Visualizing histone H4K20me1 in knock-in mice expressing the mCherry-tagged modification-specific intracellular antibody

During development and differentiation, histone modifications dynamically change locally and globally, associated with transcriptional regulation, DNA replication and repair, and chromosome condensation. The level of histone H4 Lys20 monomethylation (H4K20me1) increases during the G2 to M phases of the cell cycle and is enriched in facultative heterochromatin, such as inactive X chromosomes in cycling cells. To track the dynamic changes of H4K20me1 in living cells, we have developed a genetically encoded modification-specific intracellular antibody (mintbody) probe that specifically binds to the modification. Here, we report the generation of knock-in mice in which the coding sequence of the mCherry-tagged version of the H4K20me1-mintbody is inserted into the Rosa26 locus. The knock-in mice, which ubiquitously expressed the H4K20me1-mintbody, developed normally and were fertile, indicating that the expression of the probe does not disturb the cell growth, development, or differentiation. Various tissues isolated from the knock-in mice exhibited nuclear fluorescence without the need for fixation. The H4K20me1-mintbody was enriched in inactive X chromosomes in developing embryos and in XY bodies during spermatogenesis. The knock-in mice will be useful for the histochemical analysis of H4K20me1 in any cell types. Supplementary Information The online version contains supplementary material available at 10.1007/s00418-024-02296-8.

To track the changes in the levels and intranuclear localization of histone modifications during the cell cycle and during differentiation, we have developed a genetically encoded modification-specific intracellular antibody (mintbody) probe, which is the single-chain variable fragment (scFv) of a specific antibody tagged with a fluorescent protein (Kimura et al. 2015;Sato et al. 2021).Using the H4K20me1-specific mintbody (H4K20me1mintbody), we have demonstrated H4K20me1's cell cycle oscillation in HeLa cells and accumulation on inactivating X chromosomes in differentiating embryonic stem cells (ESCs) (Sato et al. 2016;Tjalsma et al. 2021).The mintbody probes can be expressed in model organisms, including yeast, nematodes, flies, frogs, and plants, indicating that mintbody expression does not affect development, differentiation, and fertility (Sato et al. 2021).However, the expression of mintbody in mammals has never been demonstrated.Here, we generated mice in which the H4K20me1-mintbody (a red fluorescent protein, mCherry version) is knocked into the Rosa26 locus (Soriano et al. 1999).Homozygous knock-in mice that exhibit H4K20me1-mintbody expression in various tissues developed normally and were fertile.The mintbody-expressing mice will be useful for visualizing and tracking the specific modification in any given cell type.

Generation of mouse embryonic stem cells and knock-in mice
Mouse care and experimental procedures were approved by the Institutional Animal Experiment Committee of the Tokyo Institute of Technology and the Animal Care and Use Committee of the Research Institute for Microbial Diseases, Osaka University.All animal experiments were conducted in accordance with institutional and governmental guidelines.Mice were maintained on a 12:12 dark/light cycle at a constant temperature of 22-23 ℃ in ventilated cages and were housed in a specific pathogen-free facility with free access to food and water.
HeLa cells were routinely maintained in Dulbecco's modified Eagle's medium, high glucose (DMEM; nacalai tesque) with 1% penicillin/streptomycin/glutamine solution (Sigma), and 10% fetal bovine serum (Thermo Fisher Scientific).Cells grown on a coverslip were transfected with Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's instructions and further grown for a day, before fixation, permeabilization, blocking, and staining with antibodies, as describe previously (Sato et al. 2016).Confocal images were acquired using a laser illumination system (LDI-NIR; Chroma Technology Japan) as described above.

Generation of mice expressing H4K20me1-mintbody
To generate mice expressing the H4K20me1-mintbody, we established ESC lines in which a FLP recombinaseexercisable Neo-resistant (Neo r ) gene cassette, flanking the H4K20me1-mintbody (mCherry version) coding sequence, was knocked into a Rosa26 allele (Fig. 1a).From chimeric mice generated by microinjecting knock-in ESCs into blastocysts, two independent germline-transmitted lines were cloned and then crossed with mice expressing FLP recombinase to delete the Neo r gene.The resulting homozygous H4K20me1-mintbody knock-in C57BL/6N mice (Fig. 1b) have been maintained over 5 years (tens of generations), indicating that H4K20me1-mintbody mice develop normally and were fertile.
We investigated whether H4K20me1-mintbody signals could be detected in knock-in mice.In a variety of tissues, including the ovary, kidney, brain, heart, spleen, liver, pancreas, and lung, H4K20me1-mintbody signals were detected without the need for fixation and thin-sectioning (Fig. 1c).Note that the distribution of H4K20me1mintbody was the same in the two independent knock-in lines.In most cells, H4K20me1-mintbody exhibited little specific concentrations in nuclei, consistent with the observation that H4K20me1 is not concentrated in constitutive heterochromatin.The fluorescence intensity of H4K20me1-mintbody in the nucleus varies among tissues, from the lowest in the brain to the highest in the pancreas.The difference in intensity might reflect different levels of H4K20me1, or it could be due to slight differences in the expression levels even the mintbody gene is knocked in to Rosa26 locus.Cytoplasmic spotty signals observed in some tissues, such as the brain and pancreas, may represent autofluorescence of cellular components such as lipofuscin (Schnell et al. 1999).The fluorescence of these spotty signals was also detected across multiple fluorescence channels using different laser lines, not just the mCherry channel with 555-nm excitation (Supplementary Fig. S1), consistent with the broad emission spectrum of autofluorescence (Schnell et al. 1999).
H4K20me1 is known to be concentrated in the inactive X chromosome (Xi) in cultured cells, including differentiating ESCs.However, no Xi-like foci were observed in adult female mouse tissues (Fig. 1c).To examine whether the distribution of H4K20me1 detected by a specific antibody is similar to that of H4K20me1-mintbody and is not concentrated on H3K27me3-rich Xi in adult tissues, tissue sections were prepared and stained with anti-H4K20me1 (Cy5), anti-H3K27me3 (Alexa Fluor 488), and anti-RFP (Cy3) for mCherry (Supplementary Fig. S2).Anti-H4K20me1 signals were not enriched on Xi, suggesting that H4K20me1 is no longer concentrated on Xi in non-dividing, differentiated cells, probably because monomethylation is converted to dimethylation by Suv420H1 during the cell cycle arrest  (Schotta et al. 2008;van Nuland and Gozani 2015;Corvalan and Coller 2021).Under the experimental conditions used in this study, neither H4K20me1-mintbody nor anti-RFP signals were observed in tissue sections (Supplementary Fig. S2).The antigen retrieval process, which is required for detecting histone modifications in tissue sections (Eberhart et al. 2012;Goto et al. 2024), may disrupt the retention of H4K20me1mintbody and/or its antigenicity to anti-RFP antibody.This disruption is suggested by the detection of H4K20me1mintbody in formaldehyde-fixed samples without antigen retrieval in meiotic cell spreads (see below), HeLa cells (Supplementary Fig. S3), and mouse embryonic carcinoma cells where H4K20me1-mintbody on Xi is still observed (Sato et al. 2016).Optimizing fixation and antigen retrieval conditions may enable the detection of H4K20me1mintbody in fixed tissues with antibody staining.

H4K20me1-mintbody highlights inactive X chromosomes in embryos
To visualize the distribution of H4K20me1-mintbody during development, we prepared preimplantation and postimplantation embryos, from 3.5 days post coitum (dpc) blastocysts to E14.5.As H4K20me1 is enriched in Xi in dividing cells (Kohlmaier et al. 2004;Calabrese et al. 2012;Sato et al. 2016;Tjalsma et al. 2021), it was expected to find H4K20me1-mintbody foci in female, but not in male, embryos.Indeed, H4K20me1-mintbody was concentrated in single foci in nuclei in some blastocysts, while it was distributed more homogenously in others (Fig. 2a), implying that H4K20me1 is concentrated on Xi in female embryo nuclei.In E6.5 and E7.5 post-implantation female embryos, most nuclei exhibit single foci (Fig. 2b and c, top; and Movie S1), while in male embryos, foci were not observed (Fig. 2b and  c, bottom; and Movie S2).In some nuclei in female embryos, foci of H4K20me1-mintbody were not clearly identifiable, which may be due to the background fluorescence from chromatin-free mintbody molecules.In later-stage female embryos, nuclear foci were still observed in most nuclei in E10.5, but disappeared in E14.5 (Fig. 2d).To confirm the H4K20me1-mintbody foci correspond to Xi and that its concentration decreases by E14.5, we stained wild-type mouse embryos using antibodies specific for H4K20me1 and H3K27me3, which is an Xi marker (Fig. 2e, f).As expected, H4K20me1 was concentrated in H3K27me3-erinched foci in most cells in E7.5 (Fig. 2e), but the co-localization of H4K20me1 and H3K27me3 was less clear in E14.5 (Fig. 2f).These data support the view that H4K20me1-mintbody represents the intranuclear distribution of H4K20me1, although signal-to-background ratios of H4K20me1-mintbody might be lower than those of antibody staining in fixed cells, because chromatin-free mintbody molecules could increase background fluorescence.

H4K20me1-mintbody highlights XY bodies in pachytene cells during spermatogenesis
To demonstrate that H4K20me1-mintbody represents the dynamic changes of H4K20me1 in vivo, we next visualized male mouse testis because H4K20me1 distribution is reported to dynamically change during the spermatogenesis (van der Heijden et al. 2007;Wang et al. 2021).At the pachytene stage, in particular, H4K20me1 is reported to be enriched in XY body, which consists of unsynapsed X and Y chromosomes and is subject to meiotic sex chromosome inactivation.When confocal sections were acquired for the P17.5 immature mouse testis, just isolated and placed on a glass bottom dish without fixation, a variety of H4K20me1-mintbody distribution patterns were observed in different cell nuclei (Fig. 3a and Movie S3).Near the basal membrane (z = − 3.0 μm), relatively large nuclei with higher fluorescence intensity and small interphase nuclei were observed (Fig. 3a, 1 and 2).Although additional characterizations are required for identifying the stages of these nuclei, the nuclei with higher intensity might be at the zygotene stage, arrested at G2 phase, because H4K20me1 levels are increased during G2 in somatic cells (Rice et al. 2002;Pesavento et al. 2008;Sato et al. 2016).The smaller nuclei might possibly represent spermatogonial cells or spermatocytes (van der Heijden et al. 2007;Nakata et al. 2015;Ueda et al. 2014).At a deeper section (z = 33 μm), pachytene cell nuclei with single foci were observed in addition to smaller interphase nuclei (Fig. 3a, 3 and 4).When a seminiferous tubule was pulled out of a P15.5 immature testis, a gradual change in H4K20me1mintbody's subnuclear distribution during differentiation was observed along with the tubule (Fig. 3b), from dividing cells showing condensed mitotic chromosomes (1) to cells showing interphase nuclei (2), and pachytene nuclei showing intense nuclear foci (3), possible zygotene nuclei (4).In a 1.5-month-old mouse, both in intact testis (Fig. 4a) and isolated tubules (Fig. 4b), pachytene nuclei with foci, and round and elongating spermatid nuclei, which are probably still at the premature stages before most histones are replaced with protamine (Hazzouri et al. 2000;Shirakata et al. 2014), were observed.These observations are consistent with the progression of spermatogenesis over months.To confirm whether H4K20me1-mintbody foci in pachytene nuclei correspond to XY bodies, meiotic cell spreads were stained with specific antibodies directed against synaptonemal complex protein SCP3 and γ-H2AX, the phosphorylated form of H2AX induced by DNA double strand breaks and a marker of XY body (Fernandez-Capetillo et al. 2003;van der Heijden et al. 2007).H4K20me1-mintbody signals were indeed enriched in γ-H2AX foci in SCP3-positive cells, although not all XY bodies exhibited H4K20me1minbtody concentration either in P15.5 and 4.5-month-old mice (Fig. 5).Thus, the distribution of H4K20me1 in differentiating testicular cells can be accurately detected by the H4K20me1-mintbody.

Conclusions
We demonstrate here that H4K20me1-mintbody allows us to visualize the localization of H4K20me1 in mouse tissues without the need for fixation.While any protein can, in principle, be visualized in mice by tagging with a fluorescent protein, visualizing posttranslational modifications in vivo has been challenging.Live-cell probes that detect specific modifications, such as mintbodies, have addressed this issue, yet only non-mammal organisms like flies and nematode expressing mintbodies have been developed.This is the first report to show that mammals expressing the mintbody throughout the entire body developed normally and were fertile.Although any ectopic probe that binds to specific modification could interfere with the binding of endogenous proteins, this suggest that the epigenetic readout system during development and differentiation is tolerant to a certain level of expression of a low-affinity binding probe.There are some limitations to using the current H4K20me1mintbody in mice.Firstly, the fluorescence level is not very high, and the signals sometimes have comparable levels to cellular autofluorescence in some tissues.Background fluorescence from diffused molecules may also hamper the sensitive detection of H4K20me1-enriched foci.A higher expression controlled by an exogenous promoter in the Rosa26 loci may be more suitable for high-resolution and time-lapse analyses.Secondly, H4K20me1-mintbody may not be detected in fixed and sectioned mouse tissues.For comparing the localization of H4K20me1 with other proteins or modifications, it may still be necessary to use a specific antibody directed against H4K20me1.Optimizing the fixation and antigen retrieval conditions may overcome this issue.In conclusion, we anticipate that mice expressing H4K20me1-mintbody will be useful for future work on the dynamics of this modification in vivo and also ex vivo histochemical analysis.

Fig. 1
Fig. 1 Generation of H4K20me1-mintbody knock-in mice.a Schematic drawing of the knock-in strategy.The targeting vector contains a splicing acceptor (SA), Neo resistant (Neo r ) gene flanked by two FRT sites for excision by FLP recombinase, and the H4K20me1mintbody positioned between the short and long arms of Rosa26 locus.Mouse ESCs harboring the knock-in allele were selected for Neo resistance.By crossing knock-in with FLP-expressing mice, H4K20me1-mintbody expression is achieved following the removal of Neo r .Sites for primers used to validate the knock-in and Neo r removal are indicated.b Validation of homozygous knock-in using

Fig. 2 Fig. 3 Fig. 4
Fig. 2 H4K20me1-mintbody in mouse embryos.a-d H4K20me1mintbody in unfixed mouse embryos at the blastocyst (a), E6.5 (b), E7.5 (c), and E10.5, E12.5, and E14.5 (d) stages were visualized using a confocal microscope.(a) The 3.5-dpc blastocyst-stage preimplantation embryos.Maximum intensity projections of three confocal sections are presented alongside bright-field images of embryo (top left insets) and magnified views of selected nuclei (top right and bottom left insets).Nuclei in probable female embryos exhibit single foci (open arrowheads; top panel).b E6.5 embryos.Single confocal sections are shown with magnified views of selected nuclei (insets).An open arrowhead indicates a focus (top panel).c E7.5 embryos.Low-and high-power views of single confocal sections are shown on the left and right, respectively.See Movie S1 and S2 for the full z-stack images of probable female and male embryos, respectively.d E10.5, E12.5, and E14.5 embryos.The sex of each embryo was determined by genomic PCR.The number of nuclei with foci appears to gradually decrease from E10.5 to E14.5.e, f Embryos were fixed and stained with antibodies specific for H3K27me3 and H4K20me1.DNA was counterstained with Hoechst 33342 before acquiring confocal images.The sex of each embryo was determined by genomic PCR.e E7.5 female embryo.Merged and magnified single-color views are shown on the left and right, respectively.H4K20me1 foci overlap with H3K27me3 foci.(f) E14.5 female embryo section.H4K20me1 is not concentrated in H3K27me3 foci ◂

Fig. 5
Fig. 5 H4K20me1-mintbody enriched foci correspond to XY bodies.Cells in seminiferous tubules from P15.5 and 2.5-month-old mice expressing H4K20me1-mintbody were spread, fixed, and stained with antibodies specific for γH2AX and SCP3.Max intensity projection images of confocal sections are shown