In silico Analysis and Site-directed Mutagenesis of Promoters

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Mar 2016



In normal as in cancerous cells, gene expression is tightly regulated by transcription factors, which are responsible for up- or down-regulation of thousands of targets involved in different cell processes. Transcription factors can directly regulate the expression of genes by binding to specific DNA sequences known as response elements. Identification of these response elements is important to characterize targets of transcription factors in order to understand their contribution to gene regulation. Here, we describe In silico analysis coupled to selected mutagenesis and promoter gene reporter assay procedures to identify and analyze response elements in the proximal promoter sequence of genes.

Keywords: Site-directed mutagenesis (定点诱变), in silico analysis (计算机模拟分析), Gene expression (基因表达), Promoter gene-reporter assay (启动子基因 - 报告基因测定), Response elements (响应元素), Transcription factors (转录因子)


The impact of a transcription factor on global gene expression can be studied through its knockdown using shRNA or CRISPR-Cas9 methods followed by microarray analysis which can provide significant data on hundreds of dysregulated genes. This analysis, although very useful, lacks information about the direct control of the dysregulated genes. To further investigate these genes, In silico analysis using bioinformatics provides additional information to identify direct targets of the transcription factor studied. Additionally, the functionality of these response elements can be analyzed using site-directed mutagenesis and promoter-gene-reporter assays. We have shown recently that the oncogenic transcription factor MYC (Cellular Myelocytomatosis Oncogene) controls the expression of the ITGA1 (integrin alpha 1 subunit) gene in colorectal cancer, and that their expression correlates in 72% of colorectal tumors. This protocol describes a procedure for the analysis of the ITGA1 promoter and the identification of response elements for the MYC oncogene. The latter is known to be a member of the MYC/MAX/MAD network. Interactions between these factors lead to gene activation or repression depending on upstream signalization and cell condition.

Materials and Reagents

  1. 1.5 ml tubes
  2. 10 cm dish
  3. 12-well plates (Corning, Falcon®, catalog number: 353043 )
  4. White 96-well assay plate (Corning, catalog number: 3912 )
  5. Ice
  6. HEK293T cells (ATCC, catalog number: CRL-3216 ) at low passage
  7. DH5α competent cells
  8. Primers (Sequences of the oligonucleotide primers used for the site directed mutagenesis):
  9. The promoter of interest, as for example here, the ITGA1 proximal promoter sequence inserted in the pLightSwitch_prom plasmid vector (SwitchGear Genomics, catalog number: S706788 )
  10. Plasmid constructs carrying the activator or repressor to be characterized, as for example here: pcDNA-Empty vector, pcDNA-MYC and pCMV-MAD, as described in Ni et al., 2005
  11. The pGL4.13 plasmid (luc2/SV40) as a control of transfection efficiency
  12. GeneArt Site-Directed Mutagenesis Kit (Thermo Fisher Scientific, InvitrogenTM, catalog number: A13282 )
  13. DNase and RNase free water
  14. Distilled water
  15. 0.5 M EDTA, pH 8
  16. SOC medium
  17. LB agar plates
  18. Trypan blue
  19. Effectene transfection reagent (QIAGEN, catalog number: 301425 )
  20. PBS
  21. DMEM medium (Thermo Fisher Scientific, GibcoTM, catalog number: 11995073 )
  22. Fetal bovine serum (FBS) (BOLLE COMMUNICATION, WISENT, catalog number: 080-150 )
  23. GlutaMAX (Thermo Fisher Scientific, GibcoTM, catalog number: 35050061 )
  24. HEPES (BOLLE COMMUNICATION, WISENT, catalog number: 330-050-EL )
  25. Dual Luciferase Reporter Assay System (Promega, catalog number: E1980 )
    Luciferase Assay substrate
    Luciferase Assay buffer
  26. DMEM culture medium for HEK293T cells (see Recipes)
  27. LAR II solution (see Recipes)
  28. 1x Stop & Glo (see Recipes)


  1. Orion Microplate Luminometer (Berthold, Bad Wildbad, Germany)
  2. MyCycler Personal Thermal Cycler for PCR reactions (Bio-Rad Laboratories)
  3. Water bath
  4. Cell culture incubator


  1. MatInspector web based software. Genomatix (Munich, Germany)
  2. Blast Global Align


  1. Promoter analysis
    1. The proximal promoter sequence of the promoter of interest, the ITGA1 gene, can be downloaded from the reference human genome or from the switchdb website:
    2. Genomatix website: upload the promoter sequence and use the default parameters or use the promoter search.
    3. Guideline detailed in:
    4. The identified response elements are compared to the consensus sequence (which binds the transcription factor). For instance, for MYC, possible response elements are compared to the E-box element CACGTG. In addition to the results obtained with the software, visual analysis of the promoter sequence is highly recommended.
  2. Site directed mutagenesis
    Design primers manually and change nucleotides in order to completely modify the sequence of the identified response elements (avoid creating a response element for another transcription factor). The mutations should be centrally located in both the forward and reverse primers. The primers should be complementary and 35 to 45 nucleotides in length to insure proper annealing.
    1. Prepare the PCR mix (excepting primers, all reagents are included in the GeneArt Site-Directed Mutagenesis Kit)

    2. PCR reaction

    3. Recombination reaction
      This step allows the conversion of the plasmid from a linear to a circular form.
      In a 1.5 ml tube add: 4 µl of reaction buffer (5x), 2 µl of enzyme mix (10x), 4 µl of PCR sample and complete to 20 µl with RNase-free water. Incubate 10 min at room temperature (RT). Stop the reaction with 1 µl of 0.5 M EDTA. The next step should be started immediately after recombination. Prepare for the transformation step before beginning the recombination reaction.
    4. Transformation
      1. Thaw vials of DH5α cells on ice for 5 to 7 min.
      2. Transfer 2 μl of the recombination reaction directly to the vial of DH5α cells and mix by tapping gently (Reaction A).
      3. Incubate the tubes on ice for 12 min.
      4. Immediately incubate the vials at 42 °C for 30 sec (do not exceed 30 sec) and then re-incubate on ice for 2 min.
      5. Add 250 μl of SOC medium to each vial (Reaction A) and then incubate at 37 °C for 1 h with 225 rpm shaking (Reaction B).
      6. Add 10 μl from Reaction B to 90 μl of SOC medium, then spread 100 μl onto LB plates and incubate overnight.
      7. Select 5 to 10 colonies and isolate DNA from the bacteria.
      8. Analyze and confirm, by DNA sequencing, the presence of the induced mutations in the promoter.
      9. Store DNA at -20 °C.
  3. Transient transfection
    1. Culture HEK293T cells in DMEM media supplemented with 10% FBS (without antibiotics).
    2. Use trypsin to dissociate HEK293T cells (1 ml per 10 cm dish) and incubate at 37 °C for 2 min. Add DMEM and count live cells (using trypan blue) in suspension and then plate 50,000 cells per well in 12-well plates. Add a total of 1 ml of DMEM 10% FBS media to each well.
    3. Begin the transient transfection the following day.
    4. For transfection, prepare plasmids in 1.5 ml tubes as for the following example:

      Table 1. Plasmid preparation for transient transfection into HEK293T cells

      Table 2. Plasmid mix for the comparison between wild type and mutated ITGA1 promoter

      Plasmid quantities are in ng
      X: no plasmid transfection
      Transfect with the same total quantity of plasmid for every well.
      Amounts are for one well, calculate quantities for triplicates.

    5. Add 50 µl of buffer EC and 1.5 µl of enhancer, mix gently and incubate for exactly 5 min.
    6. Add Effectene reagent (2 µl/well), mix gently and then incubate for 10 min.
    7. Change the media in the 12-well plates and then add the plasmid mix to each well. Incubate for 48 h.
    8. Add one volume of passive lysis buffer to four volumes of distilled water.
    9. Remove the media from the wells and rinse once gently with cold 1x PBS.
    10. Add 250 µl of 1x passive lysis buffer for each well of the 12-well plates and then shake gently for 15 min.
    11. Luminometer settings: set injectors 1 and 2 to dispense 50 µl of LARII and Stop & Glo solutions.
    12. For measurement, use 10 sec for reading time and 2 sec as a delay.
    13. Transfer 10 µl from each PLB lysate and put in 96 well-plate (avoid pipetting the cell debris).
    14. Run the measurement of luciferase activity.

Data analysis

  1. Response elements identified using MatInspector© software should be analyzed and selected according to their similarity to the consensus sequence. Response elements with the same sequence as the consensus response element (for the transcription factor studied) as incomplete sequences should be taken into account for the mutagenesis analysis (Boudjadi et al., 2016; Chen et al., 2011; Teye et al., 2008).
  2. After plasmid mutation, the sequence of the new plasmid should be aligned to the original sequence to confirm that the introduced mutations are the only differences between the wild type promoter sequence and the mutated sequence (Figure 1).

    Figure 1. Alignment of the promoter sequences. Comparison of the mutated ITGA1 promoter sequence to the wild type promoter. When present, the vertical bars indicate identical nucleotides between the two sequences. The sequence of the forward primer is underlined in blue. The red square points out the mutated response element (mutation of the first response element for MYC), as reported in Boudjadi et al., 2016.

  3. After transient transfection and cell lysis, the enzymatic activity of luciferase (which reflects the promoter activity) will be quantified using a luminometer. The data obtained will be used to analyze the promoter activity under each condition. Two values are obtained: the first one indicates the activity of the promoter of interest and the second indicates the activity of the control promoter (reflecting the transfection efficiency). For each well, the final value is the ratio between the first and the second values.
    Data may be presented as fold change as indicated in Figure 2.

    Figure2. Analysis of ITGA1 promoter activity. Results of the promoter gene reporter assay showing the relative luciferase activity presented as fold change compared to the empty vector (EV). The wild type ITGA1 promoter vector was transfected into HEK293T cells, together with the empty vector, a MYC expressing vector (MYC), the dominant negative MAD (MAD) or both (MYC-MAD). The dark gray columns indicate the activity of the mutated ITGA1 promoter (mutation of the first response element for MYC) after transfection with the empty vector or the MYC expressing vector. Experiments were repeated three times and performed in triplicate. t-test. **P < 0.01, ***P < 0.001. Ns: non-significant.


  1. The methylation of the template plasmid is performed in order to allow its digestion by endonucleases in the host cells after transformation. Therefore, the template will be eliminated and only the mutated plasmid will be present in the transformed cells.
  2. HEK293T cells should not be allowed to reach confluency during the transfection.
  3. HEK293T cells form a fragile monolayer, therefore the media change should be done in a gentle manner.
  4. The MAD/MAX heterodimer recognizes the same response elements as MYC/MAX. The fact that the transfection of MAD alone or together with MYC reduced the activity of the ITGA1 promoter is an indication that this activity was enhanced when MYC was introduced alone.
  5. Store DMEM at 4 °C; heat to 37 °C before use, avoid keeping warm for a prolonged time.
  6. Do not mix the bacterial vials by pipetting up and down.
  7. Invert LB plate during incubation to avoid condensation and contamination.
  8. Prepare plasmids at 100 ng/μl to facilitate the transfection procedure.


  1. DMEM culture medium for HEK293T cells
    10% FBS
    2 mM GlutaMAX
    10 mM HEPES
  2. LAR II solution
    Resuspend lyophilized Luciferase Assay substrate in Luciferase Assay buffer II (105 ml)
    Aliquot and store at -20 °C
  3. 1x Stop & Glo
    Dilute the appropriate amount of Stop & Glo substrate (stock 50x) in Stop & Glo reagent (Always prepare a fresh solution)


We thank Elizabeth Herring for reviewing the manuscript. This work was supported by the Canadian Institute of Health Research Grant MOP-123415 (JFB is a member of the Centre de Recherche of the Centre Hospitalier Universitaire de Sherbrooke funded by the Fonds de la Recherche du Québec-Santé. This protocol was adapted from Boudjadi et al. (2016), originally published in Oncogene.


  1. Boudjadi, S., Carrier, J. C., Groulx, J. F. and Beaulieu, J. F. (2016). Integrin α1β1 expression is controlled by c-MYC in colorectal cancer cells. Oncogene 35(13): 1671-1678.
  2. Chen, Y., Xu, J., Borowicz, S., Collins, C., Huo, D. and Olopade, O. I. (2011). c-Myc activates BRCA1 gene expression through distal promoter elements in breast cancer cells. BMC Cancer 11: 246.
  3. Ni, H., Dydensborg, A. B., Herring, F. E., Basora, N., Gagne, D., Vachon, P. H. and Beaulieu, J. F. (2005). Upregulation of a functional form of the β4 integrin subunit in colorectal cancers correlates with c-Myc expression. Oncogene 24(45): 6820-6829.
  4. Teye, K., Okamoto, K., Tanaka, Y., Umata, T., Ohnuma, M., Moroi, M., Kimura, H. and Tsuneoka, M. (2008). Expression of the TAF4b gene is induced by MYC through a non-canonical, but not canonical, E-box which contributes to its specific response to MYC. Int J Oncol 33(6): 1271-1280.



背景 转录因子对全球基因表达的影响可以通过其使用shRNA或CRISPR-Cas9方法的敲低来进行研究,然后进行微阵列分析,可以提供数百个失调基因的重要数据。这种分析虽然非常有用,但缺乏关于失调基因直接控制的信息。为了进一步研究这些基因,使用生物信息学的电子分析分析提供了额外的信息来鉴定研究的转录因子的直接靶标。另外,可以使用定点突变和启动子 - 基因 - 报道分析来分析这些响应元件的功能。我们最近已经表明,致癌转录因子MYC(细胞性骨髓细胞瘤癌基因)控制结肠直肠癌中ITGA1(整联蛋白α1亚基)基因的表达,并且它们的表达在72%的结肠直肠肿瘤中相关。该协议描述了用于分析ITGA1启动子的程序和用于MYC致癌基因的响应元件的鉴定。后者已知是MYC / MAX / MAD网络的成员。这些因素之间的相互作用导致基因激活或抑制,这取决于上游信号和细胞条件。

关键字:定点诱变, 计算机模拟分析, 基因表达, 启动子基因 - 报告基因测定, 响应元素, 转录因子


  1. 1.5 ml管子
  2. 10厘米盘
  3. 12孔板(Corning,Falcon ®,目录号:353043)
  4. 白色96孔分析板(Corning,目录号:3912)

  5. HEK293T细胞(ATCC,目录号:CRL-3216)在低通道
  6. DH5α感受态细胞
  7. 引物(用于定点诱变的寡核苷酸引物的序列):
  8. 感兴趣的启动子,如这里,插入pLightSwitch_prom质粒载体(SwitchGear Genomics,目录号:S706788)中的 ITGA1 近端启动子序列
  9. 携带要表征的活化剂或阻遏物的质粒构建体,例如如下所述:pcDNA-空载体,pcDNA-MYC和pCMV-MAD,如Ni等人所述,2005
  10. pGL4.13质粒(luc2/SV40)作为转染效率的对照
  11. GeneArt定点诱变试剂盒(Thermo Fisher Scientific,Invitrogen TM,目录号:A13282)
  12. DNase和RNase游离水
  13. 蒸馏水
  14. 0.5 M EDTA,pH 8
  15. SOC媒体
  16. LB琼脂板
  17. 台盼蓝色
  18. 有效转染试剂(QIAGEN,目录号:301425)
  19. PBS
  20. DMEM培养基(Thermo Fisher Scientific,Gibco TM,目录号:11995073)
  21. 胎牛血清(FBS)(BOLLE COMMUNICATION,WISENT,目录号:080-150)
  22. GlutaMAX(Thermo Fisher Scientific,Gibco TM ,目录号:35050061)
  24. 双荧光素酶报告分析系统(Promega,目录号:E1980)
  25. 用于HEK293T细胞的DMEM培养基(参见食谱)
  26. LAR II解决方案(参见食谱)
  27. 1x停止& Glo(见配方)


  1. 猎户座微孔板发光计(Berthold,Bad Wildbad,Germany)
  2. MyCycler个人热循环仪用于PCR反应(Bio-Rad Laboratories)
  3. 水浴
  4. 细胞培养箱


  1. MatInspector基于Web的软件。 Genomatix(慕尼黑,德国)
  2. Blast Global对齐
    https: //


  1. 启动子分析
    1. 目的启动子的近端启动子序列,即ITGA1基因,可以从参考人类基因组或从switchdb网站下载:
    2. Genomatix网站:上传启动子序列并使用默认参数或使用启动子搜索。
    3. 指引详见:
    4. 将鉴定的响应元件与共有序列(其结合转录因子)进行比较。例如,对于MYC,将可能的响应元素与E盒元素CACGTG进行比较。除了用软件获得的结果之外,强烈推荐对启动子序列的视觉分析。
  2. 定点诱变
    1. 准备PCR混合物(引物除外,所有试剂均包含在GeneArt Site-Directed Mutagenesis Kit中)

    2. PCR反应

    3. 重组反应
      在1.5ml管中加入4μl反应缓冲液(5x),2μl酶混合物(10x),4μlPCR样品,并用无RNase的水完成20μl。在室温(RT)下孵育10分钟。停止与1微升0.5 M EDTA反应。复合后应立即开始下一步。在开始重组反应之前准备转化步骤。
    4. 转型
      1. 在冰上解冻小瓶DH5α细胞5至7分钟。
      2. 将2μl重组反应直接转移到DH5α细胞小瓶中,轻轻敲打混合(反应A)。
      3. 在冰上孵育管12分钟。
      4. 立即将小瓶在42℃孵育30秒(不超过30秒),然后在冰上再孵育2分钟。
      5. 向每个小瓶中加入250μlSOC培养基(反应A),然后在225rpm摇动下在37℃下孵育1小时(反应B)。
      6. 从反应B中加入10μl到90μlSOC培养基,然后将100μl扩增到LB平板上并孵育过夜
      7. 选择5至10个菌落,并从细菌中分离出DNA
      8. 通过DNA测序分析和确认启动子中诱导突变的存在。
      9. 将DNA存储在-20°C。
  3. 瞬时转染
    1. 培养补充有10%FBS(不含抗生素)的DMEM培养基中的HEK293T细胞
    2. 使用胰蛋白酶解离HEK293T细胞(每10cm皿1ml),并在37℃下孵育2分钟。加入DMEM并计数活细胞(使用台盼蓝)悬浮液,然后在12孔板中每孔铺板50,000个细胞。向每个孔中加入1ml DMEM 10%FBS培养基。
    3. 第二天开始瞬时转染。
    4. 对于转染,在1.5ml试管中制备质粒,如下例所示:




    5. 加入50μl缓冲液EC和1.5μl增强剂,轻轻混匀,孵育5 min
    6. 加入Effectene试剂(2μl/孔),轻轻混合,然后孵育10 min
    7. 更换12孔板中的培养基,然后将质粒混合物加入每个孔中。孵育48小时。
    8. 将一体积的被动裂解缓冲液加入到四体积的蒸馏水中。
    9. 从孔中取出培养基,并用冷的1x PBS轻轻冲洗一次
    10. 向12孔板的每个孔中加入250μl1x被动裂解缓冲液,然后轻轻摇动15分钟。
    11. 发光计设置:设置注射器1和2以分配50μl的LARII和Stop& Glo解决方案
    12. 对于测量,使用10秒的读取时间和2秒作为延迟。
    13. 从每个PLB裂解液中转移10μl,放入96孔板(避免移液细胞碎片)
    14. 运行荧光素酶活性的测量。


  1. 应使用MatInspector © 软件识别的响应元素应根据其与共有序列的相似性进行分析和选择。作为不完全序列的共有响应元件(对于转录因子研究)具有相同序列的响应元件应考虑到诱变分析(Boudjadi et al。,2016; Chen et al。 al。,2011; Teye等人,2008)。
  2. 质粒突变后,新质粒的序列应与原始序列比对,以确认引入的突变是野生型启动子序列和突变序列之间的唯一差异(图1)。


  3. 瞬时转染和细胞裂解后,荧光素酶的酶活性(反映启动子活性)将用发光计量化。所获得的数据将用于分析每种条件下的启动子活性。获得两个值:第一个表示感兴趣的启动子的活性,第二个表示对照启动子的活性(反映转染效率)。对于每个井,最终值是第一个和第二个值之间的比率。

    图2。分析ITGA1启动子活性。与空载体(EV)相比,启动子基因报告物测定的结果显示相对荧光素酶活性为倍数变化。将野生型ITGA1启动子载体与空载体,MYC表达载体(MYC),显性阴性MAD(MAD)或两者(MYC-MAD)一起转染到HEK293T细胞中。在用空载体或MYC表达载体转染后,深灰色柱表示突变的ITGA1启动子(MYC的第一反应元件的突变)的活性。重复实验三次,一式三份进行。 t检验。 ** 0.01,*** 0.001。 Ns:不重要。


  1. 进行模板质粒的甲基化以便在转化后允许其通过内源核酸酶在宿主细胞中消化。因此,模板将被消除,只有突变的质粒将存在于转化的细胞中
  2. HEK293T细胞在转染期间不能达到融合水平
  3. HEK293T细胞形成脆弱的单层,因此介质的变化应以温和的方式进行
  4. MAD/MAX异二极体识别与MYC/MAX相同的响应元件。单独或与MYC一起转染MAD的事实降低了ITGA1启动子的活性,这表明当单独引入MYC时,该活性增强。
  5. 将DMEM储存在4°C;使用前请加热至37°C,避免长时间保暖。
  6. 不要通过上下移动来混合细菌小瓶。
  7. 孵化期间倒置LB板,以避免冷凝和污染。
  8. 准备100ng /μl的质粒以促进转染过程


  1. 用于HEK293T细胞的DMEM培养基
    2 mM GlutaMAX
    10 mM HEPES
  2. LAR II解决方案
  3. 1x停止& Glo
    稀释适量的Stop& Glo底物(库存50x)在Stop& Glo试剂(始终准备新的溶液)


我们感谢伊丽莎白鲱鱼审查手稿。这项工作得到了加拿大卫生研究所资助项目MOP-123415的支持(JFB是由魁北克圣地亚哥教会资助的Sherbrooke大学中心医院中心的成员,该协议改编自Boudjadi < (2016),最初出版于癌基因。


  1. Boudjadi,S.,Carrier,JC,Groulx,JF和Beaulieu,JF(2016)。  整联蛋白α1β1表达由结肠直肠癌细胞中的c-MYC控制。癌基因<35>(13):1671-1678。
  2. Chen,Y.,Xu,J.,Borowicz,S.,Collins,C.,Huo,D. and Olopade,OI(2011)。  c-Myc通过乳腺癌细胞中的远端启动子元件激活BRCA1 基因表达。 BMC癌症 11:246。
  3. Ni,H.,Dydensborg,AB,Herring,FE,Basora,N.,Gagne,D.,Vachon,PH和Beaulieu,JF(2005)。< a class ="ke-insertfile"href ="http: //"target ="_ blank">结直肠癌中β4整联蛋白亚基的功能形式的上调与c-Myc表达相关。癌基因 24(45):6820-6829。
  4. Teye,K.,Okamoto,K.,Tanaka,Y.,Umata,T.,Ohnuma,M.,Moroi,M.,Kimura,H。和Tsuneoka,M。(2008)。表达的TAF4b 基因是由MYC通过非 - 而不是规范的E-box,它有助于其对MYC的具体回应。 Int J Oncol 33(6):1271-1280。
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引用:Boudjadi, S. and Beaulieu, J. (2017). In silico Analysis and Site-directed Mutagenesis of Promoters. Bio-protocol 7(6): e2181. DOI: 10.21769/BioProtoc.2181.