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TBX2 and TBX3, which function as repressors, are members of the T-Box transcription factor family which are conserved throughout the metazoan lineage. TBX2 is highly expressed in rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children, and many other cancers. Previously, our lab dissected the oncogenic properties of TBX2 and its regulation of p14, p21 and PTEN. TBX3 is also expressed in some cancer types, however, its expression profile in RMS is severely down-regulated. TBX3 is shown to repress TBX2 in chondrocytes, but the characterization and regulation of TBX3 is poorly understood in the muscle lineage. Polycomb Repressive Complex 2 (PRC2), a gene silencing complex, acts to methylate histone H3 lysine 27 (H3K27me) of target gene promoters. The catalytic subunit of PRC2, EZH2, is up-regulated in RMS and data from our lab has shown that depletion of EZH2 up-regulated TBX3 and down-regulated TBX2 in C2C12 cells, an immortalized murine cell line. The hypothesis of this project was that there would be a PRC2-TBX3-TBX2 axis in RMS cells. To examine if TBX3 represses TBX2, TBX3 was transiently expressed in RMS cells representing both subtypes of RMS and we found that TBX2 was downregulated in each cell line. In a stable RH30 cell line with ectopic TBX3, TBX2 was down-regulated and PTEN expression was up-regulated. To determine if TBX2 repression by TBX3 was direct, a TBX3 ChIP assay was performed on the TBX2 promoter as well as the PTEN promoter. We found a strong enrichment of TBX3 on the TBX2 promoter but not on the PTEN promoter. Accordingly, we also observed that TBX3 over-expression impaired tumorigenesis through reduced cell proliferation, migration, and anchorage dependent growth. Also, we found that a stable RD cell line with ectopic TBX3 could promote differentiation, strongly suggesting that these results could have therapeutic value. Next, a shEZH2 plasmid was transfected into RMS cell lines ask if TBX3 was regulated by the PRC2 complex as we had observed in C2C12 cells. Just as we hypothesized, TBX3 was up-regulated and TBX2 was down-regulated. Similar to the previous TBX3 overexpression experiments, the EZH2 depleted RMS cell lines also showed decreased cell proliferation and migration rate. Also, an EZH2 knock down treatment induced differentiation in RMS cell lines. Therefore, understanding this potent regulation axis could provide an excellent opportunity for treatment of RMS cancer in the future.

In this work, MC1R, ASIP and TBX3 gene were tested on a sample of 118 Hucul horse mares included in Genetic Resources of Animals in the Czech Republic. We want to determine the genetic structure of mares and to analyse phenotypic data compared to the genotype and to identify possible differences between it. Genetic analysis showed a solid state for all alleles (HW for ASIP P = 0.9360, for MC1R P = 0.1661 and for TBX3 P = 0.4444). The frequency of the allele was as follows: E (0.6780), A (0.5254), and (0.4746), d2 (0.4323), d1 (0.3542), e (0.3220) D (0.2135). The most common genotype was AaEed1d2 and AaEEd1d2. There were very few or no genotypes based on recessive homozygotes in the genes of basic coat colours in the population, we didn´t identify any individual with genotype AaEed1d1. We have publicised genotype dependence within the TBX3 gene with primitive markings, confirming the previous work of other. Alele D was always associated with the occurrence of primitive markings, but primitive markings occur even without allele D in coincidence with the d1 allele. The d2d2 genotype is associated with a phenotype without primitive markings, or with phenotype where we can´t say if the horse has primitive markings or not.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第20536号 / 生博第378号 / 新制||生||50(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 豊島 文子, 教授 松本 智裕, 教授 井垣 達吏 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DGAM

Background: Biopacemaking is the attempt to replicate sinoatrial node (SAN)-like pacemaker activity in other areas of the heart by manipulating genes involved in pacemaking. Application of this could emulate the electronic pacemaker without the need for implantation of permanent hardware, or directly repair dysfunctional SAN tissue in human disease. We upregulated the transcription factors Tbx18, Tbx3 and the membrane ion exchanger NCX1 in bradycardic subsidiary atrial pacemaker (SAP) tissue which we used as a model of SAN dysfunction. We aimed to show that one or more of these gene targets could improve pacemaker function and alter the molecular character of SAP tissue and thus could potentially be used for the repair of dysfunctional SAN tissue. Methods: SAP tissue was isolated from the right atria of rats and kept beating in culture at 37°C for 48 hours. Recombinant adenoviruses were injected into SAP preparations to upregulate Tbx18, Tbx3 and NCX1 individually. Beating rate, overdrive suppression and pharmacological response to If blockade and β-adrenergic stimulation were measured along with molecular changes in pacemaker and atrial genes and proteins using RT-qPCR and immunohistochemistry. Results: Tbx18 upregulation significantly increased SAP beating rate after 48 hours of culture (a final rate of 141 ± 9 bpm in uninfected SAP tissue versus 215 ± 16 bpm in Ad-Tbx18 infected SAP tissue, p<0.01). It induced upregulation of HCN2 (p<0.01) and RYR2 (p<0.05), downregulation of HCN4 (p<0.05) and no change HCN1, Tbx3, Kv1.5, Kir2.1, Nav1.5, NCX1, Cx43, Cx45, Cav1.2 or Cav3.1. There was also no change in overdrive suppression and no change in response to pharmacology. No increase in beating rate was seen with either Tbx3 or NCX1 upregulation. Tbx3 preparations induced downregulation of the atrial genes Kir2.1 (p<0.01) and Nav1.5 (p<0.05), along with HCN1 (p<0.05), HCN4 (p<0.01), Tbx18 (p<0.05) and NCX1 (p<0.01), upregulated Cx43 (p<0.05) and showed no change in Cx45, RYR2, Kv1.5. NCX1 preparations demonstrated reduced overdrive suppression (p<0.05). Conclusion: Tbx18 showed the most potential for biopacemaking in SAP tissue, however both Tbx3 and NCX1 could be applied as secondary targets to fine tune biopacemaker function. Future work would focus on applying these targets to dysfunctional SAN tissue in larger animals.

Background: The canonical Wnt/β-catenin pathway is activated in a variety of heart diseases, such as myocardial infarction and cardiac hypertrophy, that are associated with altered ion channel expressions and increased risk of cardiac arrhythmias. Previous work from our lab has demonstrated that the Wnt/β-catenin signalling (Wnt signalling) inhibits sodium (Na+) current in rat cardiomyocytes. In this project, we aim to investigate the mechanisms that underlie the inhibition of Na+ current by Wnt signalling in both rat and human cardiomyocytes. Results: In both neonatal rat ventricular myocytes (NRVMs) and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), activation of the Wnt/β-catenin signalling led to reduced level of Na+ channel gene transcript (Scn5a), channel protein (Nav1.5) and channel current density. This suggests that reduced Scn5a expression is likely the primary mechanism for reduced Na+ current. In addition, we found that activation of the Wnt/β-catenin signalling in both NRVMs and iPSC-CMs upregulated Tbx3 transcript and protein levels, which is a transcription factor that is known to suppress Scn5a transcription. In NRVMs, siRNA-mediated Tbx3 knockdown attenuated (by ~30%) Wnt-induced reductions in Scn5a and Nav1.5 levels. Conclusions: Our findings are consistent with the conclusion that Wnt/β-catenin signalling inhibits Na+ current in both rat and human cardiomyocytes by reducing Scn5a levels, with Tbx3 as one of the mediators.

Domestic animals have rich phenotypic diversity that can be explored to advance our understanding of the relationship between molecular genetics and phenotypic variation. Since the advent of second generation sequencing, it has become easier to identify structural variants and associate them with phenotypic outcomes. This thesis details studies on three such variants associated with monogenic traits. The first studies on Rose-comb in the chicken were published over a century ago, seminally describing Mendelian inheritance and epistatic interaction in animals. Homozygosity for the otherwise dominant Rose-comb allele was later associated with reduced rooster fertility. We show that a 7.38 Mb inversion is causal for Rose-comb, and that two alleles exist for Rose-comb, R1 and R2. A novel genomic context for the gene MNR2 is causative for the comb phenotype, and the bisection of the gene CCDC108 is associated with fertility issues. The recombined R2 allele has intact CCDC108, and normal fertility. The dominant phenotype Greying with Age in horses was previously associated with an intronic duplication in STX17. By utilising second generation sequencing we have examined the genomic region surrounding the duplication in detail, and excluded all other discovered variants as causative for Grey. Dun is the ancestral coat colour of equids, where the individual is mostly pale in colour, but carries intensely pigmented primitive markings, most notably a dorsal stripe. Dun is a dominant trait, and yet most domestic horses are non-dun in colour and intensely pigmented. We show that Dun colour is established by radially asymmetric expression of the transcription factor TBX3 in hair follicles. This results in a microscopic spotting phenotype on the level of the individual hair, giving the impression of pigment dilution. Non-dun colour is caused by two different alleles, non-dun1 and non-dun2, both of which disrupt the TBX3-mediated regulation of pigmentation. Non-dun1 is associated with a SNP variant 5 kb downstream of TBX3, and non-dun2 with a 1.6 kb deletion that overlaps the non-dun1 SNP. Homozygotes for non-dun2 show a more intensely pigmented appearance than horses with one or two non-dun1 alleles. We have also shown by genotyping of ancient DNA that non-dun1 predates domestication.