Exploring Histone Variants’ Microenvironment With Proximity Labeling

As an important carrier of DNA folding and assembly into chromatin,histones play significant roles in these processes.Histone variants,the variants of conventional histones with different amino acid sequences,expression patterns,and localizations,regulate highly diverse chromatin structures and participate in various biological processes.Histone variants can recruit the microenvironment containing a variety of interacting proteins,to regulate DNA replication,damage repair and transcription,and to participate in chromatin structure architecture and biological processes.Therefore,the systematic analysis of the microenvironment around histone variants is vital for understanding the composition,dynamic changes and biological functions of chromatin.Affinity purification is a common technique to capture protein interactions,however,the low solubility and high dynamic of histone variants limits the application of this method to resolve the histone variant microenvironments.We applied the proximity labelling of histone variant(PAINTER)method to capture the interactome in vivo and analyze the microenvironments of histone vatiants.Using the PAINTER method,we could not only capture the interaction network of H3 variant CENP-A with low content on chromosomes,the microenvironment with high content of H2 A variant H2 A.X can also be specifically detected.Then PAINTER was used to research the chromatin dynamics of CENP-A microenvironment during the cell cycle which revealed the dynamic changes of CENP-A microenvironment over time.Subsequent systematic detection and quantitative analysis of the microenvironment around all histone variants with high specificity plus affinity revealed that H1,H2 A,and H3 histone variants recruit quite divergent candidate proteins,however,these proteins come from the same biological pathways to regulate common biological processes such as histone modification,cell cycle,chromatin remodeling,and RNA splices.Further analysis of the functions of different H1 variants in chromosome structural regulation showed that H1.0,H1.4,and H1.5 tended to be localized in dense chromatin,while H1.1,H1.3,and H1.10 tended to be localized in unfolded chromatin,and H1.3 played a role in regulating transcriptional activation.Finally,we demonstrate that PAINTER can decipher unknown chromatin functions by identifying and quantifying novel candidate proteins in microenvironment around histone variant in a high spatio and time resolution.We found that various ribosomal proteins would be recruited to DNA damage sites after DNA damage and RPS3 plays an important role in regulating homologous recombination(HR)repair.In summary,we believe that PAINTER will be a powerful strategy for studying chromatin organization and function associated with histone variation in living cells or organisms.Additionally,the application of PAINTER enlights a new path to research the histone variant microenvironments in a high spatio and time resolution.