comments | Li Guohong Researcher (Institute of Biophysics, Chinese Academy of Sciences)
Editor | Enzyme
As the core of gene expression regulation , the transcription start process occurs in the gene promoter region, and the nucleosome is removed by chromatin remodeling complex to expose the promoter, allowing transcription initiation complex (preinitiation complex, assemble the PIC-Mediator transcription start supercomplex with the help of the mediator (Mediator) . Xu Yanhui team has made a series of important breakthroughs around the research on the initiation mechanism of human transcription, and some of the results have been selected as one of the top ten progress in China's life sciences in 2021.
PIC-Mediator is the core of transcription initiation regulation. gene expression is mainly regulated by transcription factors and epigenetic . Among them, transcription factor binding promotes the recruitment and assembly of PIC-Mediator near enhancers and promoters, while epigenetic regulation of transcription is concentrated on the first nucleosome downstream of the promoter of (called +1 nucleosome ) . Unlike other nucleosomes, +1 nucleosomes contain histone acetylation and H3K4 trimethylation modifications, characteristic histone variants H2A.Z and H3.3. The formation of these characteristics is dynamically regulated by the modification enzyme and chromatin remodeling complex, affecting the expression level of the gene where they are located, and is the core of epigenetic regulation. Most models at present show that the transcription complex is isolated from the +1 nucleosome. The flexible end modification of histones in the +1 nucleosome can bind to TFIID to help PIC recruitment. At the same time, the transcription machine enters an extended state before encountering and bypassing the +1 nucleosome. Previously, all transcriptional initiation complex structures were based on the DNA template, and it was unable to show the structural functional correlation between +1 nucleosomes and transcriptional initiation complex.
On October 7, 2022, Fudan University Biomedical Research Institute /Fudan University Affiliated Cancer Hospital Xu Yanhui Research group published a research paper titled "Science" online in the journal Science" online. This study analyzed the structure of the PIC-Mediator complex containing +1 nucleosomes, and for the first time demonstrated the close binding of the transcription initiation complex and the +1 nucleosome. showed that the important regulatory effect of +1 nucleosome on the assembly of the transcription initiation complex on chromatin (Fig. 1) , established a direct association between epigenetics and transcription initiation.
Figure 1: PIC-Mediator structure bound to +1 nucleosome
Previous sequencing studies have shown that most +1 nucleosomes are located about 40 bp downstream of the gene transcription start site (TSS) . Inspired by this phenomenon, the research team built a template containing nucleosomes and promoter . According to the distance between nucleosomes and TSS, it was named T40N (T represents TSS, N represents nucleosomes, and numbers represent distance) , T50N, and T70N. Using in vitro reconstruction method, the PIC-Mediator transcription initiation super complex (84 proteins, molecular weight 4.3MD) , which binds three +1 nucleosomes, was successfully assembled. Through structural and biochemical analysis, found that the +1 nucleosome and the PIC-Mediator complex have multiple direct interactions and multiple modes of action. By combining TFIIH and Mediator multiple subunits , the assembly and transcription initiation activity of PIC-Mediator on chromatin is promoted.
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+1 nucleosome binding to PIC-Mediator also shows the following characteristics closely related to function:
(Figure 2) . PIC-Mediator is more inclined to bind to T40N nucleosomes to form relatively stable interactions. T50N nucleosomes can also be bound, and at this time some nucleosomes tend to dissociate, because the distance between the nucleosomes and the complex increases, and the binding will cause tension to cause the bending of the DNA to partially offset the bonding attraction. When it comes to T70N nucleosomes, it will be gone after direct contact. In vitro transcription initiation activity experiments showed that T40N nucleosomes can enhance transcriptional activity, while T70N did not. These results not only explain the results of in vivo sequencing (+1 nucleosomes are about 40bp from TSS) , also prompts the reason behind its occurrence . That is to say, the PIC-Mediator complex needs to integrate the sequence information on the promoter and the position information of the nucleosome to determine its binding site and transcription start site. Most promoters in human body do not contain strong localization sequences such as TATA box. PIC-Mediator uses +1 nucleosome as the main reference point to produce an appropriate binding method. The location of this binding determines the transcription start site.
Figure 2. Structure of PIC-Mediator and three different localized nucleosomes. A. Pattern diagram of three promoter templates: T40N, T50N and T70N. B, C, D. PIC-Mediator complex structures that bind T40N, T50N, T70N. E. Four different binding states of T50N nucleosomes (yellow).
interaction inclusiveness. nucleosomes bind to PIC-Mediator through electrostatic interaction without obvious sequence specificity. For example, the basic regions of TFIIH and Mediator subunits bind to the acidic backbone of nucleosome DNA and the acidic surface of H2A.Z-H2B under the framework of the whole complex. This binding is characterized by low specificity and is attractive as long as the charge is opposite and the position is close. Therefore, the high tolerance level can allow a certain degree of conformation changes and nucleosome position changes, so that the transcription machine can bind to nucleosomes of various promoters.
Currently, the mainstream concept believes that transcription machines will encounter nucleosomes only when they enter the extension stage. This work shows the close binding of the transcription initiation complex and the +1 nucleosome at the molecular level for the first time, and establishes a direct correlation between epigenetic signals near the transcription initiation site and transcription initiation. From the transcription start site to the dozens of nucleotides (about 14 nanometers) between the transcription start site and the +1 nucleosome, a series of transcription start events that occur (RNA synthesis, polymerase advance, pause, release, cap, extension, etc.) , and the dynamic dissociation and binding of dozens of different transcriptional regulatory complexes (hundreds of proteins) , all need to consider the effects of +1 nucleosome and its epigenetic regulation (Figure 3) . This study will change our traditional view on the transcription initiation process and the relationship between chromatin represented by +1 nucleosomes, and provide a new guiding framework and structural basis for studying epigenetics and gene expression regulation.
Figure 3. +1 nucleosomes may play a role in both the initiation of transcription and the extension series of dynamics in the early stages of transcription. . From TSS to +1 nucleosomes, it is about 40bp, about 14nm. A series of early transcriptional events will occur within this range, and it is also the core area of gene expression regulation.
Fudan University School of Life Sciences Youth Researcher Chen Xizi, School of Life Sciences, Fudan University, Wang Xinxin, PhD student in the 2020 class of the Institute of Biomedical Research, and Liu Weida, a student in the 2019 class of Direct Doctoral , are the co-first authors of this article, and Xu Yanhui is the corresponding author of .
Expert comments
Li Guohong Researcher (School of Biophysics, Chinese Academy of Sciences)
In eukaryotes, the precise regulation of gene transcription plays a crucial role in many important biological processes. Gene transcription includes three processes: transcription initiation, transcription extension and transcription termination. The transcription initiation is the core of gene transcription regulation, and the transcription initiation process occurs in the gene promoter region. We know that in eukaryotes, genomic DNA wraps histone octamers to form nucleosomes, forming various advanced chromatin structures under the action of connecting histone H1 and other chromatin architecture proteins. Therefore, the chromatin structure of the promoter region plays an important regulatory role in the initiation of gene transcription. Various biochemical and genomic experiments in the early stage found that the nucleosome localization and chromatin structure of the gene promoter region have very distinct characteristics, such as the nucleosomes in the promoter region are removed or shifted to form an open chromatin region, so that the transcription machine can recruit the promoter region and assemble to form a transcription initiation complex. In addition, it is very interesting that in the promoter region of most genes, the first nucleosome (called +1 nucleosome) located very regularly downstream of the transcription start site, generally located about 40 bp downstream of the gene transcription start site (TSS). Moreover, epigenetic studies have found that the +1 nucleosome of highly expressed genes are rich in various important histone chemical modifications (such as H3K4me3 and histone acetylation) and specific histone variants (H2A.Z and H3.3). The function of +1 nucleosomes and their epigenetic characteristics in transcription initiation regulation is still not very clear in the field, such as the interaction between the transcription initiation complex and the +1 nucleosome and its epigenetic regulatory mechanism. In recent years, with the development of cryo-electron microscopy technology, teams such as Xu Yanhui and Patrick Cramer have achieved a series of important breakthroughs and achievements in the recruitment and assembly of transcriptional initiation complex (PIC) and PIC-Mediator transcription initiation supercomplexes. However, all the above-mentioned transcription initiation complex structures are based on naked DNA templates and cannot demonstrate structural functional correlation between +1 nucleosomes and transcription initiation complexes. The work published by Xu Yanhui's team on
Today, the work published on Science answered the above key questions. This study analyzed the structure of the PIC-Mediator complex containing +1 nucleosomes, and demonstrated the close binding of the transcription initiation complex with +1 nucleosomes for the first time, indicating that the +1 nucleosomes have an important regulatory role in the assembly of the transcription initiation complex on chromatin, establishing a direct association between epigenetics and transcription initiation. The experimental system of this work is complex and very difficult, the experimental design is very clever, and the research ideas are very novel.
1) In order to prove the important function of +1 nucleosome localization, the research team designed and constructed three different DNA templates by changing the distance between +1 nucleosome and TSS (40 bp, 50 bp and 70 bp). This experimental design is very clever and can answer why +1 nucleosome is generally located at 40 bp downstream of TSS. The results of this study show that the binding of +1 nucleosome to PIC-Mediator has two significant characteristics: the preference of nucleosome location (~40 bp) and the inclusiveness of interaction, which explains well how the PIC-Mediator complex determines its binding site and transcription start site by integrating the sequence information on the promoter and the location information of nucleosomes, and accurately regulates the transcription initiation of various different genes.
2) Using in vitro reconstruction method, the research team successfully assembled the PIC-Mediator transcription-initiating supercomplex (84 proteins, molecular weight 4.3MD) that bind three +1 nucleosomes respectively, and analyzed their cryo-electron microscopy structure. This system is very complex, involving many types of proteins (including nearly 100 proteins), and its structural form is highly dynamic and very difficult. I did similar work during my postdoctoral work in Danny Reinberg's laboratory. We used the in vitro transcription system to study the transcription process of RNA Pol II on 30-nm chromatin fibers, and we knew the difficulty and difficulty of this in vitro reconstructive system.A few years ago, in the epigenetics retreat organized by Shi Yang, Shi Yujiang and Lan Fei, Mr. Xu Yanhui had in-depth exchanges with me. At that time, I was still skeptical about the regulatory mechanism of using cryo-electron microscopy to analyze the transcription initiation complex and the transcription extension process. Recently, Xu Yanhui's team, Patrick Cramer from the MaxPokém Institute in Germany, and Hitoshi Kurumizaka from the University of Tokyo, Japan (Two articles on science丨 RNA polymerase II how to transcribe through nucleosomes? ; Science | Molecular mechanism of eukaryotic gene transcription extension complex passing through nucleosomes ) have made a series of breakthrough progress in the recruitment and assembly of transcription initiation complexes and the regulation mechanism of RNA Pol II on transcription extension on nucleosomes. I would like to congratulate the progress and achievements made by Teacher Xu Yanhui's team, and also admire the students and staff who have completed these tasks. At the same time, I would also be a great encouragement to our own research work.
3) In this study, Xu Yanhui's research team constructed the histone variant H2A.Z nucleosome in +1 nucleosome. +1 nucleosome is rich in histone variant H2A.Z downstream of the promoter of most highly expressed or inducible genes, but now there is still a lot of controversy about the biological function of H2A.Z nucleosomes. The work of Xu Yanhui's team shows that the basic regions of TFIIH and Mediator subunits can bind to the acidic backbone of nucleosome DNA and the acidic region of H2A.Z-H2B. The acidic region of H2A.Z nucleosome is more acidic than the acidic region of conventional nucleosomes, so the PIC-Mediator complex has a stronger effect with H2A.Z nucleosomes, which may be of great significance to regulate the functional interaction of the transcription initiation complex and the +1 nucleosome. Recently, Patrick Cramer and Hitoshi Kurumizaka (Science | Molecular mechanism of eukaryotic gene transcriptional extension complex passing through nucleosomes ) used cryoelectron microscopy to analyze the RNA Pol II transcriptional extension complex (including transcriptional extension factor DSIF (Spt4/5), Spt6, PAF1, and FACT (Spt16/SSRP1) and TFIIS) perform transcriptional extension mechanism on nucleosomes, capturing the details of the extension complex advancing on nucleosome DNA. It is found that the extension complex can mediate downstream nucleosome depolymerization and upstream reassembly, in which histone chaperone FACT or Spt6 promotes the process, thereby maintaining the integrity of nucleosomes during transcription. This also verifies the research results of our research team's FACT-nucleosome single-molecule magnetic tweezers a few years ago (Mol Cell丨Li Guohong's group revealed the molecular mechanism of histone chaperone regulating nucleosome structure ). It is worth mentioning that the above two teams use conventional H2A nucleosomes to study the transcriptional extension mechanism, but in most genes, the +1 nucleosomes are H2A.Z nucleosomes. In the future, cryo-electron microscopy and single-molecule technology are used to study the structural details and kinetic processes of the +1 nucleosomes containing H2A.Z, which will have more physiological significance.
Summary, the experimental design of this work by Xu Yanhui's team is clever, the experimental system is complex, difficult, rich content, novel research ideas, and very beautiful structure. This study not only changed our traditional view on the transcription initiation process and the relationship between chromatin represented by +1 nucleosomes, but also provided a research system and guiding framework for future research on the epigenetic regulatory mechanism of (RNA synthesis, polymerase advancement, transcriptional arrest and release, RNA capping and transcription extension, etc.) . is here. I would like to congratulate Xu Yanhui's team on making another important progress in the research direction of transcription initiation regulation mechanism, and look forward to their team making more breakthroughs in subsequent research.
original link:
http://doi.org/10.1126/science.abn8131
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