How to develop a comprehensive strategy for the analysis of transcription factors

Transcription factors as master regulators of physiological and pathological processes

Decoding the genome and transcribing genetic information from DNA into messenger RNA (mRNA) are fundamental components of virtually all biological processes. In humans, approximately 1,600 transcription factors regulate gene transcription, including its initiation and elongation. The underlying mechanism involves binding of transcription factors to specific DNA sequences, known as promoters or enhancers, via their DNA-binding domains.2,3 Moreover, transcription factors recruit co-regulators, adding an additional level of complexity of gene transcription regulation. Further, the chromatin state also affects the function of transcription factors.1 Finally, the dynamics of transcription factor binding to DNA sequences has important implications for the effects of transcription factors on gene transcription.

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The complex process of gene transcription regulation contributes to the differential gene expression across different cell and tissue types and across developmental stages. Notably, transcription factors are involved in virtually all major biological processes, including cell division, differentiation, migration, and death as well as cellular responses to environmental stimuli. Moreover, dysregulation of transcription factors has been implicated in numerous diseases, including cancer, inflammation, autoimmune disorders, cardiovascular disorders, and neurological conditions.

Techniques to analyze the activation and functions of transcription factors 

The key role of transcription factors in physiological and pathological processes and the complexity of their regulation have led to active research on the establishment of techniques for their analysis. Thus, a wide range of methods have been developed to assess the function of transcription factors in vitro or in vivo.6 Traditional techniques, including electrophoretic mobility shift assays (EMSA) and reporter-coupled promoter deletion assays, evaluate the function of single transcription factors. More recently, high-throughput assays for the simultaneous analysis of multiple transcription factors have also been established, including ChIP-chip and ChIP-seq techniques, DNA microarrays, binding site enrichment-based methods, and microfluidic devices.6 Signosis has also developed a comprehensive range of assays for individual or multiplex transcription factor evaluation to advance and streamline research in this field.

Assays for the analysis of individual transcription factors

EMSA Kits We offer over 100 non-radioactive, highly sensitive EMSA kits based on chemiluminescent detection. The kits developed by our talented scientific team are easy-to-use and include biotin-prelabeled, optimized probes in combination with a complete set of reagents.

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TF Filter Plate Assays

Signosis has also developed high-throughput, quantitative “EMSA on a plate” assays that enable the simultaneous monitoring of a single transcription factor activation in up to 96 samples. 

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Luciferase Reporter Vectors and Vector Sets

We have developed more than 100 luciferase reporter vectors that can be used in quantitative, functional assays to evaluate the activation of transcription factors in cells. In addition, our expert team or scientists can create a luciferase reporter vector for any transcription factor. Moreover, using luciferase reporter vectors, we can establish Luciferase Stable Cell Lines to facilitate research on transcription factors of interest.

TF ELISAs have been developed by our team to profile the DNA-binding activity of specific transcription factors. These assays are high-throughput, effective, specific, and easy-to-use.

Profiling plate arrays for the simultaneous evaluation of multiple transcription factors

To gain insight into the complexity of regulation of different transcription factors under a variety of biological conditions, we have developed innovative profiling plate arrays that can simultaneously assess multiple transcription factors in a quantitative and convenient manner.

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TF Activation Profiling Plate Arrays simultaneously and quantitatively assess the activation of up to 96 transcription factors. They are based on an easy-to-use, ELISA-like procedure and can be applied on nuclear extracts from humans, rats, and mice. We have developed arrays  to assess key transcription factors most frequently implicated in the regulation of cellular functions as well as transcription factors relevant for specific diseases or biological processes, such as oxidative stress, endoplasmic reticulum stress, cancer stem cells, and neurodegenerative conditions.

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Transcriptional Interaction TF Plate Arrays broaden our understanding of the complexity of transcription factor functions by simultaneously monitoring the interactions among 48 or 96 transcription factors in relation to a specific transcription factor or coregulator. 

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Promoter-Binding TF Profiling Arrays simultaneously evaluate the binding of 48 or 96 transcription factors to a certain promoter. The underlying technology is based on magnetic bead capture of immunoprecipitated transcription factor complexes, and the output can be  detected with standard chemiluminescence plate readers.

Strategies for the comprehensive evaluation of transcription factors 

Elucidating the mechanisms implicated in the complex regulation of transcription requires a comprehensive strategy for the evaluation of transcription factors’ activity and functions. A robust approach would include the assessment of both individual and multiple transcription factors in vitro and in vivo. At Signosis, we have developed a comprehensive range of assays that evaluate different aspects of transcription factors’ activities and functions. In addition, our team of talented and experienced scientists provides personalized support to our customers to develop custom assays for the assessment of transcription factors of interest.

Resources

1. Lambert SA, Jolma A, Campitelli LF, Das PK, Yin Y, Albu M, Chen X, Taipale J, Hughes TR, Weirauch MT. The human transcription factors. Cell. 2018;172(4):650-665. doi: 10.1016/j.cell.2018.01.029. Erratum in: Cell. 2018;175(2):598-599.

2. https://www.frontiersin.org/research-topics/18211/structure-and-function-of-transcription-factors-and-coregulators

3. https://www.nature.com/scitable/definition/transcription-factor-167/

4. Lickwar CR, Mueller F, Hanlon SE, McNally JG, Lieb JD. Genome-wide protein-DNA binding dynamics suggest a molecular clutch for transcription factor function. Nature. 2012;484(7393):251-5. doi: 10.1038/nature10985.

5. Lee TI, Young RA. Transcriptional regulation and its misregulation in disease. Cell. 2013;152(6):1237-51. doi: 10.1016/j.cell.2013.02.014. 

6. Geertz M, Maerkl SJ. Experimental strategies for studying transcription factor-DNA binding specificities. Brief Funct Genomics. 2010;9(5-6):362-73. doi: 10.1093/bfgp/elq023.