RNF20及其介导的组蛋白H2B单泛素化对小鼠棕色脂肪细胞分化的影响

doi:10.11843/j.issn.0366-6964.2020.04.008

摘要/Abstract

摘要： 旨在研究RNF20及其介导的组蛋白H2B第120位赖氨酸的单泛素化（H2Bub）对小鼠棕色脂肪细胞成脂分化的影响。采集1日龄和2月龄雄性C57BL/6小鼠的棕色脂肪组织（n=3），用Western blot方法检测RNF20的表达及其介导的H2Bub水平。利用胶原酶消化法分离获得1日龄小鼠的棕色前体脂肪细胞。分别诱导棕色前体脂肪细胞和C3H10T1/2细胞系成脂分化，通过油红O染色检测其分化效果，进一步通过Western blot检测细胞分化前后（0和8 d）RNF20的表达及其介导的H2Bub水平。通过siRNA干扰Rnf20基因在C3H10T1/2细胞系中的表达，油红O染色方法观察Rnf20基因对成脂分化的影响，利用qPCR和Western blot技术检测Rnf20基因的干扰效率及其介导的H2Bub水平。结果显示，2月龄小鼠棕色脂肪组织中RNF20表达量及其介导的H2Bub水平均显著高于1日龄小鼠。脂肪细胞分化标记蛋白PPARγ和CEBPα的表达水平，RNF20表达量及其介导的H2Bub水平在棕色前体脂肪细胞及C3H10T1/2细胞成脂分化后均显著增加。此外，在C3H10T1/2细胞中敲降Rnf20基因后，与阴性对照组相比，RNF20及其介导的H2Bub水平显著降低，成脂分化后脂滴明显减少。综上表明，RNF20对小鼠棕色脂肪细胞的分化是必需的，敲降Rnf20基因导致组蛋白H2Bub水平显著降低，且降低了C3H10T1/2细胞的成脂分化效率。本研究丰富了小鼠棕色脂肪细胞分化过程中的表观遗传调控研究，为深入理解动物脂肪细胞分化提供了新的基因素材。

关键词: Rnf20基因, H2Bub, 棕色脂肪细胞, 成脂分化, 小鼠

Abstract: The objective of this study was to investigate the effect of RNF20 and it-mediated monoubiquitination of histone H2B at lysine120 (H2Bub) on brown adipocytes differentiation in mice. In this study, the expression of RNF20 and it-mediated H2Bub level in brown adipose tissue (BAT) from 1-day-old and 2-month-old male C57BL/6 mice were measured by Western blot (n=3). The brown preadipocytes were primarily isolated by collagenase digestion from 1-day-old mice. The brown preadipocytes and C3H10T1/2 cell lines were differentiated into mature adipocytes, respectively. Oil Red O staining was used to detect the adipogenic differentiation efficiency. The expression of RNF20 and it-mediated H2Bub level were detected in non-differentiated and differentiated cells (0 and 8 d) by Western blot. Furthermore, the siRNAs were applied to knockdown the expression of Rnf20 gene in C3H10T1/2 cells and the Oil Red O staining was used to detect adipogenic differentiation efficiency. qPCR and Western blot were used to detect the efficiency of interfering Rnf20 gene and it-mediated H2Bub level. The results showed that the expression of RNF20 and it-mediated H2Bub level were significantly higher in BAT from 2-month-old mice than those from 1-day-old mice. The expression of adipocyte differentiation marker proteins, PPARγ and CEBPα, and RNF20 and H2Bub level significantly increased in differentiated brown adipocytes and C3H10T1/2 cells. In addition, knockdown of Rnf20 gene in C3H10T1/2 cells significantly decreased the expression of RNF20 and it-mediated H2Bub level and reduced lipid droplets deposition in differentiated cells compared to those from negative control group. In summary, RNF20 was essential for mouse brown adipocyte differentiation. Knockdown of Rnf20 gene resulted in the decrease of H2Bub level and decreased the adipogenic differentiation efficiency of C3H10T1/2 cells. The study result adds more data to the epigenetic regulation of mouse brown adipocytes differentiation and provides new genetic material for further understanding the differentiation of animal brown adipocytes.

Key words: Rnf20 gene, H2Bub, brown adipocytes, adipogenic differentiation, mice

中图分类号:

Q953

梁小娟, 陶聪, 赵莹, 王超, 刘璐璐, 王彦芳. RNF20及其介导的组蛋白H2B单泛素化对小鼠棕色脂肪细胞分化的影响[J]. 畜牧兽医学报, 2020, 51(4): 722-731.

LIANG Xiaojuan, TAO Cong, ZHAO Ying, WANG Chao, LIU Lulu, WANG Yanfang. Effect of RNF20 and It-mediated Monoubiquitination of Histone H2B at Lysine 120 (H2Bub) on Brown Adipocyte Differentiation in Mice[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(4): 722-731.

参考文献 40

[1]	JUNG S M,SANCHEZ-GURMACHES J,GUERTIN D A.Brown adipose tissue development and metabolism[J].Handb Exp Pharmacol, 2019,251:3-36.
[2]	CAROBBIO S,GUÉNANTIN A C,SAMUELSON I,et al.Brown and beige fat:from molecules to physiology and pathophysiology[J]. Biochim Biophys Acta Mol Cell Biol Lipids,2019,1864(1):37-50.
[3]	LIANG X J,PAN J F,CAO C W,et al.Transcriptional response of subcutaneous white adipose tissue to acute cold exposure in mice[J].Int J Mol Sci,2019,20(16):3968.
[4]	DANYSZ W,HAN Y,LI F,et al.Browning of white adipose tissue induced by the ß3 agonist CL-316,243 after local and systemic treatment-PK-PD relationship[J].Biochim Biophys Acta Mol Basis Dis,2018,1864(9):2972-2982.
[5]	PHILLIPS K J.Beige fat,adaptive thermogenesis,and its regulation by exercise and thyroid hormone[J].Biology,2019,8(3):57.
[6]	STILLMAN B.Histone modifications:insights into their influence on gene expression[J].Cell,2018,175(1):6-9.
[7]	FIERZ B,CHATTERJEE C,MCGINTY R K,et al.Histone H2B ubiquitylation disrupts local and higher-order chromatin compaction[J].Nat Chem Biol,2011,7(2):113-119.
[8]	CAO J,YAN Q.Histone ubiquitination and deubiquitination in transcription,DNA damage response,and cancer[J].Front Oncol, 2012,2:26.
[9]	MAO P,SMERDON M J.Rescue of DNA damage-stalled RNA Pol II:histone H2B in action[J].RNA Dis,2014,1(1):e422.
[10]	KARPIUK O,NAJAFOVA Z,KRAMER F,et al.The histone H2B monoubiquitination regulatory pathway is required for differentiation of multipotent stem cells[J].Mol Cell,2012,46(5):705-713.
[11]	SADEGHI L,SIGGENS L,SVENSSON J P,et al.Centromeric histone H2B monoubiquitination promotes noncoding transcription and chromatin integrity[J]. Nat Struct Mol Biol,2014,21(3):236-243.
[12]	GLICKMAN M H,CIECHANOVER A.The ubiquitin-proteasome proteolytic pathway:destruction for the sake of construction[J]. Physiol Rev,2002,82(2):373-428.
[13]	WEST M H,BONNER W M.Histone 2B can be modified by the attachment of ubiquitin[J].Nucleic Acids Res,1980,8(20):4671-4680.
[14]	ROBZYK K,RECHT J,OSLEY M A.Rad6-dependent ubiquitination of histone H2B in yeast[J].Science,2000, 287(5452):501-504.
[15]	WOOD A,KROGAN N J,DOVER J,et al.Bre1,an E3 ubiquitin ligase required for recruitment and substrate selection of Rad6 at a promoter[J].Mol Cell,2003,11(1):267-274.
[16]	ZHU B,ZHENG Y,PHAM A D,et al.Monoubiquitination of human histone H2B:the factors involved and their roles in HOX gene regulation[J].Mol Cell,2005,20(4):601-611.
[17]	XU Z,SONG Z,LI G,et al.H2B ubiquitination regulates meiotic recombination by promoting chromatin relaxation[J].Nucleic Acids Res,2016,44(20):9681-9697.
[18]	LIANG Q L,XIA W L,LI W,et al.RNF20 controls astrocytic differentiation through epigenetic regulation of STAT3 in the developing brain[J].Cell Death Differ,2018,25(2):294-306.
[19]	REN P,SHENG Z,WANG Y,et al.RNF20 promotes the polyubiquitination and proteasome-dependent degradation of AP-2α protein[J]. Acta Biochim Biophys Sin,2014,46(2):136-140.
[20]	LEE J H,LEE G Y,JANG H,et al.Ring finger protein20 regulates hepatic lipid metabolism through protein kinase A-dependent sterol regulatory element binding protein1c degradation[J].Hepatology,2014,60(3):844-857.
[21]	VETHANTHAM V,YANG Y,BOWMAN C,et al.Dynamic loss of H2B ubiquitylation without corresponding changes in H3K4 trimethylation during myogenic differentiation[J].Mol Cell Biol,2012,32(6):1044-1055.
[22]	任玲,胡鑫,邢义珅,等.S100a10基因对小鼠前体脂肪细胞白色和棕色成脂分化的影响[J].畜牧兽医学报, 2019,50(6):1171-1178.REN L,HU X,XING Y S,et al.Effects of S100a10 on white and brown adipogenic differentiation of mice preadipocytes[J].Acta Veterinaria et Zootechnica Sinica,2019,50(6):1171-1178.(in Chinese)
[23]	何长晟,王永,许晴,等.KLF2对山羊肌内前体脂肪细胞分化的影响[J].畜牧兽医学报,2020,51(1):64-73.HE C S,WANG Y,XU Q,et al.The effect of KLF2 on the differentiation of goat intramuscular preadipocyte[J].Acta Veterinaria et Zootechnica Sinica,2020,51(1):64-73.(in Chinese)
[24]	许晴,李倩,王永,等.过表达FGF10促进山羊皮下前体脂肪细胞分化[J].畜牧兽医学报,2019,50(10):1972-1984.XU Q,LI Q,WANG Y,et al.Overexpression of FGF10 promotes the differentiation of subcutaneous preadipocyte in goat[J].Acta Veterinaria et Zootechnica Sinica,2019,50(10):1972-1984.(in Chinese)
[25]	张宁芳,成志敏,乐宝玉,等.猪肌源性前体脂肪细胞的分离培养和成脂诱导分化研究[J].畜牧兽医学报, 2018,49(12):2612-2621.ZHANG N F,CHENG Z M,LE B Y,et al.Isolation,culture and adipogenic differention of pig myogenic preadipocytes cell[J].Acta Veterinaria et Zootechnica Sinica,2018,49(12):2612-2621.(in Chinese)
[26]	WORDEN E J,WOLBERGER C.Activation and regulation of H2B-Ubiquitin-dependent histone methyltransferases[J].Curr Opin Struct Biol,2019,59:98-106.
[27]	MIKKELSEN T S,XU Z,ZHANG X L,et al.Comparative epigenomic analysis of murine and human adipogenesis[J].Cell, 2010, 143(1):156-169.
[28]	GALMOZZI A,MITRO N,FERRARI A,et al.Inhibition of class I histone deacetylases unveils a mitochondrial signature and enhances oxidative metabolism in skeletal muscle and adipose tissue[J].Diabetes,2013,62(3):732-742.
[29]	OKUNO Y,OHTAKE F,IGARASHI K,et al.Epigenetic regulation of adipogenesis by PHF2 histone demethylase[J].Diabetes, 2013,62(5):1426-1434.
[30]	WANG L F,XU S,LEE J E,et al.Histone H3K9 methyltransferase G9a represses PPARγ expression and adipogenesis[J].EMBO J,2013,32(1):45-59.
[31]	ZHUANG L N,JANG Y,PARK Y K,et al.Depletion of Nsd2-mediated histone H3K36 methylation impairs adipose tissue development and function[J].Nat Commun,2018,9(1):1796.
[32]	CHEN S,LI J,WANG D L,et al.Histone H2B lysine 120 monoubiquitination is required for embryonic stem cell differentiation[J]. Cell Res,2012,22(9):1402-1405.
[33]	FUCHS G,SHEMA E,VESTERMAN R,et al.RNF20 and USP44 regulate stem cell differentiation by modulating H2B monoubiquitylation[J].Mol Cell,2012,46(5):662-673.
[34]	ZHANG K Q,WANG J H,TONG T R,et al.Loss of H2B monoubiquitination is associated with poor-differentiation and enhanced malignancy of lung adenocarcinoma[J].Int J Cancer,2017,141(4):766-777.
[35]	KIM J,HAKE S B,ROEDER R G.The human homolog of yeast BRE1 functions as a transcriptional coactivator through direct activator interactions[J].Mol Cell,2005,20(5):759-770.
[36]	DICKSON K A,COLE A J,GILL A J,et al.The RING finger domain E3 ubiquitin ligases BRCA1 and the RNF20/RNF40 complex in global loss of the chromatin mark histone H2B monoubiquitination (H2Bub1) in cell line models and primary high-grade serous ovarian cancer[J].Hum Mol Genet,2016,25(24):5460-5471.
[37]	CASTELLANO-CASTILLO D,DENECHAUD P D,FAJAS L,et al.Human adipose tissue H3K4me3 histone mark in adipogenic,lipid metabolism and inflammatory genes is positively associated with BMI and HOMA-IR[J].PLoS One,2019, 14(4):e0215083.
[38]	WAKI H,NAKAMURA M,YAMAUCHI T,et al.Global mapping of cell type-specific open chromatin by FAIRE-seq reveals the regulatory role of the NFI family in adipocyte differentiation[J].PLoS Genet,2011,7(10):e1002311.
[39]	PAN D N,HUANG L,ZHU L J,et al.Jmjd3-mediated H3K27me3 dynamics orchestrate brown fat development and regulate white fat plasticity[J].Dev Cell,2015,35(5):568-583.
[40]	JEON Y G,LEE J H,JI Y,et al.RNF20 Functions as a transcriptional coactivator for PPARγ by promoting NCoR1 degradation in adipocytes[J].Diabetes,2020,69(1):20-34.