Transcription Factor Response Elements on Tip: A Sensitive Approach for Large-Scale Endogenous Transcription Factor
Wenhao Shi1,2, Kai Li2,3, Lei Song1,2
1School of Life Sciences, Tsinghua University , Beijing 100084, China.
Analytical Chemistry
|February 15, 2017
Summary
Researchers developed a new method, transcription factor response elements on tip-mass spectrometry (TOT-MS), to quantitatively measure endogenous transcription factors (TFs). This rapid, high-throughput technique can identify hundreds of TFs from small samples, advancing biological process understanding.
Area of Science:
- Proteomics
- Molecular Biology
- Biochemistry
Background:
- Understanding endogenous transcription factor (TF) DNA binding activity at the proteome scale is crucial for deciphering biological processes.
- Existing methods may lack the sensitivity, throughput, or quantitative accuracy needed for comprehensive TF analysis.
Purpose of the Study:
- To introduce a novel, highly sensitive, rapid, and high-throughput approach for the quantitative measurement of endogenous transcription factors.
- To demonstrate the capability of the new method in analyzing dynamic changes in TF activity.
Main Methods:
- Developed transcription factor response elements on tip-mass spectrometry (TOT-MS), a method for quantitative measurement of endogenous TFs.
- Utilized TOT-MS coupled with on-tip small size reverse-phase liquid chromatography for enhanced TF identification.
- Applied the method to analyze TF dynamics within the epidermal growth factor (EGF) signaling pathway.
Main Results:
- Quantified 150 TFs from just 1 μg of nuclear extracts within 1 hour of machine time using TOT-MS.
- Identified up to 755 TFs, achieving a depth comparable to RNA-sequencing.
- Successfully demonstrated the method's utility by interrogating dynamic TF changes in the EGF signaling pathway.
Conclusions:
- TOT-MS provides a sensitive, rapid, and high-throughput solution for quantitative measurement of endogenous TFs.
- The approach enables deep TF profiling from limited biological material, comparable to RNA-seq depth.
- This method holds significant potential for broad applications in elucidating the TF landscape across various biological contexts.
More Related Videos
06:38High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy
Published on: February 7, 2019
9.3K
06:43A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents
Published on: August 31, 2013
19.2K
Related Concept Videos
02:16Transcription Factors
83.2K
Tissue-specific transcription factors contribute to diverse cellular functions in mammals. For example, the gene for beta globin, a major component of hemoglobin, is present in all cells of the body. However, it is only expressed in red blood cells because the transcription factors that can bind to the promoter sequences of the beta globin gene are only expressed in these cells. Tissue-specific transcription factors also ensure that mutations in these factors may impair only the function of...
83.2K
01:30General Transcription Factors
7.4K
Tissue-specific transcription factors contribute to diverse cellular functions in mammals. For example, the gene for beta globin, a major component of hemoglobin, is present in all cells of the body. However, it is only expressed in red blood cells because the transcription factors that can bind to the promoter sequences of the beta globin gene are only expressed in these cells. Tissue-specific transcription factors also ensure that mutations in these factors may impair only the function of...
7.4K
02:36RNA Polymerase II Accessory Proteins
11.2K
Proteins that regulate transcription can do so either via direct contact with RNA Polymerase or through indirect interactions facilitated by adaptors, mediators, histone-modifying proteins, and nucleosome remodelers. Direct interactions to activate transcription is seen in bacteria as well as in some eukaryotic genes. In these cases, upstream activation sequences are adjacent to the promoters, and the activator proteins interact directly with the transcriptional machinery. For example, in...
11.2K
02:23Master Transcription Regulators
8.0K
Master transcription regulators are regulatory proteins that are predominantly responsible for regulating the expression of multiple genes. Often these genes work in concert to drive a complex process. Activation of a master transcription regulator can lead to a cascade of transcriptional activation necessary for that outcome. These regulators can directly bind to the regulatory sequences of the various genes involved, or they can indirectly regulate transcription by binding to regulatory...
8.0K
02:57Real Time RT-PCR
66.0K
Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...
The real-time quantification of the number of amplified products is...
66.0K
02:04Co-activators and Co-repressors
8.8K
Gene transcription is regulated by the synergistic action of several proteins that form a complex at a gene regulatory site. This is observed in eukaryotes, where the regulation of gene expression is a complex process. Regulatory proteins in eukaryotes can broadly be classified into two types – regulators that bind directly to specific DNA sequences and co-regulators that associate with regulatory proteins but cannot directly bind to the DNA. These co-regulators are further divided into...
8.8K