猪Nhlh2基因启动子活性及其表达调控的初步研究

doi:10.11843/j.issn.0366-6964.2018.04.002

Abstract

Abstract:

The present study aimed to explore the promoter activity of Nhlh2 gene and the relationship between the promoter activity and transcription factors in cells, and further provide a theoretical basis for the regulating mechanism of Nhlh2 gene in pigs. Based on the template of porcine ovarian genomic DNA, PCR was used to amplify different length deletion fragments of Nhlh2 gene promoter. These fragments were cloned into pGL3-basic to build the promoter recombinant plasmid, and then were transfected into the porcine ovarian granulosa cells. The relative luciferase activities of these plasmids were further measured by using the dual luciferase assay system. Chromatin immunoprecipitation assay (ChIP) was used to confirm the interaction between YY1, C/EBPβ and the promoter of Nhlh2. Finally, the overexpression and mutant vectors, small interfering RNA (siRNA) of YY1 and C/EBPβ were constructed, the luciferase activity was detected by dual luciferase reporter assay system. Results showed that transcription activity of P7 (—238-+129) region was the highest, —654-—238 was the potential region containing the negative regulatory element, and —238-—20 was the potential region containing the positive regulatory element; After the bioinformatic analysis and ChIP test, we found the binding sites of YY1 and C/EBPβ at Nhlh2 gene promoter were —101-—85 and —153-—140, respectively. After mutating the binding sites of YY1 and C/EBPβ, the transcription activity of P7 was significantly up-regulated; The overexpression of YY1 and C/EBPβ significantly reduced the transcription activity of P7, and the transcription activity was significantly up-regulated by YY1-siRNA and C/EBPβ-siRNA. These results indicate that the core promoter region of Nhlh2 is —238-+129. Furthermore, YY1 and C/EBPβ respectively bound at —101-—85 and —153-—140 of Nhlh2 gene promoter to inhibit the transcription of Nhlh2 gene in pigs.

CLC Number:

S828.2

WEN Li-juan, LIU Fa-wei, LIN Chang-guang, CHEN Zi-tao, ZHANG Zhe, ZHANG Hao, LI Jia-qi, YUAN Xiao-long. Analysis of the Promoter Activity and Expression Regulation of Porcine Nhlh2 Gene[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(4): 667-674.

0
/ / Recommend

Add to citation manager EndNote|Reference Manager|ProCite|BibTeX|RefWorks

URL: https://www.xmsyxb.com/EN/10.11843/j.issn.0366-6964.2018.04.002

https://www.xmsyxb.com/EN/Y2018/V49/I4/667

References

[1] GÖBEL V, LIPKOWITZ S, KOZAK C A, et al. NSCL-2:a basic domain helix-loop-helix gene expressed in early neurogenesis[J]. Cell Growth Differ, 1992, 3(3):143-148.
[2] HERBISON A E, PORTEOUS R, PAPE J R, et al. Gonadotropin-releasing hormone neuron requirements for puberty, ovulation, and fertility[J]. Endocrinology, 2008, 149(2):597-604.
[3] KRVGER M, RUSCHKE K, BRAUN T. NSCL-1 and NSCL-2 synergistically determine the fate of GnRH-1 neurons and control necdin gene expression[J]. EMBO J, 2004, 23(21):4353-4364.
[4] GOOD D J, PORTER F D, MAHON K A, et al. Hypogonadism and obesity in mice with a targeted deletion of the Nhlh2 gene[J]. Nat Genet, 1997, 15(4):397-401.
[5] COGLIATI T, DELGADO-ROMERO P, NORWITZ E R, et al. Pubertal impairment in Nhlh2 null mice is associated with hypothalamic and pituitary deficiencies[J]. Mol Endocrinol, 2007, 21(12):3013-3027.
[6] JOHNSON S A, MARÍN-BIVENS C L, MIELE M, et al. The Nhlh2 transcription factor is required for female sexual behavior and reproductive longevity[J]. Horm Behav, 2004, 46(4):420-427.
[7] NONNEMAN D, LENTS C, ROHRER G, et al. Genome-wide association with delayed puberty in swine[J]. Anim Genet, 2014, 45(1):130-132.
[8] TSUNODA T, TAKAGI T. Estimating transcription factor bindability on DNA[J]. Bioinformatics, 1999, 15(7-8):622-630.
[9] MATHELIER A, FORNES O, ARENILLAS D J, et al. JASPAR 2016:a major expansion and update of the open-access database of transcription factor binding profiles[J]. Nucleic Acids Res, 2016, 44(D1):D110-D115.
[10] 高萍, 钟玉宜, 张爱玲. 母猪卵巢颗粒细胞的分离培养及鉴定[J]. 广东农业科学, 2014, 41(4):131-135.
GAO P, ZHONG Y Y, ZHANG A L. Separation, culture and identification of sow ovarian granulose cells[J]. Guangdong Agricultural Sciences, 2014, 41(4):131-135. (in Chinese)
[11] LIU J Y, DU X, ZHOU J L, et al. MicroRNA-26b functions as a proapoptotic factor in porcine follicular Granulosa cells by targeting Sma-and Mad-related protein 4[J]. Biol Reprod, 2014, 91(6):146.
[12] GADE P, KALVAKOLANU D V. Chromatin immunoprecipitation assay as a tool for analyzing transcription factor activity[M]//VANCURA A. Transcriptional Regulation:Methods and Protocols. New York, NY:Springer, 2012:85-104.
[13] RODRÍGUEZ-UBREVA J, BALLESTAR E. Chromatin immunoprecipitation[M]//STOCKERT J C, ESPADA J, BLÁZQUEZ-CASTRO A. Functional Analysis of DNA and Chromatin. Totowa, NJ:Humana Press, 2014:309-318.
[14] MUELLER J K, KOCH I, LOMNICZI A, et al. Transcription of the human EAP1 gene is regulated by upstream components of a puberty-controlling Tumor Suppressor Gene network[J]. Mol Cell Endocrinol, 2012, 351(2):184-198.
[15] ROTH C L, MASTRONARDI C, LOMNICZI A, et al. Expression of a tumor-related gene network increases in the mammalian hypothalamus at the time of female puberty[J]. Endocrinology, 2007, 148(11):5147-5161.
[16] GRIFFITH G J, TRASK M C, HILLER J, et al. Yin-yang1 is required in the mammalian oocyte for follicle expansion[J]. Biol Reprod, 2011, 84(4):654-663.
[17] MAALOUF S W, SMITH C L, PATE J L. Changes in MicroRNA expression during maturation of the bovine corpus luteum:regulation of luteal cell proliferation and function by microRNA-34a[J]. Biol Reprod, 2016, 94(3):71.
[18] SANDISON H E, USHER S, KARIMIANI E G, et al. PLK1 and YY1 interaction in follicular lymphoma is associated with unfavourable outcome[J]. J Clin Pathol, 2013, 66(9):764-767.
[19] MANDAL K, BADER S L, KUMAR P, et al. An integrated transcriptomics-guided genome-wide promoter analysis and next-generation proteomics approach to mine factor(s) regulating cellular differentiation[J]. DNA Res, 2017, 24(2):143-157.
[20] NALBANT D, WILLIAMS S C, STOCCO D M, et al. Luteinizing hormone-dependent gene regulation in Leydig cells may be mediated by CCAAT/enhancer-binding protein-β[J]. Endocrinology, 1998, 139(1):272-279.
[21] STERNECK E, TESSAROLLO L, JOHNSON P F. An essential role for C/EBPβ in female reproduction[J]. Genes Dev, 1997, 11(17):2153-2162.
[22] FAN H Y, LIU Z L, JOHNSON P F, et al. CCAAT/enhancer-binding proteins (C/EBP)-α and -β are essential for ovulation, luteinization, and the expression of key target genes[J]. Mol Endocrinol, 2011, 25(2):253-268.
[23] ZHEN Y H, WANG L, RIAZ H, et al. Knockdown of CEBPβ by RNAi in porcine granulosa cells resulted in S phase cell cycle arrest and decreased progesterone and estradiol synthesis[J]. J Steroid Biochem Mol Biol, 2014, 143:90-98.
[24] CHRISTENSON L K, JOHNSON P F, MCALLISTER J M, et al. CCAAT/enhancer-binding proteins regulate expression of the human steroidogenic acute regulatory protein (StAR) gene[J]. J Biol Chem, 1999, 274(37):26591-26598.
[25] PALL M, HELLBERG P, BRÄNNSTRÖM M, et al. The transcription factor C/EBP-β and its role in ovarian function; evidence for direct involvement in the ovulatory process[J]. EMBO J, 1997, 16(17):5273-5279.
[26] REN Y A, LIU Z L, MULLANY L K, et al. Growth Arrest Specific-1(GAS1) is a C/EBP target gene that functions in ovulation and corpus luteum formation in mice[J]. Biol Reprod, 2016, 94(2):44.
[27] DUPUIS L, SCHUERMANN Y, COHEN T, et al. Role of leptin receptors in granulosa cells during ovulation[J]. Reproduction, 2014, 147(2):221-229.

Analysis of the Promoter Activity and Expression Regulation of Porcine Nhlh2 Gene

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	KANG Jiawei, HUANG Xuankai, WANG Zhipeng, ZHANG Aizhen, MENG Fangrong, GAI Peng, BAO Junfu, SUN Kexin, SONG Shaokang, SUN Pan, CHEN Yichuan, ZHANG Lei, GAO Shengyue, CHANG Minghang. Estimation of Genetic Parameters for Growth, Reproduction, and Body Measurements Traits in Large White Pigs [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 1936-1944.
[2]	CUI Shengdi, WANG Kai, ZHAO Zhenjian, CHEN Dong, SHEN Qi, YU Yang, WANG Junge, CHEN Ziyang, YU Shixin, CHEN Jiamiao, WANG Xiangfeng, TANG Guoqing. Identification of Candidate Genes for Pork Texture Traits Using GWAS Combined with Co-localisation of DNA Methylation [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 1945-1957.
[3]	SUN Wenli, WANG Haoqi, ZE Licuo, GAO Yufan, ZHANG Feifan, ZHANG Jian, DUAN Mengqi, SHANG Peng, QIANG Bayangzong. Polymorphism of Pro-Inflammatory Factors (IL-1β, IL-6, TNF-α) in Tibetan Pigs and Its Association Analysis with Immune Traits [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 1958-1969.
[4]	ZHANG Shuai, CHEN Kuirong, XU Di, JIANG Shan, WANG Mengying, ZHANG Kun, XU Yupei, LEI Guofeng, ZHANG Zhicheng, GUO Meng, ZHAO Yunxiang, LAN Ganqiu, LIANG Jing. Analysis of Microbial Composition Differences in High and Low Feed Conversion Rates Pig Feces based on 16S rRNA Sequencing [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(4): 1605-1614.
[5]	SHEN Qi, WANG Kai, ZHAO Zhenjian, CHEN Dong, YU Yang, CUI Shengdi, WANG Junge, CHEN Ziyang, WU Pingxian, TANG Guoqing. Regulation of NO Concentration by NOS2 Gene DNA Methylation Editing Affects the Expression of Muscle Development Pathway Genes [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(3): 984-994.
[6]	SONG Kelin, YAN Zunqiang, WANG Pengfei, CHENG Wenhao, LI Jie, BAI Yaqin, SUN Guohu, GUN Shuangbao. Analysis on Genetic Diversity and Genetic Structure Based on SNP Chips of Huixian Qingni Black Pig [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(3): 995-1006.
[7]	NIU Naiqi, ZHAO Runze, ZONG Wencheng, LIU Xiance, LIU Hai, SHI Guohua, JING Xitao, ZHANG Longchao. Association of Polymorphisms of GREB1L and MIB1 Genes with Rib Number and Carcass Traits in Beijing Black Pigs [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 79-86.
[8]	ZHU Xueli, ZHANG Longchao, WANG Lixian, PU Lei, LIU Xin. Association Analysis of AQP9 and RPS10 Gene Polymorphisms with Backfat Thickness in Beijing Black Pigs [J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 87-98.
[9]	YANG Kai, LU Zhuoda, HE Jian, ZHANG Ruiqi, WANG Suqing, LI Kebiao, ZHAO Yunxiang, ZHU Xiaoping, GUO Jinbiao. The Population Effect of Commercial Pigs on the Accuracy of Genomic Selection for Carcass Traits in Duroc Pigs [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 4943-4951.
[10]	XU Tingting, QI Fenfang, HUANG Shihui, NIU Xi, LI Sheng, RAN Xueqin, WANG Jiafu, XIE Jian. Study of the Polymorphisms of Structural Variation and the Expression of MAP3K4 gene in Xiang Pig [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5046-5055.
[11]	HU Ziping, WANG Ligang, ZONG Wencheng, HOU Renda, SU Yanfang, NIU Naiqi, WANG Lixian, WANG Yuan, ZHNAG Longchao. Genetic Structure Analysis of Jianbai Xiang Pig Population Based on Genomic SNP and ROH [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(10): 4117-4125.
[12]	JI Zhengyu, NI Mengru, ZHANG Zhaobo, ZHAO Gan, HUANG Zan, LI Pinghua, HUANG Ruihua, HOU Liming. Research on Adipogenic Capacity and Gene Expression Pattern of in Vitro FAPs Cells Derived from Longissimus Dorsi Muscle of Suhuai Pig [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(10): 4126-4142.
[13]	BI Huan, QIN Hai, YUAN Wei, ZHANG Yudan, ZHANG Yiyu, CHEN Wei. Effect of Knocking down TYRP1 Gene on Melanin Production in the Xiang Pig Epidermal Melanocytes [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(10): 4143-4153.
[14]	WANG Hui, FENG Baoliang, WU Dan, XIANG Guangming, WANG Nan, MU Yulian, LI Kui, LIU Zhiguo. Research Progress of CD163 Gene and Disease-Resistant Breeding on Porcine Reproductive and Respiratory Syndrome [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3127-3138.
[15]	ZHANG Wanfeng, ZHAO Tianzhi, LI Jiao, YOU Ziwei, YANG Yang, CAI Chunbo, GAO Pengfei, CAO Guoqing, GUO Xiaohong, LI Bugao. Study on NR2F2 Gene Regulating Proliferation and Apoptosis of Porcine PK15 Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(8): 3242-3251.