BAF/PBAF complexes are members of the human chromatin regulator family that act like “gatekeepers” and influence the final manifestation of human genes. In recent years, scientists have found that mutations in the complex are linked to more than 20 percent of cancers and multiple neurological developmental defects.
Professor Chen Zhucheng’s team of Tsinghua University used the advanced method of cryo-EM to reveal the assembly method of PBAF complex and its mechanism of identifying nucleosomes, which provided a theoretical framework for studying the pathogenesis of mutations associated with human diseases.
At 23:00 beijing time on April 27, 2022, the relevant results were published in the journal Nature.
Structural analysis “breaking the problem”
Chromatin is a carrier of eukaryotical genetic material, a dense structure formed by high compression of DNA wrapped around histone octamers to form nucleosomes. This form and structure guarantees the stability of the genome on the one hand, and hinders life activities such as replication, transcription, and DNA damage repair on the other hand.
So, when the chromosomes “remain calm” and when they “open their hearts” depends on a series of regulatory mechanisms developed by eukaryotic cells. For example, during the occurrence of the regulatory mechanism of “chromatin remodeling”, some proteins called “remodeling complexes” use the energy of ATP to slide, exchange or depolymerize nucleosomes, by changing the binding position of nucleosomes and DNA, thereby achieving dynamic regulation of chromatin.
The complexes, named BAF and PBAF, are two typical chromatin remodeling complexes belonging to mammals that regulate chromatin structure and gene expression and are widely involved in the developmental differentiation of animal cells. As “gatekeepers” of genes, these two complexes are also considered potential drug targets for the treatment of diseases such as cancer.
According to researchers, as early as 1996, these two complexes were identified. Moreover, thanks to the development of cryo-EM technology, a high-resolution structure of one of the BAF complexes was also released. However, for the way PBAF complexes are assembled, the mechanisms of identifying nucleosomes, and their differences with BAF complexes are still at the forefront of science.
The team of researchers who have long been working on the mechanism of chromosome remodeling believes that understanding the structure of the PBAF complex will help us “see” what it looks like, so that it can help understand its working process.
The three-dimensional structure is baked
To solve these puzzles, it is first necessary to obtain a complete PBAF complex for study. But the complex is a very large complex of 12 subunits with a molecular weight of more than 1 trillion daltons, which brings the team the first technical difficulty that needs to be solved: how to get a high-quality complete PBAF complex?
Professor Chen Zhucheng led the team to fight together in the laboratory, constantly looking for a way out to solve the problem. After five years of research, after a series of explorations on the protein expression system, protein building boundary conditions, purification methods, etc., the team explored a new protocol for biochemical recombinant experiments of PBAF complexes, and finally obtained high-quality recombinant PBAF complexes.
Subsequently, the team used a cryo-electron microscope from Tsinghua University to image and reconstruct the complex in three dimensions. The second difficulty arises, how to build a three-dimensional model of the complex?
The team first thought of the “amino acid residue feature fuzzy recognition algorithm”. Based on the high-resolution electron microscopy density map, the prediction of the secondary structure of proteins and the chemical properties of amino acid residues, they successfully resolved the structure of PBAF-binding nucleosomes and revealed the assembly mode of PBAF complexes.
The three-dimensional structure shows that the 12 subunits of PBAF can be divided into three modules according to their different functions, including the “motor module” responsible for exerting catalytic activity, the “ARP module” with regulatory function, and the “SRM module” that plays the function of chromatin targeting.
Interestingly, the “SRM module” consists of 9 auxiliary subunit shuttles intertwined to form a three-lobed shape. The research team also named these leaves based on their main biological functions, including nucleosome-binding leaves (NBL), histone tail-bound leaves (HBL), and DNA-bound leaves (DBL).
Chen Kangjing, co-first author of the paper and a doctoral student at Tsinghua University, told China Science Daily that compared with BAF, PBAF has a special HBL, including some specific subunits. “This is equivalent to a superhistemic recognition submodule.”
The researchers believe that it is this particular domain that allows the PBAF complex to sense chromatin signals more efficiently in the body.
The PBAF chromatin remodeling complex binds to the structure of nucleosomes and disease-associated mutations. (a) Cryo-EM density plot of PBAF-nucleosome complex. (b) Structural model of the PBAF-nucleosome complex. (c) PBAF complexes recognize nucleosomes, regulating models of chromatin structure. (d) 3703 cancer-associated SMARCA4 missense mutation sites, as well as multiple smarCA2 malocusion mutation site distributions associated with neurodevelopmental defects (data from COSMIC, cBioPortal, and published literature). With the SMARCA4 sequence as a reference, the high-frequency mutation site is labeled; the corresponding amino acid residue of the SMARCA2 mutation is invillage. (e) Disease-associated mutation sites are mainly located around the “ATP pocket” and the “Brace interface”, the local magnification plot shows the distribution of high-frequency mutation sites at the Brace-helix interface, and the biochemical experiments on the right panel show that mutations in R973 and R1243 greatly reduce chromatin remodeling activity. (Courtesy of the research team)
Powering Precision Medicine
After elucidating the PBAF complex assembly and chromatin remodeling mechanism, the team began to think about the next step: How can the study serve the study of human diseases?
In previous studies, it was difficult to provide pathogenic mechanisms for disease-related mutations due to the inactive state of the core “motor subunit” of the BAF complex.
In studies around PBAF, the researchers found that the motor subunits in the PBAF complex were in an active state. “We clearly saw a large number of disease-related mutations distributed at two key active interfaces, and mutations at these sites also significantly reduced chromatin remodeling activity, suggesting that disease occurrence may be related to the loss of BAF/PBAF complex function.” Researchers introduced.
In addition, they revealed for the first time the mechanism by which motor subunits recognize nucleosomes.
“Our findings provide the basis for future precision medicine.” Yuan Junjie, co-first author of the paper and a doctoral student at Tsinghua University, told China Science News that “through the study of PBAF complexes and their active subunits, it provides a structural basis for the development of new drugs targeting BAF/PBAF complexes.”
Industry experts believe that this work not only elucidates the PBAF complex assembly, nucleosome recognition, and chromatin remodeling mechanisms, but also provides a theoretical basis for understanding the pathogenesis of BAF/PBAF-related mutations. This finding is useful for understanding the mechanism of chromatin remodeling at the chromatin level and will also promote the development of drugs targeted at related diseases. (Source: China Science Daily, Sun Danning, Gan Xiao)
Related paper information:https://doi.org/10.1038/s41586-022-04658-5