Investigating the Epigenetic Mechanisms of Trophoblast Giant Cells

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Biology ︱ Assistant Professor Koji Hayakawa NUCLEOSOME STRUCTURE OF TROPHOBLAST GIANT CELL (TGC) Diploid TSC Polyploid TGC H2A H2B H2AX/ Investigating the H2AZ epigenetic mechanisms Entry into DNA Endocycle H3 H4 H3.3 of trophoblast giant cells TGCs possess a loose chromatin structure owing to alterations in the histone composition of the nucleosomes, which involves the replacement of canonical histones with histone variants such as H2AX, H2AZ, and H3.3 during differentiation.

Trophoblast giant cells (TGCs) ucleosome is a large molecule in the placenta of rodents, are a unique days, and that certain histone variants Many polyploid cells identified in plants are found in the placental in the cell which is primarily cell type that replicate their DNA until were associated with differentiated walls of rodents and play a Nmade up of DNA and proteins. the cell contains thousands of copies, cells. Overall, there was much less and animals appear to have a secretory role in maintaining pregnancy. The major protein in nucleosome is unlike most cells which normally contain variation in TGCs compared to the In contrast to most cell types called a histone, around which DNA two sets of chromosomes (diploid cells). undifferentiated, diploid cells. They or nutritive function. which contain two copies of wraps. These proteins are classified into The reasons for this condition are not found the histone profile to be very from undifferentiated cells showed distinct concentrations of salt buffer to disrupt each chromosome (diploid), canonical histones and non-canonical clear. However, it has been suggested similar in differentiated TSCs at day six bands when digested, demonstrating that DNA-protein bonds. They then viewed TGCs can contain more histones, which are also known as histone that polyploidy (cells with multiple copies of differentiation compared with in vivo DNA becomes more loosely bound to them by single-cell imaging using a than 1000 copies of their variants, based on the similarity of amino of genes) may increase a cell’s capacity TGCs isolated from a placenta. The histones the more the cell differentiates. machine called a microfluidic device genomic deoxyribonucleic acid sequences and their cell cycle- for protein synthesis, allowing for faster scientists also compared the expression To confirm this, the scientists exposed which manipulated individual cells acid (DNA), a condition known dependent or independent expression. tissue growth than a diploid cell. Many of histone variants in embryonic cells TSCs expressing H4-GFPs to increasing using optical tweezers. As the TSCs as polyploidy. In a recent Nucleosome has a number of functions, polyploid cells identified in plants (diploid) with placental TGCs and found paper published in the journal including organising and condensing and animals appear to have a secretory certain variants were much higher in LOCATION OF TGC IN PLACENTA Scientific Reports, Assistant DNA, controlling its replication, or nutritive function. placental TGCs than embryonic cells. Professor Koji Hayakawa of the and protecting DNA from damage. PLACENTA Trophoblast giant cells University of Tokyo Graduate The array of nucleosome is called a Due to the enormous amount of genomic Chromatin is found in the nucleus, School of Agricultural and chromatin. Chromatin is assembled DNA packaged into its nucleus (the the organ in the cell that acts as a Life Sciences unravelled the Decidua and disassembled by usage of histone command centre of the cell), it is thought command centre. Dr Hayakawa’s group molecular mechanisms that (maternal tissue) stained the cells and were able to visualise allow so much DNA to be Labyrinth packaged into TGCs, a function Polyploidy may increase a cell’s capacity chromatin dynamics by attaching one accomplished by structures of the core histones, H4, to a green known as nucleosomes. for protein synthesis, allowing for faster fluorescent protein (GFP) molecule and monitoring movement under a tissue growth than a diploid cell. microscope during the differentiation process. They found that histones types and their post-translational that the chromatin of TGCs may behave occupied different parts of the nucleus modifications as the cell faces various in a different manner than that of diploid during differentiation. To test the mobility Spongiotrophoblast metabolic demands and challenges. cells. In a series of experiments recently of histones, one half of the nucleus of cells CELL CYCLE OF TGC published in the journal Scientific Reports, that contained H4 attached to a green During DNA replication and transcription Dr Hayakawa and his collaborators fluorescence molecule was bleached. (the beginning of protein synthesis), set out to determine the dynamics of Fluorescence intensity was then measured G1 S G1 S the DNA becomes more loosely wrapped chromatin structure in TGCs and deduce in the presence of cycloheximide, a drug around the histone, which allows crucial the function and location of their histones that prevents protein synthesis. It was Mitosis Endocycle enzymes easier access during the differentiation of trophoblast found that the histones in the TGCs did to its structure. stem cells (TSCs) (immature trophoblast not move around as much as in the TSCs. 2-4n >4n In general, histones cells which are still diploid) into TGCs. tend to bind VARIANT HISTONES MAINTAIN more strongly HISTONE PROFILE AND ‘LOOSE’ NUCLEOSOMES M G2 to inactive genes CHROMATIN STRUCTURE IN TGC IN MATURE TGC G2 and more loosely The researchers first stimulated In another experiment, the scientists Diploid cell to active ones. trophoblast stem cells to cause them to digested genomic DNA at day six of Polyploid TGC mature (differentiate) and collected them differentiation using a special protein Rodent placenta consists of three layers, i.e. labyrinth-, spongiotrophoblast-, and TGC-layer. TGC can be polyploidized by non-typical cell cycles such as endoreduplication (endocycle), which is a system to Trophoblast every other day for 10 days. It was found known as a DNase enzyme and showed replicate DNA without M-phase. Polyploidization results in an increase of the amount of DNA content giant cells, found that the cells were differentiated after six that bands became more blurred. DNA accompanied by the enlargement of nuclei and the expansion of cell size.

www.researchfeatures.com www.researchfeatures.com INTERCELLULAR LOCALISATION OF HISTONE VARIANT H3.3 IN Behind NUCLEUS OF TGC the Research Assistant Professor Koji Hayakawa

E: [email protected] T: +81 3-5841-7540 W: www.vm.a.u-tokyo.ac.jp/seika/ Research Objectives

Assistant Professor Koji Hayakawa of the University of Tokyo investigates cell- type specific epigenetic regulation.

Histone variant H3.3 was predominantly expressed in TGC, suggesting that this variant Detail constitutes the unique chromatin structures in their nucleus by replacing canonical H3.1/3.2. Koji Hayakawa, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, Japan Bio differentiated into TGCs, a decrease in Koji Hayakawa earned a BS in animal reproduction from Hirosaki University and binding DNA with histones was observed a MS from Hokkaido University. Eventually, he moved to the University of Tokyo in the nucleus. and received his PhD. He is currently an Assistant Professor in the Department of Animal Resource Sciences at the University of Tokyo.

Based on Dr Hayakawa’s research, it was Funding found that histone variants H2AX, H2AZ, Lotte Shigemitsu Prize and H3.3 were potentially responsible Key lab members for the formation of the loose nucleosome • Kanae Terada structure that was unique to TGCs. • Prof. Satoshi Tanaka When differentiated TSCs were prevented Collaborators for nucleosome stability analysis using microfluidic device from making H3.3, DNA remained tightly • Tomohiro Takahashi (Department of Mechanical Engineering, The University wound around histones but when H3.3 of Tokyo) protein levels were raised again in the • Assoc. Prof. Hidehiro Oana (Department of Mechanical Engineering, cells, the nucleosomes became more The University of Tokyo) ‘loose.’ In contrast, the research showed • Prof. Masao Washizu (Department of Mechanical Engineering, the converse effect with canonical The University of Tokyo) histone H3.1.

FUTURE DIRECTIONS References Dr Hayakawa believes that H3.3 may be a key player in maintaining the www.nature.com/articles/s41598-018-23832-2 polyploid state of the cell. In future, his https://currentprotocols.onlinelibrary.wiley.com/doi/ team hope to analyse H3.3 distribution abs/10.1002/9780470151808.sc01e04s32 in the genome of TGCs. In addition, https://orcid.org/0000-0003-0453-1303 the researchers hope to analyse the distribution of H2AZ and H2AX within the www.u-tokyo.ac.jp/focus/en/people/k0001_01650.html genome of TGCs and determine which histone variants make up the chromatin Personal Response regions observed in the nucleus of TGCs. What do you find most satisfying about your work? From our study using TGCs, the nucleus of polyploid cells contains quite different chromatin structures, compared to those of well-studied diploid cells. These structures involve the replacement of canonical histones with histone variants such as H2AX, H2AZ, and H3.3. Therefore, other polyploid cells such as megakaryocytes and cancer giant cells may also have a unique chromatin structure to allow an enormous amount of genomic DNA to fit in a nucleus and to express their specific functions including high cellular invasive activity.