Credit: Imke Mandemaker. Photo taken in the lab of Andreas Ladurner in Munich. 9 November 2023 New fluorescence-based assay sheds light on histones and their chaperones Back to news Histone chaperones are responsible for placing histones onto the DNA and therefore play an important role in packaging the DNA of cells and regulating its accessibility. Researchers from the Mattiroli group developed a new fluorescence-based assay and used this to identify ANP32B as the histone chaperone responsible for regulating a specific histone, macroH2A. The results were published on October 18th 2023 in Cell Reports and provide a basis for future studies into the interaction between these two proteins and their role in development and cancer. Each cell in the human body contains about two meters of DNA. In order to fit this into the cell nucleus, the DNA is tightly packaged by wrapping it around proteins called histonesProteins in the nucleus around which the DNA is wound. This way, all the DNA fits inside the nucleus. Through modifications of the histones, the DNA is wound tighter or looser, which changes the accessibility of the genes in that area.. Apart from compacting the DNA, this also protects it and regulates which parts of the DNA are accessible. The histones are placed onto the DNA by histone chaperones. These ‘helper’ proteins ensure that histones are positioned at the right location at the right time. In this study, researchers from the Mattiroli group aimed to track down the chaperone that is responsible for placing a specific histone protein, macroH2A, onto the DNA. Silencing DNA regions Imke Mandemaker, first author of the study, explains the function of macroH2A: “It is the largest of all histones and is mainly present at inaccessible DNA regions, where it provides extra compaction. The more compact a region is packaged, the less accessible it is to enzymesProteins that accelerate a specific chemical reaction, for example the breakdown of a molecule. The enzyme itself remains intact in such a reaction, which enables the molecule to exert its function over and over again. Enzymes are present both in- and outside the cell. that use the DNA information to determine the behavior of the cell. Therefore, macroH2A is mainly associated with silencing parts of the DNA, meaning that the information is not used. For example, macroH2A is important in silencing the stem cell-like properties of cells, thereby preventing specialized cells of the body from going back into stem cellsCells that are not specialized yet, but from which various different specialized cells can emerge.. As this transition to a stem cell-like state also happens during the development of cancer, macroH2A may play an important role in preventing cancer. Therefore, both macroH2A itself and its regulators are interesting therapeutic targets that we want to understand.” Schematic representation of the fluorescence-based assay used to measure the amount of macroH2A on the DNA. The dark blue ribbon represents the DNA, which is wrapped around histones indicated in light blue. Together, this is called the nucleosome. One of the histones is macroH2A (orange). GFP is the fluorescent protein, which only emits green light when the two parts (N-GFP and C-GFP) come together. This happens when macroH2A is on the DNA, close to the other histone (right image). Credit: Imke Mandemaker. Copyright: Hubrecht Institute. One plus one equals light The researchers therefore set out to find the histone chaperone in charge of macroH2A. To make this possible, they first needed to develop a new strategy, which Mandemaker did during her time in Munich in the lab of Andreas Ladurner. Mandemaker: “We developed a new fluorescence-based assay that allowed us to measure the amount of macroH2A in living human cells, by looking at the amount of light emitted. For this we took advantage of a fluorescent protein that can be split into two parts. Only when these two parts come close enough together, can they form the full protein that emits light. We fused one half to macroH2A and the other half to a histone that is always present on the DNA. Therefore, when we measure light, we know that macroH2A is on the DNA, close to the other histone. The amount of light we measure is relative to the amount of macroH2A on the DNA.” Microscopy image of human cells to which the purified histone chaperone ANP32B was added, showing the subsequent deposition of the histone macroH2A (green) onto the DNA (magenta). Credit: Imke Mandemaker. Photo taken in the lab of Andreas Ladurner in Munich. Histone chaperone identified Mandemaker and colleagues from the laboratory of Lucas Jae then used this new assay to perform a large genetic screening, where they could see the effect of disrupting genesSmall pieces of DNA with a specific function. For example, genes determine which color our eyes have and whether we have curly or straight hair. In a human cell, the DNA contains about 30.000 genes, each having a specific function. Genes are hereditary and can therefore be passed on to offspring. on the amount of macroH2A deposited onto the DNA. “If you disrupt a gene and the amount of light is reduced, it means less macroH2A is present. So that is an indication that the disrupted gene encodes a protein involved in placing macroH2A onto the DNA,” says Mandemaker. “This allowed us to identify the histone chaperone ANP32B as a potential candidate. Using several other techniques, including a genome-wide analysis of macroH2A localization in collaboration with the lab of Marcus Buschbeck, we confirmed that this is indeed the protein responsible for delivering macroH2A to the DNA. Both macroH2A and ANP32B were known for a very long time, but thanks to the fluorescence-based screen, we now identified the link between them. This work serves as the basis for future studies aimed at understanding why ANP32B specifically binds to macroH2A and what the role of these proteins is in cancer and development,” concludes Mandemaker. Publication The histone chaperone ANP32B regulates chromatin incorporation of the atypical human histone variant macroH2A. Imke K. Mandemaker, Evelyn Fessler, David Corujo, Christiane Kotthoff, Andreas Wegerer, Clément Rouillon, Marcus Buschbeck, Lucas T. Jae, Francesca Mattiroli and Andreas G. Ladurner. Cell Reports, 2023. Francesca Mattiroli is group leader at the Hubrecht Institute.
