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To investigate the possible action of 5HT on HTR3A in the progression of tumors originating from sympathoadrenal cells, we analyzed several clones of human-derived neuroblastoma for HTR3A expression and tumorigenicity using an immunodeficient mouse model. Based on mRNA (Fig. 6a) and protein expression levels (Fig. 6b, c), the examined neuroblastoma cell lines could be characterized as either HTR3Ahigh (SH-SY5Y, CHLA-15, and CHLA-20), expressing markedly high levels of HTR3A, or HTR3Alow (NBL-28, NBL-38, and NBL-40), with only weak HTR3A expression. While all cell lines were negative for MYCN amplification (two copies of gene in the genome), HTR3A protein expression was associated with expression of major drivers of aggressive neuroblastomas, N-MYC and c-MYC44,45, or one of the core stemness factors SOX2 (Fig. 6b). Intriguingly, the same association was observed in NTERA-2 embryonal pluripotent carcinoma cells, which served as a positive control for HTR3A expression, and which are known to express high levels of N-MYC and share characteristics with early neural progenitors. To investigate the possible role of HTR3A receptor in regulation of proliferation in tumor cells, we treated the cells with the HTR3A agonists, N-methylquipazine dimaleate (NMQ) and SR57277, as well as antagonists, VUF10166 and granisetron HCl, in the presence of 5HT. HTR3A agonists dramatically limited proliferation of HTR3Ahigh cell lines, whereas they did not affect HTR3Alow cell lines, or the effects were seen only at much higher doses (Fig. 6d, e). No cleaved caspase-3 was detected after the treatment with NMQ, indicating that such treatment does not induce apoptosis (Supplementary Fig. 9). In contrast, there was no significant effect of HTR3A antagonists on cell growth of both HTR3Ahigh SH-SY5Y and HTR3Alow NBL-28 cell lines (Fig. 6f, g).
NOD/ShiLtSz-scid/Il2rγnull mice were used as a NSG model. All NSG mouse experiments were conducted in accordance with a study (21379/2011-30) approved by the Institutional Animal Care and Use Committee of Masaryk University and registered by the Ministry of Agriculture of the Czech Republic as required by national legislation.
We re-analyzed single-cell RNA-seq data of mouse adrenal gland from Furlan et al.6. Gene counts were obtained from GEO database (GSE99933). Gene count matrix was analyzed with a standard Seurat (version 3.0.2) workflow88. We used the original embeddings and clustering from ref. 6 (Figs. 5B and 5G), downloaded from the published pagoda apps: -bin/R/rook/nc.SS2_16_249-2/pathcl.json and -bin/R/rook/nc.SS2_16_250-2/pathcl.json (json slots embedding/data for the embedding and colcols/clusters/data for the cluster labels).
The raw and processed data of single-cell transcriptomic experiments generated in this study have been deposited in the GEO database under accession codes: GSE180861 (mouse), GSE195929 (human). The single-cell RNA-seq data of mouse adrenal gland from Furlan et al. (2017) used in this study are available in the GEO database under accession code GSE99933, mm10_ERCC genome used in this study is available in the RefSeq database under accession code GCF_000001635.20. The data other than RNA-seq data generated in this study are provided in the Source data file.
First we examined Xi chromatin modifications in vole TS cells described in our previous publication . The XX TS cell line (R1) was derived and maintained in the absence of FGF-4 and heparin, a condition that maintains the undifferentiated state of multipotent TS cells in mouse . In vole extraembryonic tissues, the paternal X-chromosome is inactivated ,  and the vole TS cells, like these of mice, demonstrate imprinted XCI and Xist RNA accumulation on Xi (Figure 1A) . The X-chromosome of M. levis is acrocentric and has a giant block of constitutive heterochromatin consisting of two repetitive sequences: MS3 (3265 bp) and MS4 (4088 bp), which contain mobile element fragments .
In mouse XX TS cells, Xi is known to be enriched with H3K27me3 and transiently associated with Polycomb repressive complex 2 (PRC2) which mediates H3K27 trimethylation . We expected to observe the same Xi chromatin structure in vole XX TS cells. Surprisingly, immunostaining of metaphase chromosomes with antibodies to H3K27me3 did not reveal this modification on Xi in vole undifferentiated TS cells. We also were not able to detect the EED protein, which is a subunit of PRC2 complex found on Xi in mouse (data not shown). Instead, we observed that Xi in vole undifferentiated TS cells was enriched with H3K9me3, HP1, H4K20me3, and uH2A, which were uniformly distributed along the entire Xi in contrast to somatic and XEN cells (Figure 1). These modifications were also detected in the constitutive heterochromatin of pericentromeric and telomeric regions. Interestingly, H3K9me3, H4K20me3, and HP1 were revealed as well in the heterochromatic block of the active X-chromosome but only in approximately 50 of 100 metaphase spreads analyzed (Figure 1E).
To eliminate a possibility that the differences in Xi chromatin structure between mouse and vole XX TS cells are due to different derivation and maintenance conditions, we obtained a new vole TS cell line according to the standard mouse TS cell protocol using FGF-4 and heparin. We found that these vole XX TS cell lines had the same gene expression and differentiation pattern as the R1 cell line (Figure S1). As in the R1 line, Xi in these undifferentiated TS cells was enriched with H3K9me3, HP1, H4K20me3, and uH2A, while both EED and H3K27me3 were not detected (Figure S2).
H3K27me3 enrichment typical of Xi in mouse blastocyst was not revealed at this stage in vole (Figure 6A). Surprisingly, we were able to detect H4K20me3, which demonstrated the same distribution pattern as H3K9me3 and HP1. However, the modification was shown not to be associated with Xi in mouse blastocyst . To reveal uH2A, we used FK2 antibodies to polyubiquitylated proteins that produced strong signals over the whole blastomeres so uH2A signals specific for Xi could not be observed. In case of TS cell metaphase spreads and nuclei, an accurate uH2A detection on Xi was possible due to using hypotonic treatment and cytospin technique. Thus, Xi chromatin in vole preimplantation development is enriched with H3K9me3, HP1, and H4K20me3 but not with H3K27me3 and is very similar to that in undifferentiated vole XX TS cells.
Next, the chromatin modification pattern in vole postimplantation development was established. In mouse, implantation is accompanied by reactivation of the paternal X-chromosome in inner cell mass and maintenance of imprinted XCI in trophectoderm and its derivatives . Later stages of imprinted XCI in vole were analyzed in extraembryonic ectoderm of 7,5-day cryosectioned embryos. H3K27me3 accumulation was revealed in all extraembryonic ectoderm cells of some embryos and allowed males to be distinguished from females. In female extraembryonic ectoderm, there were no foci of H3K9me3, HP1, and H4K20me3. Therefore, the vole Xi at postimplantation stages was characterized by H3K27me3 recruitment and reduced levels of H3K9me3, HP1, and H4K20me3 (Figure 6C). This pattern was different from that during preimplantation development but reminiscent of that in differentiated vole XX TS cells.
Xi in vole undifferentiated TS cells derived under various conditions was found to contain repressive chromatin markers H3K9me3, HP1, H4K20me3, and uH2A distributed uniformly, while H3K27me3, a well known component of Xi chromatin in mouse TS cells, was not detected. It has been previously shown that two distinct types of facultative heterochromatin are present on the vole Xi at late stages of both imprinted XCI in XEN cells and random XCI in adult cells . One Xi heterochromatin type is enriched with H3K27me3 and uH2A and located in gene-rich regions, whereas the other one consists of H3K9me3, HP1, and H4K20me3 and is present in gene-poor repeat containing regions. The two heterochromatin types are also found on Xi in adult cells of humans and cattle , . Thus, Xi chromatin organization in undifferentiated TS cells differs from that in cells demonstrating later stages of imprinted XCI and random XCI. The distribution pattern of the Xi repressive modifications (except for H4K20me3) in undifferentiated vole TS cells was similar to mammalian XY-body chromatin during MSCI , , , . This is confirmed by our study of the chromatin modifications during spermatogenesis in M. levis. Three of the modifications, H3K9me3, HP1, and H4K20me3, are known to be permanent components of constitutive heterochromatin and observed not only on Xi but also in heterochromatic centromeric and telomeric regions of the other chromosomes in vole TS cells. Surprisingly, uH2A was also revealed together with H3K9me3, HP1, and H4K20me3 in the repeat containing heterochromatic regions of chromosomes, suggesting that histone ubiquitylation is not specific for X-chromosome silencing only but can also take part in constitutive heterochromatin formation at certain developmental stages. Thus, it appears that heterochromatin repression with H3K9me3, HP1, H4K20me3, and uH2A at early developmental stages in vole is a genome-wide mechanism, rather than being specific to the paternal X-chromosome.
Imprinted XCI in mouse was demonstrated to be established in two steps. The first step is a silencing of repeats which occurs independently of Xist at the 2-cell stage, while the genes of the paternal X-chromosome remain active , , , , . The second step is Xist-dependent gene silencing which occurs gradually in preimplantation development. The inactive state of repeat elements is proposed to be inherited from the paternal germline, whereas the genic silencing is established de novo in imprinted XCI , . Two different states of Xi heterochromatin found during imprinted XCI in vole might correspond to the two steps of imprinted XCI revealed in mouse. If so, the pattern of Xi modifications at early stages of imprinted XCI may represent a mechanism required for silencing that involves repeat elements, and Xi chromatin at later stages of imprinted XCI may correspond to the gene silencing. Interestingly, the repressive chromatin modifications are uniformly distributed along the vole Xi including both gene- and repeat-rich regions, although only repeats are silenced at first steps of imprinted XCI. Taking into account that the maternal X-chromosome is two-fold up-regulated beginning at the zygote stage , it is tempting to speculate that the set of modifications also take part in decreasing gene expression on the paternal X-chromosome to achieve a balance between expression levels of X-linked and autosomal genes, which is important during mammalian ontogenesis . 2b1af7f3a8