Fudan University has developed a chromatin open region transcription protein machine toolkit

The research team of Fudan University has developed a "toolkit" for precise analysis of protein-DNA transcription machinery in the open region of chromatin

The research team of Fudan University has developed a "toolkit" for precise analysis of the open region of chromatin protein-DNA transcription machinery

Tao Weishuo Science and Technology Daily reporter Wang Chun

On October 23, the international authoritative academic journal "Science" magazine "Science Progress" published online the latest research results of the team of Fudan University Biomedical Research Institute, Human Phenotype Research Institute Ding Chen/He Fuchu team on protein-DNA transcription machinery
.

The results show that the Fudan University team has realized the identification and functional research of the transcriptional protein machinery of the open chromatin region for the first time in the world

The transcriptional regulatory protein machinery is composed of transcription factors (transcirption factor, TF), transcription cofactor (transcirptional coregulator, TC), etc.
, which are involved in almost all biological processes (such as differentiation, development, cell cycle control, and apoptosis).
Play a key role
.

The process of gene transcription relies on the combination of transcriptional regulatory protein machinery and DNA to perform corresponding functions.

At the genome level, in 2013, Professor Howard Chang of Stanford University developed the ATAC-seq technology (Assay for Transposase-Accessible Chromatin with highthroughput sequencing), which can obtain chromatin open regions and active transcriptional regulatory sequences in the genome through high-throughput sequencing.
Key information
.

However, at the proteome level, due to the low expression abundance of transcription factors and transcription co-regulators and the lack of relevant enrichment techniques, the scientific community is still difficult to make a panoramic and fine analysis of the binding and pairing relationship between the above-mentioned protein molecules and the open regions of chromatin
.

For many years, the Ding Chen/He Fuchu team of the Institute of Biomedical Research of Fudan University and the Institute of Human Phenotype Group has focused on this problem and continued to carry out scientific research
.

As early as 2013, the Ding Chen/Qin Jun team developed the catTFRE technology and designed and synthesized multi-copy double-stranded DNA binding elements in tandem with various transcription factors for the first time, enriching endogenous transcription factors with DNA binding activity and their The complex is of great value for carrying out transcriptional regulation

In the latest results published this time, based on ATAC-seq technology, researchers have developed a new method ATAC-MS that directly enriches and quantifies the transcriptional protein machinery of the open region of endogenous chromatin in real-time in situ transcription
.

This technology uses biotinylated high-activity Tn5 transposon to specifically bind to the exposed DNA in the open region of chromatin, fragment the DNA and label it with biotin

This method can have good sensitivity and specificity for the identification of transcription factors and transcription co-regulators when using cells as low as 5×105
.

Different from the transcription factor binding sequence detected by ATAC-seq, ATAC-MS can directly capture transcription factor (TF) and the transcription complex that interacts with it

After stimulating HeLa cells with tumor necrosis factor (TNF-α), ATAC-MS successfully identified the changes in the transcription factor NFKB family and the TFs that interact with it
.

In addition, when the estrogen receptor 17β estradiol (E2) and its antagonist 4-Hydroxytamoxifen (4-Hydroxytamoxifen) were used to stimulate MCF7 cells, ATAC-MS not only detected changes in the core transcription factor ESR1, but also It can be observed that the stimulation of E2 leads to the enrichment of transcription co-activators, such as NCOA3, EP300, etc.

It is reported that the team further combined ATAC-MS technology with anion exchange technology to develop high resolution ATAC-MS analysis technology (fractionationATAC-MS, fATAC-MS)
.

Using salt solutions of different concentrations for elution, divide the DNA-transcription machinery complex into different components, reduce complexity, and provide a higher resolution transcription protein-DNA map

It is reported that the author further designed and expressed a new type of Tn5-dCas9 fusion protein, which upgraded ATAC-MS to ctATAC-MS (dCas9 targeted ATAC-MS)
.

In the absence of sgRNA, the Tn5-dCas9 fusion protein acts as a Tn5 transposon and binds to open chromatin, which can be used for transcriptional regulatory protein machinery at the genome-wide level; and under the guidance of sgRNA, Tn5-dCas9 targets specific targets.

The research published by the Ding Chen/He Fuchu team of Fudan University this time provides a "toolkit" for the analysis of transcriptional regulatory complexes including 3 new methods-ATAC-MS, fATAC-MS and ctATAC-MS, enabling us to both To map active transcriptional regulatory protein machinery at the genome-wide level, it is also possible to explore protein-DNA transcriptional regulatory complexes at specific genomic sites
.
The toolkit will enable researchers in this field to dig deeper into different levels of transregulation mechanisms, and has the characteristics of taking into account in vivo/in vitro reactions, and has a wide range of potential applications in clinical biopsy, detection of various tissue specimens, and testing of different species.

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