTCF in the genome lack significant correlation with the expression of their neighboring genes, suggesting that CTCF mainly influences liver gene expression through long-distance regulation, possibly by modulating higher-order chromatin structure and enhancer-promoter interactions.

Key words: CCCTC-binding factor (CTCF), AML12 cell line, lipid metabolism, transcription regulation

中图分类号:

R394.3

陈怀煌, 左武, 卞迁. CTCF调控小鼠AML12肝细胞系脂质代谢功能与基因表达[J]. 上海交通大学学报（医学版）, 2024, 44(9): 1069-1082.

CHEN Huaihuang, ZUO Wu, BIAN Qian. CTCF regulates lipid metabolism and gene expression in mouse AML12 liver cell line[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(9): 1069-1082.

图/表 11

表1 shRNA序列

Tab 1 shRNA sequences

Primer name	Sequence (5′→3′)
Mouse Ctcf-shRNA 1	GGTGCAATTGAGAACATTATA CTCGAG TATAATGTTCTCAATTGCACC
Mouse Ctcf-shRNA 2	TGGACGATACCCAGATCATAACTCGAGTTATGATCTGGGTATCGTCCA
Mouse scramble shRNA	TTCTCCGAACGTGTCACGT CTCGAG ACGTGACACGTTCGGAGAA

表2 RT-qPCR引物序列

Tab 2 Primer sequences for RT-qPCR

Gene	Forward primer (5′→3′)	Reverse primer (5′→3′)
Ctcf	AACAGTGACCCTCCTGAGGAATC	TATAACGACGATGCCGCACCA
β-actin	CATTGCTGACAGGATGCAGAAGG	TGCTGGAAGGTGGACAGTGAGG

图1 Ctcf 敲低验证及细胞增殖速率检测Note： A. Schematic diagram showing the target regions of two different shRNAs used to knock down Ctcf. B. Quantification of Ctcf transcript levels in AML12 Ctcf knockdown cells and control cells (Scramble) using RT-qPCR. C. Western blotting shows efficient depletion of CTCF protein by Ctcf shRNA2. D. Quantitative analysis of the Western blotting bands shown in Fig C. E. Line graph shows cell proliferation rates of Ctcf knockdown cells and control cells measured by the CCK-8 assay. F?H. Comparative analysis of cell cycle composition of Ctcf knockdown cells and control cells at 40% (F), 60% (G), and 80% (H) confluence.

Fig 1 Validation of Ctcf knockdown and assessment of its impact on cell proliferation

图2 Ctcf 敲低后全基因组CTCF结合变化Note： A. PCA plot of CUT & Tag data reveals significant differences in CTCF signals between Ctcf knockdown and Scramble cells. B. Volcano plot shows the changes of CTCF signals at CTCF peaks following Ctcf knockdown. C. Averaged line graph and heatmaps show the CTCF CUT&Tag signal intensities that are upregulated, unchanged, or downregulated at CTCF peaks in Ctcf knockdown cells. D. Representative regions exhibiting changes in CTCF binding after Ctcf knockdown.

Fig 2 Genome-wide changes in CTCF binding after Ctcf knockdown

图3 Ctcf 敲低后CTCF结合变化与CTCF结合不变区域的关联基因分析Note： A?C. Pie charts show the proportions of CTCF down peaks (A), CTCF up peaks (B), and CTCF unchanged peaks (C) associated with different genomic features. D?F. Dot plots show the top 10 enriched Gene Ontology (GO) terms in genes associated with CTCF down peaks (D), CTCF up peaks (E), and CTCF unchanged peaks (F).

Fig 3 Analysis of genes associated with changed or unchanged CTCF peaks after Ctcf knockdown

图4 Ctcf 敲低对脂滴堆积的影响Note： A. Representative Oil Red O staining images of control and Ctcf knockdown cells. B. Quantitative Image J analysis of Oil Red O staining signals shows increased lipid droplet areas in Ctcf knockdown cells. C. Quantitative spectrophotometry analysis of isopropanol-extracted Oil Red O shows increased lipid droplet content in Ctcf knockdown cells.

Fig 4 Increased lipid droplet accumulation after Ctcf knockdown

图5 Ctcf 敲低对转录组的影响Note： A. Volcano plot shows differentially expressed genes (DEGs) upon Ctcf knockdown. B. Dot plots show the top 10 enriched Gene Ontology (GO) terms in upregulated genes after Ctcf knockdown. C/D. Heatmaps show gene expression changes for DEGs involved in fatty acid metabolism (C) and lipid localization (D) processes. E. GSEA enrichment analysis shows that several gene sets related to lipid metabolism and localization are significantly upregulated in Ctcf knockdown cells. F/G. Genes involved in the fatty acid metabolic process (F) and lipid localization (G) exhibited overall upregulation in our study (Ctcf knockdown AML12 cells) and in a previously published study[19] (conditional Ctcf knockout in liver in vivo).

Fig 5 Transcriptome changes after Ctcf knockdown

表3 CTCF相关与不相关基因分类统计

Tab 3 Classification statistics of CTCF-associated and CTCF-non-associated genes

Gene

CTCF-associated

gene

CTCF-non-associated

gene

图6 CTCF结合变化与基因表达变化关联分析Note： A. Pie charts show the proportions of DEGs in genes associated or not associated with CTCF peaks. B. Pie charts show the proportions of DEGs in genes associated with CTCF down peaks, unchanged peaks, or up peaks. C/D. Box plots summarize the fold changes in expression changes for the upregulated (C) or downregulated (D) genes associated with CTCF down peaks, unchanged peaks, or up peaks. E?H. Dot plots show the top 10 enriched Gene Ontology (GO) terms in DEGs associated with CTCF up peaks (E), CTCF down peaks (F), CTCF unchanged peaks (G) and genes not associated with CTCF peaks (H).

Fig 6 Analysis of the correlation between CTCF binding changes and gene expression changes

表4 CTCF差异结合峰注释基因和AML12细胞差异基因统计

Tab 4 Statistics of genes annotated with differential CTCF binding peaks and differentially expressed genes in AML12 cells

Gene	CTCF up peak annotation	CTCF unchanged peak annotation	CTCF down peak annotation
Downregulated gene	14	338	117
Unchange gene	294	5 323	1 803
Upregualated gene	48	461	158

图7 CTCF结合变化调控基因表达的复杂性Note： A?C. CTCF binding and RNA-seq tracks of representative regions show that the changes in CTCF signals do not necessarily correlate with changes in gene expression direction. A. Mlxipl gene; B. Ppara gene; C. Sirt1 gene.

Fig 7 Complex effects of CTCF binding changes on gene expression

参考文献 23

1	ARZATE-MEJÍA R G, RECILLAS-TARGA F, CORCES V G. Developing in 3D: the role of CTCF in cell differentiation[J]. Development, 2018, 145(6): dev137729.
2	KUBO N, ISHII H, XIONG X, et al. Promoter-proximal CTCF binding promotes distal enhancer-dependent gene activation[J]. Nat Struct Mol Biol, 2021, 28(2): 152-161.
3	LIU Y T, WAN X, LI H, et al. CTCF coordinates cell fate specification via orchestrating regulatory hubs with pioneer transcription factors[J]. Cell Rep, 2023, 42(10): 113259.
4	BISSERIER M, MATHIYALAGAN P, ZHANG S H, et al. Regulation of the methylation and expression levels of the BMPR2 gene by SIN3a as a novel therapeutic mechanism in pulmonary arterial hypertension[J]. Circulation, 2021, 144(1): 52-73.
5	NUEBLER J, FUDENBERG G, IMAKAEV M, et al. Chromatin organization by an interplay of loop extrusion and compartmental segregation[J]. Proc Natl Acad Sci U S A, 2018, 115(29): E6697-E6706.
6	XIANG J F, CORCES V G. Regulation of 3D chromatin organization by CTCF[J]. Curr Opin Genet Dev, 2021, 67: 33-40.
7	DAVIDSON I F, BARTH R, ZACZEK M, et al. CTCF is a DNA-tension-dependent barrier to cohesin-mediated loop extrusion[J]. Nature, 2023, 616(7958): 822-827.
8	ROWLEY M J, CORCES V G. Organizational principles of 3D genome architecture[J]. Nat Rev Genet, 2018, 19(12): 789-800.
9	DEHINGIA B, MILEWSKA M, JANOWSKI M, et al. CTCF shapes chromatin structure and gene expression in health and disease[J]. EMBO Rep, 2022, 23(9): e55146.
10	HYLE J, ZHANG Y, WRIGHT S, et al. Acute depletion of CTCF directly affects MYC regulation through loss of enhancer-promoter looping[J]. Nucleic Acids Res, 2019, 47(13): 6699-6713.
11	RAHME G J, JAVED N M, PUORRO K L, et al. Modeling epigenetic lesions that cause gliomas[J]. Cell, 2023, 186(17): 3674-3685.e14.
12	POULOS R C, THOMS J A I, GUAN Y F, et al. Functional mutations form at CTCF-cohesin binding sites in melanoma due to uneven nucleotide excision repair across the motif[J]. Cell Rep, 2016, 17(11): 2865-2872.
13	RIBEIRO DE ALMEIDA C, STADHOUDERS R, DE BRUIJN M J, et al. The DNA-binding protein CTCF limits proximal Vκ recombination and restricts κ enhancer interactions to the immunoglobulin κ light chain locus[J]. Immunity, 2011, 35(4): 501-513.
14	HIRAYAMA T, TARUSAWA E, YOSHIMURA Y, et al. CTCF is required for neural development and stochastic expression of clustered Pcdh genes in neurons[J]. Cell Rep, 2012, 2(2): 345-357.
15	CHRISTOV M, CLARK A R, CORBIN B, et al. Inducible podocyte-specific deletion of CTCF drives progressive kidney disease and bone abnormalities[J]. JCI Insight, 2018, 3(4): e95091.
16	GOMEZ-VELAZQUEZ M, BADIA-CAREAGA C, LECHUGA-VIECO A V, et al. CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart[J]. PLoS Genet, 2017, 13(8): e1006985.
17	DUBOIS-CHEVALIER J, OGER F, DEHONDT H, et al. A dynamic CTCF chromatin binding landscape promotes DNA hydroxymethylation and transcriptional induction of adipocyte differentiation[J]. Nucleic Acids Res, 2014, 42(17): 10943-10959.
18	WANG R R, QIU X Y, PAN R, et al. Dietary intervention preserves β cell function in mice through CTCF-mediated transcriptional reprogramming[J]. J Exp Med, 2022, 219(7): e20211779.
19	CHOI Y, SONG M J, JUNG W J, et al. Liver-specific deletion of mouse CTCF leads to hepatic steatosis via augmented PPARγ signaling[J]. Cell Mol Gastroenterol Hepatol, 2021, 12(5): 1761-1787.
20	WANG W, REN G, HONG N, et al. Exploring the changing landscape of cell-to-cell variation after CTCF knockdown via single cell RNA-seq[J]. BMC Genomics, 2019, 20(1): 1015.
21	PINTO P B, DOMSCH K, LOHMANN I. Hox function and specificity: a tissue centric view[J]. Semin Cell Dev Biol, 2024, 152/153: 35-43.
22	AITKEN S J, IBARRA-SORIA X, KENTEPOZIDOU E, et al. CTCF maintains regulatory homeostasis of cancer pathways[J]. Genome Biol, 2018, 19(1): 106.
23	DAVIDSON I F, PETERS J M. Genome folding through loop extrusion by SMC complexes[J]. Nat Rev Mol Cell Biol, 2021, 22(7): 445-464.

[1]	江全鑫, 陈素贞, 刘军力. 铜蓝蛋白在脂质代谢稳态调控中作用的研究进展[J]. 上海交通大学学报（医学版）, 2024, 44(1): 124-130.
[2]	胡婵婵, 范毅, 徐源, 胡志坚, 曾奕明. 脂质代谢在肺癌发生发展及诊疗领域中的研究进展[J]. 上海交通大学学报（医学版）, 2022, 42(12): 1766-1771.
[3]	顾鹏, 孙星. 超级增强子驱动致癌转录机制的研究进展[J]. 上海交通大学学报(医学版), 2021, 41(10): 1378-1383.
[4]	刘舒婷，姚玉峰，倪进婧. 鼠伤寒沙门菌中假定转录调控因子的筛选[J]. 上海交通大学学报（医学版）, 2018, 38(10): 1174-.
[5]	闵雪洁，赵丽，赵小平. SREBP在肿瘤中的研究进展[J]. 上海交通大学学报（医学版）, 2016, 36(8): 1256-.
[6]	刘锦燕，史册，王影，等. 白假丝酵母菌唑类耐药相关的转录调控研究进展[J]. 上海交通大学学报（医学版）, 2016, 36(2): 291-.
[7]	孙珍珍，彭川，郑金英，等. 甲硫氨酸负荷致同型半胱氨酸轻度升高对大鼠肝脏脂质代谢的影响及机制研究[J]. 上海交通大学学报（医学版）, 2015, 35(7): 983-.
[8]	罗澜，陈艳娟，万远方. 急性早幼粒细胞白血病患者BAX表达水平及意义[J]. 上海交通大学学报（医学版）, 2015, 35(6): 799-.
[9]	杨科峰, 房玥晖, 张雄, 等. 植物甾醇对大鼠脂质代谢紊乱的预防作用[J]. , 2010, 30(1): 13-.

CTCF调控小鼠AML12肝细胞系脂质代谢功能与基因表达

CTCF regulates lipid metabolism and gene expression in mouse AML12 liver cell line

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 11

参考文献 23

相关文章 9

编辑推荐

Metrics