The human RNA methyltransferase like i gene(RNMTL1)is one of thirteen newly discovered geneswithin a 116 Kb segment of the chromosome 17p13.3 that suffers from a high frequent loss of heterozygosityin human hepatocell...The human RNA methyltransferase like i gene(RNMTL1)is one of thirteen newly discovered geneswithin a 116 Kb segment of the chromosome 17p13.3 that suffers from a high frequent loss of heterozygosityin human hepatocellular carcinoma in China[1-5].To understand the molecular mechanisms underlyingtranscription control of the RNMTL1 gene in human cancers,we decline using of the conventional approachwhere the cis-elements bound by the known transcription factors are primary targets,and carried out thesystematic analyses to dissect the promoter structure and identify/characterize the key cis-elements thatare responsible for its strong expression in cell.The molecular approaches applied included 1,the primerextension for mapping of the transcription starts;2,the transient transfection/reporter assays on a largenumber of deletion and site-specific mutants of the promoter segment for defining the minimal promoterand the crucial elements within;and 3,the electrophoresis mobility shift assay with specific antibodies forreconfirming the nature of the transcription factors and their cognate cis-elements.We have shown that theinteraction of an ATF/CREB element(-38 to-31)and its cognate transcription factors play a predominantrole in the promoter activity of the RNMTL1 gene.The secondary DNA structures of the ATF/CREBelement play a more vital role in the protein-DNA interaction.Finally,we reported a novel mechanismunderlying the YY1 mediated transcription repression,namely,the ATF/CREB dependent transcription-repression by YY1 is executed in absence of its own sequence-specific binding.展开更多
The human RNA methyltransferase like 1 gene (RNMTL1) is one of thirteen newly discovered genes within a 116 Kb segment of the chromosome 17pl3.3 that suffers from a high frequent loss of heterozygosity in human hepato...The human RNA methyltransferase like 1 gene (RNMTL1) is one of thirteen newly discovered genes within a 116 Kb segment of the chromosome 17pl3.3 that suffers from a high frequent loss of heterozygosity in human hepatocellular carcinoma in China[1-5]. To understand the molecular mechanisms underlying transcription control of the RNMTL1 gene in human cancers, we decline using of the conventional approach where the cis-elements bound by the known transcription factors are primary targets, and carried out the systematic analyses to dissect the promoter structure and identify/characterize the key cis-elements that are responsible for its strong expression in cell. The molecular approaches applied included 1, the primer extension for mapping of the transcription starts; 2, the transient transfection/reporter assays on a large number of deletion and site-specific mutants of the promoter segment for defining the minimal promoter and the crucial elements within; and 3, the electrophoresis mobility shift assay with specific antibodies for reconfirming the nature of the transcription factors and their cognate cis-elements. We have shown that the interaction of an ATF/CREB element (-38 to -31) and its cognate transcription factors play a predominant role in the promoter activity of the RNMTL1 gene. The secondary DNA structures of the ATF/CREB element play a more vital role in the protein-DNA interaction. Finally, we reported a novel mechanism underlying the YY1 mediated transcription repression, namely, the ATF/CREB dependent transcription-repression by YY1 is executed in absence of its own sequence-specific binding.展开更多
文摘The human RNA methyltransferase like i gene(RNMTL1)is one of thirteen newly discovered geneswithin a 116 Kb segment of the chromosome 17p13.3 that suffers from a high frequent loss of heterozygosityin human hepatocellular carcinoma in China[1-5].To understand the molecular mechanisms underlyingtranscription control of the RNMTL1 gene in human cancers,we decline using of the conventional approachwhere the cis-elements bound by the known transcription factors are primary targets,and carried out thesystematic analyses to dissect the promoter structure and identify/characterize the key cis-elements thatare responsible for its strong expression in cell.The molecular approaches applied included 1,the primerextension for mapping of the transcription starts;2,the transient transfection/reporter assays on a largenumber of deletion and site-specific mutants of the promoter segment for defining the minimal promoterand the crucial elements within;and 3,the electrophoresis mobility shift assay with specific antibodies forreconfirming the nature of the transcription factors and their cognate cis-elements.We have shown that theinteraction of an ATF/CREB element(-38 to-31)and its cognate transcription factors play a predominantrole in the promoter activity of the RNMTL1 gene.The secondary DNA structures of the ATF/CREBelement play a more vital role in the protein-DNA interaction.Finally,we reported a novel mechanismunderlying the YY1 mediated transcription repression,namely,the ATF/CREB dependent transcription-repression by YY1 is executed in absence of its own sequence-specific binding.
基金This work is supported by the 973 projects of China (G1998051004) to Jingde Zhu and (G199805l200) to Dafang Wan, respectively.Thanks are due to Hongyu Zhang and other mem-bers in Jingde Zhu's lab for assistance and helps onnumerous occasions.
文摘The human RNA methyltransferase like 1 gene (RNMTL1) is one of thirteen newly discovered genes within a 116 Kb segment of the chromosome 17pl3.3 that suffers from a high frequent loss of heterozygosity in human hepatocellular carcinoma in China[1-5]. To understand the molecular mechanisms underlying transcription control of the RNMTL1 gene in human cancers, we decline using of the conventional approach where the cis-elements bound by the known transcription factors are primary targets, and carried out the systematic analyses to dissect the promoter structure and identify/characterize the key cis-elements that are responsible for its strong expression in cell. The molecular approaches applied included 1, the primer extension for mapping of the transcription starts; 2, the transient transfection/reporter assays on a large number of deletion and site-specific mutants of the promoter segment for defining the minimal promoter and the crucial elements within; and 3, the electrophoresis mobility shift assay with specific antibodies for reconfirming the nature of the transcription factors and their cognate cis-elements. We have shown that the interaction of an ATF/CREB element (-38 to -31) and its cognate transcription factors play a predominant role in the promoter activity of the RNMTL1 gene. The secondary DNA structures of the ATF/CREB element play a more vital role in the protein-DNA interaction. Finally, we reported a novel mechanism underlying the YY1 mediated transcription repression, namely, the ATF/CREB dependent transcription-repression by YY1 is executed in absence of its own sequence-specific binding.
文摘目的:探讨2型糖尿病(T2DM)小鼠骨形成变化及不同运动对T2DM小鼠骨中环磷酸腺苷(c AMP)/环磷腺苷效应元件结合蛋白(CREB)/激活转录因子4(ATF4)途径和骨形成的影响。方法:40只4周龄C57BL/6雄性小鼠,适应性喂养1周后随机分为正常对照组(ZC,10只)和T2DM造模组(30只)。T2DM造模组小鼠造模成功后随机分为T2DM对照组(TC组,9只)、T2DM游泳组(TS组,9只)和T2DM下坡跑组(TD组,9只)。利用游泳和下坡跑对TS组和TD组小鼠进行8周训练。结束后,取小鼠血清并利用ELISA法检测c AMP浓度;取右侧股骨并利用RT-PCR法检测CREB、ATF4、骨钙素(OC)、骨钙蛋白(OCN)和骨涎蛋白(BSP)的m RNA表达;取右侧胫骨并利用West-blotting法检测CREB蛋白表达;取小鼠骨髓间充质干细胞(BMSCs)并诱导其向骨细胞(OB)分化,利用碱性磷酸酶(ALP)染液对OB染色;取左侧股骨并用Micro-CT对股骨远端BMD进行扫描。结果:与ZC组相比,TC组c AMP浓度下降,CREB、ATF4、OC、OCN和BSP m RNA及CREB蛋白表达均显著下降(P<0.05或P<0.01),OB成骨能力和BMD显著下降(P<0.01)。与TC组相比,TS组OC和OCN m RNA表达上调(P<0.05或P<0.01),OB骨形成能力增强;TD组c AMP浓度下降,CREB、ATF4、OC和OCN m RNA及CREB蛋白表达均显著上调(P<0.05或P<0.01),OB骨形成能力和BMD显著增加(P<0.01)。与TS组相比,TD组c AMP浓度升高,CREB、ATF4和OC的m RNA表达显著上调(P<0.05或P<0.01),OB骨形成能力增强。结论:2型糖尿病小鼠骨形成代谢被抑制;下坡跑通过激活2型糖尿病小鼠骨中c AMP/CREB/ATF4途径,促进成骨细胞分化及骨形成能力,提高骨密度,且其作用效果优于游泳。
基金Research from the corresponding author’s laboratory was supported by grants from National Key Research and Development Program of China(No.2019YFA0802502)the National Natural Science Foundation of China(No.81925008)+1 种基金Shanghai Science and Technology Commission(19140903300)Key Laboratory of Wuliangye-flavor Liquor Solid-state Fermentation of China National Light Industry(No.2019JJ005)to Y.L。
